Complement components (C3, C4) in childhood asthma
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《美国医学杂志》
1 Departments of Laboratory Medicine, Faculty of Medicine, Al-Arab Medical University, Benghazi, Libya
2 Departments of Pediatrics, Faculty of Medicine, Al-Arab Medical University, Benghazi, Libya
Abstract
Objective: To assess the involvement of complements (C3, C4) in the pathophysiology of bronchial asthma; Methods: Selection of patients (n=64) were made according to the recommended international criteria for diagnosis and classification of asthma. Serum levels of complement components (C3, C4) were measured by radial immunodiffusion technique in 64 Libyan children (age: 1-12 years, sex: 39 males, 25 females) with mild to moderately severe asthma (Group A). Among these patients, 35 had active disease (AA) and 29 had inactive disease (NA). According to age range, 20, 21 and 23 patients were between 1-3 years (A1), > 3-5 years (A2) and > 5-12 years (A3) respectively. A1 had 9 and 11 patients with active (AA1) and inactive (NA1) disease; A2 had 10 and 11 patients with active (AA2) and inactive (NA2) disease; A3 had 16 and 7 patients with active (AA3) and inactive (NA3) disease respectively. Age matched comparisons were made with 57 healthy children (age: 1-12 years; sex: 30 males, 27 females) (Group B). Among the controls, 15, 19 and 23 children were between 1-3 years (B1), > 3-5 years (B2) and > 5-12 years (B3) respectively. Results: Mean C3 level was significantly elevated in patients, while C4 level was normal (A vs B à C3: P < 0.02, C4: P > 0.2). Serum C3 level was significantly higher in patients with active disease only, while it was normal in patients with inactive disease (AA,NA,B à P = 0.045); AA vs NA à P < 0.05, AA vs B à P < 0.02, NA vs B à P > 0.05) and C4 levels were normal in both the groups (AA,NA,B à P = 0.354). Further, C3 levels were significantly elevated in all the age groups, but in patients with active disease only (AA1, NA1, B1 à P = 0.0024; AA2, NA2, B2 à P = 0.0411; AA3, NA3, B3 à P =0.0102). Conclusion: The elevated C3 level was possibly due to induction by pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1). The probable mechanisms of C3 involvement in the pathophysiology of bronchial asthma were discussed.
Keywords: Complement; Asthma
Asthma is a clinical syndrome characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli, which may be spontaneous, allergen-related or drug-induced, and the primary pathophysiological abnormality being bronchial wall inflammation leading to airway narrowing.[1],[2],[3] The fundamental question that needs to be answered in bronchial asthma is "What are the molecular mechanisms through which all the diverse etiological factors to triggering agents are linked to the common pathophysiology of airway inflammation" Studies focusing upon inflammation of the airway and the immune responses at cellular and molecular levels have led to the proposition of a number of mechanisms such as mast cell degranulation,[3],[4] neurogenic dysfunction,[5] involvement of T-lymphocytes and eosinophils,[6],[7] altered immunosuppressive macrophages [8],[9], excessive nitric oxide (NO) through inducible nitric oxide synthase (iNOS),[10] overproduction of proinfla-mmatory cytokines[10],[11] and immunoglobulins (IgE, IgG & IgD).[12] The role of the complement system in asthma was suggested possibly through initiation and/or amplification of the inflammatory response of the complement cascade.[13],[14] If this system is involved in the pathogenesis of asthma, serum levels of the complement components are expected to be altered. Complements in asthma have since been studied to a great extent, although reports were conflicting. Some studies have reported significantly increased serum C3 levels whereas others have demonstrated no change.[15],[16],[17] Secondly, literature survey showed that no immunological studies were done or reported in childhood asthma in Libyans, although in adult Libyans some studies were reported.[18],[19] The present study was therefore undertaken to assess the serum levels of complements (C3, C4) in childhood asthma in Libyans.
Materials and methods
Patients and Healthy Controls: A group of 64 children with bronchial asthma (age:1-12 years, mean ± SD: 4.24 ± 2.25 years; sex: 39 boys, 25 girls) obtained randomly from Pediatric Allergy Clinic and Out-patients department at Al-Fatah Children′s Hospital, Benghazi, Libya, over a period of one year were included in the study (Group A). Among them, 35 patients (17 males, 18 females) were classified as having mild to moderately active disease (AA) and the remaining 29 symptom-free patients (22 males, 7 females) classified as having inactive disease (NA) according to the recommended criteria for diagnosis of asthma.[1],[20],[21] The patients were also divided into three groups according to their age range at inclusion (i.e. A1: 1-3 years, 20 patients: 15 males, 5 females; A2:> 3-5 years, 21 patients: 12 males, 9 females: A3: > 5-12 years, 23 patients: 12 males, 11 females). These patients were further classified into six groups according to age range as well as disease activity at inclusion, i.e. AA1 (9 patients: 6 males, 3 females); NA1 (11 patients: 9 males, 2 females); AA2 (10 patients: 5 males, 5 females); NA2 (11 patients: 7 males, 4 females); AA3 (16 patients: 6 males, 10 females) and NA3 (7 patients: 6 males, 1 female). None of these patients were on steroid therapy, systemic or local, for at least 4-6 weeks prior to inclusion in the study. A total of 57 normal Libyan children without family history of asthma, atopy and any of other serious illnesses (age: 1-12 years, mean ±SD: 4.08 ±2.1 years; sex: 30 males, 27 females) were recruited from Ben-Zaher Polyclinic, Benghazi, Libya as healthy controls (Group B). This group B subjects were divided into three sub-groups according to age range, i.e. B1: 1-3 years (8 males, 7 females), B2:> 3-5 years (12 males, 7 females), and B3:> 5-12 years (10 males, 13 females), for age-matched comparison.
Blood Specimens: After obtaining consent, a 10 ml aliquot of blood was extracted form the antecubital vein of each subject and collected part of it in tubes without, and part of it in tubes with, anticoagulant for routine tests as well as immunological assays. The separated sera were stored frozen at -30o C until required for analysis.
Complements (C3, C4): Serum levels of complements (C3, C4) were estimated by using commercially available immunokits of biomerieux, France based on the principle of radial immunodiffussion.[22] The inter-assay and intra-assay coefficient of variation were = 1.7 % and = 2.6 % respectively.
Statistical Analysis: The statistical significance of the results were evaluated by Student′s ′t′ test, One-way analysis of variance (ANOVA) and Chi-Square (c2) test.[23] The observed values were log transformed to normalize the skewed data and then Student′s t test, ANOVA and calculations for 95% Range and 95% Confidence interval for mean(CIM) were carried out and the results were expressed in the original unit by taking antilog.
Results
C3 & C4 Levels in Patients (Group A) and Controls (Group B): Mean serum C3 level was significantly higher in patients, whereas C4 level was not different compared to controls table1. The proportion of patients with abnormal serum levels above the normal (95%) range was also significantly higher for C3, while no difference was observed for C4 compared to controls (A vs B à C3: c2 =11.08, df=1, P< 0.001, C4: c2 =2.73, df=1, P> 0.05).
C3 & C4 Levels According to Disease Activity: Serum levels (Mean ± SD) of C3 and C4 in patients (AA, NA) and controls (B) and their statistical analysis are presented in Figure1. Serum mean level of C3 was significantly higher in AA only (AA, NA, B à C3: P= 0.04. C4: P = 0.354). In AA, NA and B, 10/35(28.5%), 5/29(17.2%) and 2/57(3.5%) subjects respectively had abnormal C3 levels above the normal (95%) range which was significant, but only 2/35 (5.7%), 1/29(3.4%) and 0/57 (0%) subjects respectively had abnormal C4 levels above the normal (95%) range which was not significant [AA, NA, B à c2 (C3): 11.67, P < 0.005 ; c2 (C4): 2.89, P> 0.1].
C3 & C4 Levels According to Age Range: Serum mean levels of C3 and C4 according to age range in patients (A1, A2, A3) and controls (B1, B2, B3) are stated in table2. Mean serum C3 levels were significantly higher in all the age ranges of patients compared to respective age- matched controls. The proportion of patients with abnormal serum levels above the normal (95%) range was also significantly higher for C3 in all the age groups (A1 vs B1 à c2 =4.04, P < 0.05; A2 v B2 à c2 = 3.82, P < 0.05; A3 vs B àc2 = 5.59, P < 0.025). No significant differences were observed for C4 levels in any of the age groups (P > 0.05).
C3 levels According to Disease Activity As Well As Age Range: Serum levels (Mean ± SD) of C3 according to age range as well as disease activity and their statistical analysis are shown in Figure2. Mean serum levels of C3 were significantly higher in patients with active disease only in all the age groups.
Discussion
Our asthmatic patients had higher serum C3 level while C4 level was within normal range. In fact C3 level was elevated in patients with active disease, while patients with non-active disease had normal C3 level. When patients were divided according to age, all age groups with active disease only had significantly raised C3 level. Some reported studies were supported by the present findings that C3 level was increased in asthmatic patients.[16], [24], [25]
Some of the complement breakdown products are known anaphylatoxins with a potential for causing mediator release from mast cell as well as potent chemo-attractant.[25], [26] Complement activation may therefore be considered as a possible contributor to bronchospasm and inflammation in asthma.[16] Several investigators studied the concentrations of C3, C3d and C3d/C3 ratio in asthmatic patients and they found elevation in C3 and C3d levels.[16], [25] They observed in some of these patients a raised C3d/ C3 ratio associated with lower C4 levels compared with those without an increased ratio. This suggested that classical pathway activation was occurring in certain asthmatic patients.[25] On the contrary, Schifferli et al observed lower level of C1q with low C4 in asthmatic patients and this suggested the greater involvement of the classical pathway compared to alternative pathway of complement cascade.[26]
Several researchers reported elevated levels of TNF-αin BALF, sputum and in serum of asthmatic patients.[11],[27],[28] These findings in patients with active disease were suggestive of possible involvement of TNF-αas the inflammatory component in active asthma. TNF-αand 1L-1 are potent inducers of endothelial leucocyte adhesion molecular-1 (ELAM-1) and intracellular leucocyte adhesion molecule-1(ICAM-1) on endothelial cells and these adhesion molecules (ELAM-1, ICAM-1) play an important role in inflammation through induced diapedesis of inflammatory cells from the blood into the tissue.[29],[30]
Barnes and Liew proposed that inducible nitric oxide synthase (iNOS) may be induced in airway epithelial cells and possibly macrophages by exposure to proinflammatory cytokines (TNF -a, IL-Ib) and the large amount of nitric oxide (NO) generated in the airways epithelium results in proliferation of TH2 type T-lymphocytes leading to availability of TH2 type cytokines and thus asthmatic inflammation is amplified and perpetuated.[10] Poulter et al proposed that a failure in the activity of an immunosuppressive T-cell regulatory macrophage may lead to excessive T-cell activation and chronic inflammation which might exacerbate bronchial hyperreacivity to a state where symptomatic asthma is generated.[8] Thus, possibly either due to deficiency of regulatory subtype of alveolar macrophage or defective over-expression of genes for inflammatory cytokines, particularly for TNF-a, on activation of airway resident macrophages, iNOS may be induced leading to increased production of NO and hence symptoms of asthma.
Conclusion
The primary site of biosynthesis for the majority of the complement components is the hepatocytes and more then 90% of plasma complements are derived from liver.[31] The present study′s finding of elevated C3 level in patients with active disease was therefore suggestive that complement biosynthesis was induced in the liver of asthmatic patients possibly due to systemic inflammatory changes in TNF-αand 1L-1. The development of inhibitors particularly specific to TNF-αor IL-I or iNOS may present a nobel therapeutic approach for asthma and possibly for other diseases as well, although cytokine network is getting increasingly complex.[4], [32], [33] It was evident from the present study and the literature that a number of cellular and molecular mechanisms including the complement cascade are linked to the common pathophysiology of bronchial asthma.[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[33]
Acknowledgement
The authors would like to thank the Faculty of Medicine, Al-Arab Medical University, Benghazi, Libya for financial support. Thanks are also due to Dr. Baker S. EIShahabi, Senior Registrar, for his help in obtaining the patients from the Allergy Clinic and Outpatients, Al- Fatah Children′s Hospital, Benghazi, Libya.
References
1. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987; 136: 225-244.
2. Pride NB. Definition and clinical spectrum. In: Barnes PJ (eds). Br Med Bull (Asthma) 1992; 48: 1-9.
3. Jeffery PK. Pathology of asthma. In Barnes PJ, ed. Br Med Bull (Asthma) 1992; 48: 23-39.
4. Chapel H, Haeney M. Mishbah J, Snowden N, eds. Essentials of Clinical Immunology, 4th edn. Oxford, Blackwell Science, 1999: 10-14.
5. Busse WW, Calhoun WF, Sedgwick JD. Mechanism of airway inflammation in asthma. Am Rev Respir Dis 1993; 147: 520-524.
6. Corrigan CJ, Key AB. T-cell and eosinophils in the pathogenesis of asthma. Immunol Today 1992; 13(2): 501-7.
7. Kline JN, Hunninghake GW. T-lymphocyte dysregulation in asthma. Proc Soc Exp Biol Med 1994; 207 (3): 243-253.
8. Poulter LW, Janossy G, Power C et al. Immunological/ Physiological relationships in asthma: Potential regulation by lung macrophages. Immunol Today 1994; 15 (6): 258-261.
9. Sipteri MA, Poulter LW. Characterization of immune inducer and suppressor macrophage from normal human lung. Clin Exp Immunol 1991; 83: 157-162.
10. Barnes PJ, Liew FV. Nitric oxide and asthmatic inflammation. Immunol Today 1991; 16 (3): 128-130.
11. FIE Najam, ASM Giasuddin, AH Shembesh. Tumour necrosis factors in childhood Asthma. Indian J Pediatr 2001; 68 (3): 217 -222.
12. FIE Najam, ASM Giasuddin, AH Shembesh. Immunoglobulin isotypes in childhood asthma. Indian J Pediatr 1999; 66 (3):337-344.
13. Golub ES, Green DR, eds. Immunology a synthesis, 2nd edn. Sunderland; Sinauer Associates Inc, 1991; 117-132.
14. Roitt V, Borstaff J, Male D, eds. Immunology , 3rd edn. St. louis; Mosby, 1993; 12.1-12.17.
15. Barnes PJ, Chung KF, Page CP. Inflammatory mediators in asthma. Pharmacol Rev 1988; 40: 49-84.
16. Lin RY, Caveliere LF, Lorenzana FG et al. Pattern of C3, C3b and C3d in patients hospitalized with acute asthma. Ann Allergy 1992; 68: 324-330.
17. Kirshchifink M, Gastrof FM, Rother U et al. Complement activation and C3 allotype distribution in patients with bronchial asthma. Int Arch Allergy Immunol 1993; 100 (2): 151- 155.
18. Giasuddin ASM, Ziu MM, Basha SA, Abusedra A. Serum immunoglobulin and complement profiles in bronchial asthma in Libyans. J Islam Acad Sci 1989; 2: 118-122.
19. Giasuddin ASM, Boryswick G, Basha A. Serum IgE levels in Libyan patients with Bronchial asthma. Garyounis Med J 1988; 11(1-2): 18-24.
20. British Thoracic Society. Guidelines on the management of asthma. Thorax 1993; 48 (Suppl 2): S1-S4.
21. Global Initiative for Asthma. Global strategy for asthma management and prevention: NHLB/WHO Workshop Report; Bethesda: National Institute of Health; 1996: 57-59.
22. Mancini G, Carbonare AQ, Hereman JF. Immunological quantitation of antigens by single radial immunodiffusion. Int J Immunochem 1965; 2: 235-254.
23. Kirkwood BR. Essentials of Medical Statistics, 1st edn. (reprinted), Oxford; Blackwell Scientific Publications, 1989.
24. Onyemelukwe GC. Complement components in Nigerians with bronchial asthma. Ann Allergy 1989; 63: 309-312.
25. Michel O, Sergysels R, Duchateau J. Complement activation in bronchial asthma evaluated by the C3d/C3 index. Ann Allergy 1986; 57(6): 405-408.
26. Schifferli JA, Ng YC, Peters DK. The role of complement and its receptors in elimination of immune complexes. N Engl J Med 1989; 315(8): 488-496.
27. Kips JC, tavernier IH, Joos GF et al. The potential role of tumor necrosis factor-a in asthma. Clin Exp Allergy 1993; 23: 247 - 50.
28. Taki F, Toriik K, Ikata N et al. Increased levles of TNF-a concentration in supta of patients with bronchial asthma. Am Rev Respir Dis 1991; 143: A13
29. Pober JS, Giarbron MA, Lupierre LA et al. Overlapping patterns of activation of human endothelial cells by interleukin-1, tumor necrosis factor and immune interferon. J Immunol 1986; 137: 1890-1896.
30. Bevilacqua MP, Pober JS, Mendrick DI et al. Identification of an inducible endothelial leucocyte adhesion molecule. Proc Natl Acad Sci USA 1987; 84: 9238-9242.
31. Morgan BP, Gasque P. Expression of complement in the brain: role in health and disease. Immunol Today 1996; 17(10): 461-466.
32. Balkwill FR, Burke F. The cytokine network. Immunol Today 1989; 10 (2): 299-302.
33. Boyton RJ, Altmann DM. Asthma: new developments in cytokine regulation. Clin Exp Immunol 2004; 136: 13-14.(Najam F I E, Giasuddin A )
2 Departments of Pediatrics, Faculty of Medicine, Al-Arab Medical University, Benghazi, Libya
Abstract
Objective: To assess the involvement of complements (C3, C4) in the pathophysiology of bronchial asthma; Methods: Selection of patients (n=64) were made according to the recommended international criteria for diagnosis and classification of asthma. Serum levels of complement components (C3, C4) were measured by radial immunodiffusion technique in 64 Libyan children (age: 1-12 years, sex: 39 males, 25 females) with mild to moderately severe asthma (Group A). Among these patients, 35 had active disease (AA) and 29 had inactive disease (NA). According to age range, 20, 21 and 23 patients were between 1-3 years (A1), > 3-5 years (A2) and > 5-12 years (A3) respectively. A1 had 9 and 11 patients with active (AA1) and inactive (NA1) disease; A2 had 10 and 11 patients with active (AA2) and inactive (NA2) disease; A3 had 16 and 7 patients with active (AA3) and inactive (NA3) disease respectively. Age matched comparisons were made with 57 healthy children (age: 1-12 years; sex: 30 males, 27 females) (Group B). Among the controls, 15, 19 and 23 children were between 1-3 years (B1), > 3-5 years (B2) and > 5-12 years (B3) respectively. Results: Mean C3 level was significantly elevated in patients, while C4 level was normal (A vs B à C3: P < 0.02, C4: P > 0.2). Serum C3 level was significantly higher in patients with active disease only, while it was normal in patients with inactive disease (AA,NA,B à P = 0.045); AA vs NA à P < 0.05, AA vs B à P < 0.02, NA vs B à P > 0.05) and C4 levels were normal in both the groups (AA,NA,B à P = 0.354). Further, C3 levels were significantly elevated in all the age groups, but in patients with active disease only (AA1, NA1, B1 à P = 0.0024; AA2, NA2, B2 à P = 0.0411; AA3, NA3, B3 à P =0.0102). Conclusion: The elevated C3 level was possibly due to induction by pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1). The probable mechanisms of C3 involvement in the pathophysiology of bronchial asthma were discussed.
Keywords: Complement; Asthma
Asthma is a clinical syndrome characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli, which may be spontaneous, allergen-related or drug-induced, and the primary pathophysiological abnormality being bronchial wall inflammation leading to airway narrowing.[1],[2],[3] The fundamental question that needs to be answered in bronchial asthma is "What are the molecular mechanisms through which all the diverse etiological factors to triggering agents are linked to the common pathophysiology of airway inflammation" Studies focusing upon inflammation of the airway and the immune responses at cellular and molecular levels have led to the proposition of a number of mechanisms such as mast cell degranulation,[3],[4] neurogenic dysfunction,[5] involvement of T-lymphocytes and eosinophils,[6],[7] altered immunosuppressive macrophages [8],[9], excessive nitric oxide (NO) through inducible nitric oxide synthase (iNOS),[10] overproduction of proinfla-mmatory cytokines[10],[11] and immunoglobulins (IgE, IgG & IgD).[12] The role of the complement system in asthma was suggested possibly through initiation and/or amplification of the inflammatory response of the complement cascade.[13],[14] If this system is involved in the pathogenesis of asthma, serum levels of the complement components are expected to be altered. Complements in asthma have since been studied to a great extent, although reports were conflicting. Some studies have reported significantly increased serum C3 levels whereas others have demonstrated no change.[15],[16],[17] Secondly, literature survey showed that no immunological studies were done or reported in childhood asthma in Libyans, although in adult Libyans some studies were reported.[18],[19] The present study was therefore undertaken to assess the serum levels of complements (C3, C4) in childhood asthma in Libyans.
Materials and methods
Patients and Healthy Controls: A group of 64 children with bronchial asthma (age:1-12 years, mean ± SD: 4.24 ± 2.25 years; sex: 39 boys, 25 girls) obtained randomly from Pediatric Allergy Clinic and Out-patients department at Al-Fatah Children′s Hospital, Benghazi, Libya, over a period of one year were included in the study (Group A). Among them, 35 patients (17 males, 18 females) were classified as having mild to moderately active disease (AA) and the remaining 29 symptom-free patients (22 males, 7 females) classified as having inactive disease (NA) according to the recommended criteria for diagnosis of asthma.[1],[20],[21] The patients were also divided into three groups according to their age range at inclusion (i.e. A1: 1-3 years, 20 patients: 15 males, 5 females; A2:> 3-5 years, 21 patients: 12 males, 9 females: A3: > 5-12 years, 23 patients: 12 males, 11 females). These patients were further classified into six groups according to age range as well as disease activity at inclusion, i.e. AA1 (9 patients: 6 males, 3 females); NA1 (11 patients: 9 males, 2 females); AA2 (10 patients: 5 males, 5 females); NA2 (11 patients: 7 males, 4 females); AA3 (16 patients: 6 males, 10 females) and NA3 (7 patients: 6 males, 1 female). None of these patients were on steroid therapy, systemic or local, for at least 4-6 weeks prior to inclusion in the study. A total of 57 normal Libyan children without family history of asthma, atopy and any of other serious illnesses (age: 1-12 years, mean ±SD: 4.08 ±2.1 years; sex: 30 males, 27 females) were recruited from Ben-Zaher Polyclinic, Benghazi, Libya as healthy controls (Group B). This group B subjects were divided into three sub-groups according to age range, i.e. B1: 1-3 years (8 males, 7 females), B2:> 3-5 years (12 males, 7 females), and B3:> 5-12 years (10 males, 13 females), for age-matched comparison.
Blood Specimens: After obtaining consent, a 10 ml aliquot of blood was extracted form the antecubital vein of each subject and collected part of it in tubes without, and part of it in tubes with, anticoagulant for routine tests as well as immunological assays. The separated sera were stored frozen at -30o C until required for analysis.
Complements (C3, C4): Serum levels of complements (C3, C4) were estimated by using commercially available immunokits of biomerieux, France based on the principle of radial immunodiffussion.[22] The inter-assay and intra-assay coefficient of variation were = 1.7 % and = 2.6 % respectively.
Statistical Analysis: The statistical significance of the results were evaluated by Student′s ′t′ test, One-way analysis of variance (ANOVA) and Chi-Square (c2) test.[23] The observed values were log transformed to normalize the skewed data and then Student′s t test, ANOVA and calculations for 95% Range and 95% Confidence interval for mean(CIM) were carried out and the results were expressed in the original unit by taking antilog.
Results
C3 & C4 Levels in Patients (Group A) and Controls (Group B): Mean serum C3 level was significantly higher in patients, whereas C4 level was not different compared to controls table1. The proportion of patients with abnormal serum levels above the normal (95%) range was also significantly higher for C3, while no difference was observed for C4 compared to controls (A vs B à C3: c2 =11.08, df=1, P< 0.001, C4: c2 =2.73, df=1, P> 0.05).
C3 & C4 Levels According to Disease Activity: Serum levels (Mean ± SD) of C3 and C4 in patients (AA, NA) and controls (B) and their statistical analysis are presented in Figure1. Serum mean level of C3 was significantly higher in AA only (AA, NA, B à C3: P= 0.04. C4: P = 0.354). In AA, NA and B, 10/35(28.5%), 5/29(17.2%) and 2/57(3.5%) subjects respectively had abnormal C3 levels above the normal (95%) range which was significant, but only 2/35 (5.7%), 1/29(3.4%) and 0/57 (0%) subjects respectively had abnormal C4 levels above the normal (95%) range which was not significant [AA, NA, B à c2 (C3): 11.67, P < 0.005 ; c2 (C4): 2.89, P> 0.1].
C3 & C4 Levels According to Age Range: Serum mean levels of C3 and C4 according to age range in patients (A1, A2, A3) and controls (B1, B2, B3) are stated in table2. Mean serum C3 levels were significantly higher in all the age ranges of patients compared to respective age- matched controls. The proportion of patients with abnormal serum levels above the normal (95%) range was also significantly higher for C3 in all the age groups (A1 vs B1 à c2 =4.04, P < 0.05; A2 v B2 à c2 = 3.82, P < 0.05; A3 vs B àc2 = 5.59, P < 0.025). No significant differences were observed for C4 levels in any of the age groups (P > 0.05).
C3 levels According to Disease Activity As Well As Age Range: Serum levels (Mean ± SD) of C3 according to age range as well as disease activity and their statistical analysis are shown in Figure2. Mean serum levels of C3 were significantly higher in patients with active disease only in all the age groups.
Discussion
Our asthmatic patients had higher serum C3 level while C4 level was within normal range. In fact C3 level was elevated in patients with active disease, while patients with non-active disease had normal C3 level. When patients were divided according to age, all age groups with active disease only had significantly raised C3 level. Some reported studies were supported by the present findings that C3 level was increased in asthmatic patients.[16], [24], [25]
Some of the complement breakdown products are known anaphylatoxins with a potential for causing mediator release from mast cell as well as potent chemo-attractant.[25], [26] Complement activation may therefore be considered as a possible contributor to bronchospasm and inflammation in asthma.[16] Several investigators studied the concentrations of C3, C3d and C3d/C3 ratio in asthmatic patients and they found elevation in C3 and C3d levels.[16], [25] They observed in some of these patients a raised C3d/ C3 ratio associated with lower C4 levels compared with those without an increased ratio. This suggested that classical pathway activation was occurring in certain asthmatic patients.[25] On the contrary, Schifferli et al observed lower level of C1q with low C4 in asthmatic patients and this suggested the greater involvement of the classical pathway compared to alternative pathway of complement cascade.[26]
Several researchers reported elevated levels of TNF-αin BALF, sputum and in serum of asthmatic patients.[11],[27],[28] These findings in patients with active disease were suggestive of possible involvement of TNF-αas the inflammatory component in active asthma. TNF-αand 1L-1 are potent inducers of endothelial leucocyte adhesion molecular-1 (ELAM-1) and intracellular leucocyte adhesion molecule-1(ICAM-1) on endothelial cells and these adhesion molecules (ELAM-1, ICAM-1) play an important role in inflammation through induced diapedesis of inflammatory cells from the blood into the tissue.[29],[30]
Barnes and Liew proposed that inducible nitric oxide synthase (iNOS) may be induced in airway epithelial cells and possibly macrophages by exposure to proinflammatory cytokines (TNF -a, IL-Ib) and the large amount of nitric oxide (NO) generated in the airways epithelium results in proliferation of TH2 type T-lymphocytes leading to availability of TH2 type cytokines and thus asthmatic inflammation is amplified and perpetuated.[10] Poulter et al proposed that a failure in the activity of an immunosuppressive T-cell regulatory macrophage may lead to excessive T-cell activation and chronic inflammation which might exacerbate bronchial hyperreacivity to a state where symptomatic asthma is generated.[8] Thus, possibly either due to deficiency of regulatory subtype of alveolar macrophage or defective over-expression of genes for inflammatory cytokines, particularly for TNF-a, on activation of airway resident macrophages, iNOS may be induced leading to increased production of NO and hence symptoms of asthma.
Conclusion
The primary site of biosynthesis for the majority of the complement components is the hepatocytes and more then 90% of plasma complements are derived from liver.[31] The present study′s finding of elevated C3 level in patients with active disease was therefore suggestive that complement biosynthesis was induced in the liver of asthmatic patients possibly due to systemic inflammatory changes in TNF-αand 1L-1. The development of inhibitors particularly specific to TNF-αor IL-I or iNOS may present a nobel therapeutic approach for asthma and possibly for other diseases as well, although cytokine network is getting increasingly complex.[4], [32], [33] It was evident from the present study and the literature that a number of cellular and molecular mechanisms including the complement cascade are linked to the common pathophysiology of bronchial asthma.[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[33]
Acknowledgement
The authors would like to thank the Faculty of Medicine, Al-Arab Medical University, Benghazi, Libya for financial support. Thanks are also due to Dr. Baker S. EIShahabi, Senior Registrar, for his help in obtaining the patients from the Allergy Clinic and Outpatients, Al- Fatah Children′s Hospital, Benghazi, Libya.
References
1. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 1987; 136: 225-244.
2. Pride NB. Definition and clinical spectrum. In: Barnes PJ (eds). Br Med Bull (Asthma) 1992; 48: 1-9.
3. Jeffery PK. Pathology of asthma. In Barnes PJ, ed. Br Med Bull (Asthma) 1992; 48: 23-39.
4. Chapel H, Haeney M. Mishbah J, Snowden N, eds. Essentials of Clinical Immunology, 4th edn. Oxford, Blackwell Science, 1999: 10-14.
5. Busse WW, Calhoun WF, Sedgwick JD. Mechanism of airway inflammation in asthma. Am Rev Respir Dis 1993; 147: 520-524.
6. Corrigan CJ, Key AB. T-cell and eosinophils in the pathogenesis of asthma. Immunol Today 1992; 13(2): 501-7.
7. Kline JN, Hunninghake GW. T-lymphocyte dysregulation in asthma. Proc Soc Exp Biol Med 1994; 207 (3): 243-253.
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