Role of Complements and Immunoglobulins in Type 1 Diabetes Mellitus

Background: The aim of the present study was to determine the role of complements and immunoglobulins in normal healthy population and patients having Type 1 diabetes mellitus (T1DM). Methods: A total of 26 patients with T1DM and 20 healthy controls were included in the study. The sample was collected from OPD of Department of Endocrinology, SS Hospital, IMS, BHU, Varanasi. Immunoglobulins and complements estimation was done by turbidometry method. Result: A total of 26 patients with T1DM between 8 yrs to 33 yrs age were studied. Out of 26 cases, 19.2% cases had low C4 and 3.9% had elevated serum C4 value. Mean value of serum C4 was significantly reduced in T1DM. The patients having low C4 value belonged to the age group less than 18 years. About 23.1% DM patient had increased serum IgM levels and 7.7% had increased serum IgA levels. But only rise of IgM was statistically significant. C3 and IgG in DM patients were within normal range. There was significant positive correlation of serum C4 with increase BMI. IgA was significantly correlated with rising of BMI and low age of onset of T1DM. Conclusion: Our study concludes that C4 deficiency associated with Type I DM. The study also showed that increased IgM which may be due to recent infection. *Corresponding author: Shailja Singh, C/O Prof. Usha, Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, UP, India Phone: +91 9415455259 E-mail: [email protected] Original Article A-55 AABS; 3(1): 2016 Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016 e-ISSN: 2349-6991; p-ISSN: 2455-0396 Introduction Type I Diabetes Mellitus (T1DM) is one of the most common chronic diseases in childhood. The incidence of T1DM in India is 3.0 per 100000 populations (1). It is divided into two subtypes, i.e. autoimmune type-1 (type1A or T1A) diabetes and idiopathic (type-1B or T1B) diabetes (2). Several types of autoantibody to pancreatic islet cells have been recognized as markers of T1A diabetes. These islet-related autoantibodies include islet cell antibodies (ICA), anti-GAD antibodies (GADAb), insulin autoantibodies (IAA), and anti-insulinomaassociated antigen 2 antibodies (IA-2Ab). Patients with T1B diabetes who do not have islet autoantibodies at the time of diagnosis are classified as having idiopathic. Prevalence of T1DM is highest in Finland 57.6 per 100000 population. Contrary to this incidence of TI DM in China and India is 0.6 and 3.0 per 100000 population (3, 4). The incidence of T1DM is increasing, either because of environmental factors accelerating onset of the disease or because of inducement of autoimmune diabetes. A major environmental risk factor disclosed by epidemiological studies is infection in the development of type-1 diabetes (5, 6, 7, 8, 9). The DM patients are very sensitive for infection. The mortality rate of DM patients with an infection and ketoacidosis is 43% (10, 11). Gillani, et al. (2011) conducted the study in Penang, Malaysia and found that every third patient with diabetes mellitus had infectious exposure (11). Although the mechanism of this increased incidence is less well established, studies of the immune cells of DM patients have demonstrated significant defects in both humoral and cellular function (12, 13). Immunoglobulins mediated humoral adaptive immune system and provide protection against infections. Hence, the aim of the present study was to determine the complements and immunoglobulins in normal healthy population and patients with T1DM and also to identify their deficiency and elevation related to the GAD positivity. Materials and Methods A total of 26 patients with T1DM and 20 healthy controls were included in the study. The sample was collected from OPD of Department of Endocrinology, SS Hospital, IMS, BHU, Varanasi. • Characteristics of the patients: Patients (26) Control (20) Males: Females 17: 9 12:8 Patients (26) Control (20) Age (years)* 21.31±6.33 31.45±11.02 Diabetes Mellitus (DM): T1DM Body Mass Index (BMI) (kg/m2) 17.77±3.40 (10.42-23.78) Disease Duration (Years) 2.06± 3.23 (0.0816) Age at onset (Years) 19.25± 6.37 (630.92) *Results expressed as mean ± SD Laboratory Analysis Immunoglobulins and complements estimation was done by turbidometry method. The kit was supplied by Spinrect, Spain through Avadh Scientific, Lucknow. Glutamic acid decarboxylase (GAD) estimation was done by indirect enzyme linked immunosorbent assay (ELISA). The kit was supplied by Medyzyn, through Vishat Diagnostic , Lucknow. Data Analysis All data were analyzed using Statistical Package for Social Sciences (SPSS, Chicago, Illinos, USA version 16),. Statistical tests such as, Student ‘t test’ and one way ANOVA, were done to compare the mean values of different groups. Bivariate correlation was done to compare the multiple groups. P values less than 0.05 was taken as significant for the analysis. Result The mean value of complement C4 was significantly decreased in T1DM as compared to controls (17.47±11.59 vs 23.67±5.70 mg/dl; P 0.0336). Although only 19.2% T1DM patient had decreased C4 value as compared to control (5%) (Table-I). All reduced cases of C4 were observed in below 18 years of age (Table-II). IgG in T1DM patients was within normal range (Table-II) and there was no association with age of the patient (TableIV). The mean value of IgM was significantly increased in T1DM patients as compared to controls. Out of 26 cases, 23.1% had increased serum IgM levels and all controls were within normal range (Table V). Correlation between age of T1DM patients and IgM showed that elevated value of IgM was varied in various age group, about 11.11% in below 18 years; 33.33% in between 18-25 years and 20% in patients age greater than 25 years (Table-VI). The mean value of IgA was within normal in majority of cases (92.3%) (Table VII). Correlation between age of T1DM patients with serum IgA showed no significant change (Table-VIII). Original Article A-56 http://www.pacificejournals.com/aabs Significant positive correlation was observed among C4 and IgA with increased BMI and serum IgA with age of onset of disease. There was no correlation among IgG and IgM with age of onset, family history, disease duration and BMI (Table-IX). Mean values of complement and immunoglobulins was decreased in GAD positive patients but only IgA reduction was statistically significant in GAD positive patienrs from GAD negative cases (Table-X). Table I: Serum C4 level in patients with T1DM and control group Groups <10 mg/dl No (%) 10-40 mg/dl No (%) >40 mg/dl No (%) Mean ± SD mg dl p-value A T1DM (26) 5(19.2) 20(76.9) 1 (3.9) 17.47±11.59 0.0336 B Control (20) 1(5.0) 19(95.0) 0(0.0) 23.67±5.70 Table II: Correlation of Age of T1DM patients and C4 level. Age of the patients Total (26) No (%) <10 mg/dl No (%) 10-40 mg/dl No (%) >40 mg/dl No (%) Mean±SD mg/dl p-value <18 9 5 (55.56) 4 (44.44) 0 (0.0) 10.92±8.89 0.014* 18-25 12 0 (0.0) 11 (91.67) 1(8.33) 24.38±12.31 >25 5 0 (0.0) 5 (100) 0 (0.0) 13.73±4.19 *statistically significant (p<0.05) Table III: Serum IgG level in patients with T1DM and control group Groups <600 mg/dl No (%) 600-1600 mg/dl No (%) >1600 mg/dl No (%) Mean±SD mg/dl p-value A T1DM (26) 0 (0.0) 26 (100) 0 (0.0) 1176.30±206.67 0.215 B Control (20) 0 (0.0) 20 (100) 0 (0.0) 1216.20±178.21 Table IV: Correlation of Age of T1DM patients and Serum IgG level. Groups Total (26) No (%) <700 mg/dl No (%) 600-1600 mg/dl No (%) >1600 mg/dl No (%) Mean±SD mg/dl p-value <18 9 0 (0.0) 9(100) 0 (0.0) 998.43±371.79 0.180 18-25 12 0 (0.0) 12(100) 0 (0.0) 1221.15±168.67 >25 5 0 (0.0) 5(100) 0 (0.0) 1137.66±205.46 *statistically significant (p<0.05) Table V: Serum IgM level in patients with T1DM and control group Groups <40 mg/dl No (%) 40-230 mg/dl No (%) >230 mg/dl No (%) Mean±SD mg/dl p-value A T1DM (26) 0 (0.0) 20 (76.9) 6 (23.1) 174.59±66.15 0.036* B Control (20) 0 (0.0) 20 (100.0) 0 (0.0) 135.02±55.25 *statistically significant (p<0.05) Table VI: Correlation of Age of T1DM patients and Serum IgM level. Groups Total (26) No (%) <40 mg/dl No (%) 40-230 mg/dl No (%) >230 mg/dl No (%) Mean±SD mg/dl p-value <18 9 0 (0.0) 8 (88.89) 1 (11.11) 175.39±53.65 0.821 18-25 12 0 (0.0) 8 (66.67) 4 (33.33) 180.91±75.80 >25 5 0 (0.0) 4 (80) 1(20.0) 158.01±72.96 A-57 AABS; 3(1): 2016 Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016 e-ISSN: 2349-6991; p-ISSN: 2455-0396 Table VII: Serum IgA level in patients with T1DM and control group Groups <70 mg/dl No (%) 70-400 mg/dl No (%) >400 mg/dl No (%) Mean±SD mg/dl p-value A T1DM (26) 0 (0.0) 24 (92.3) 2 (7.7) 255.39±105.65 0.201 B Control (20) 0 (0.0) 20 (100) 0 (0.0) 216.55±93.71 Table VIII: Correlation of Age of T1DM patients and Serum IgA level. Groups Total (26) No (%) <70 mg/dl No (%) 70-400 mg/dl No (%) >400 mg/dl No (%) Mean±SD mg/dl p-value <18 9 0 (0.0) 9 (100) 0 (0.0) 204.99±70.47 0.114 18-25 12 0 (0.0) 11 (91.67) 1 (8.33) 264.22±120.79 >25 5 0 (0.0) 4 (80.0) 1(20.0) 324.95±87.80 Table IX : Correlation of complement and immunoglobulins with clinical detail of T1DM BMI Disease duration Age of onset Family history C4 r 0.404* 0.057 0.007 0.067 p .040* .781 .972 0.747 IgG r 0.039 -0.006 -0.197 -0.189 p 0.851 0.978 0.335 0.356 IgM r 0.134 0.002 -0.146 -0.237 p 0.513 0.991 0.477 0.244 IgA r 0.584** 0.013 0.525** 0.066 p 0.002* 0.949 0.006* 0.747 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed). Table X: Correlation of GAD positivity with complement and immunoglobulins in T1DM Patients. Group Total (26) C4 IgG IgM IgA No (%) (mg/dl) (mg/dl) (mg/dl) (mg/dl) GAD Ab Positive 9 13.35±10.81 1114.8±280.85 161.95±54.21 172.74±48.76 GAD Ab Negative 17 19.65±11.69 1208.85±155.07 181.29±72.32 299.16±101.85 t value 1.3401 1.1090 0.7020 3.4930 p value 0.1928 0.2784 0.4894 0.0019* *statistically significant (p<0.05) Discussion The complement proteins play an important role in both innate and adaptive immune responses (14). Dysregulation of the complement system has been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus erythematosus, vasculitides, Sjogren’s syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and rheumatoid arthritis (15). In present study the mean value of C4 was decreased in T1DM patients as compared to controls (17.47±11.59 vs 23.67±5.70 mg/ dl; P 0.0336). Previous studies were also found the low concentration of serum complement C4 in T1DM (16, 17, 18, 19, 20). We observed that 19.2% cases were below the normal range in T1DM whereas in control 5% below the normal range. Analysis of C4 with reference to age and GAD positivity showed that all C4 reduced cases were present in younger age (below 18 years) and they were GAD positive. Chiarelli et al. (1988) were demonstrated that the mean value of C4 was significantly lower in T1DM Original Article A-58 http://www.pacificejournals.com/aabs than in control (27.99 +/8.01 vs 32.03 +/8.91 mg/dl; P < 0.01). They found 25% low serum levels of C4 in T1DM (20). The lack of serum concentrations may help to identify a subgroup (diabetic microangiopathy) of T1DM patients at high risk of developing diabetic nephropathy (16, 17, 20, 21). There are very few studies on Immunoglobulin in T1DM patients. In our study, we found that the mean value of IgM and IgA was increased in T1DM patients as compared to controls whereas IgG was within normal range. Out of 26 cases of T1DM, about 23.1% had increased serum IgM levels and 7.7% had high concentration of serum IgA. Similar to our study, other researchers also found high concentration of IgM and IgA in T1DM patients than in control subjects or siblings(22). Correlation between age of T1DM patients and immunoglobulin showed that concentration of IgM and IgA were increased with increasing the age of patients. IgA deficiency is relatively common in T1DM patients (23, 24, 25). However, immunoglobulin deficiency was not present in our study. Previously, it has been demonstrated that concentration of IgM increased in 21% cases and IgG decreased in 11% cases in age of onset between 20 and 40 years, whereas in our study IgM was increased in 30.77% and IgG was normal in onset between 20-40 years (22). Svensson et al (2012) found low concentration of IgG and higher concentration of IgA levels, whereas no differences in IgE or IgM value in newly diagnosed T1DM (26). Elevated levels of serum IgMand IgA suggested that diabetic patients suffered from bacterial infections (27, 28, 29). The changes in total Immunoglobulin concentrations at onset were largely reversed under insulin therapy. Rise of immunoglobulin may be due to environmental causes, such as viral infections, or insulinopenia prior to clinical disease onset (22). Patients with T1DM have infections more often than those without DM (10, 11, 12, 30). In our study, we found that serum concentration of complement C4 and serum IgA was increased with rising of BMI and rising of disease onset. There was no correlation among IgG and IgM with age of onset, family history, disease duration and BMI. Mean values of complement and immunoglobulins were decreased in GAD positive patients. Conclusion From our study, we conclude that C4 deficiency was present in T1DM and it was related with lower age and GAD positivity. The study also showed that recent infection was the reason for higher concentration of IgM in T1DM. Thus our study concluded that T1DM patients are very sensitive for infection. Acknowledgements We are thankful to UGC Advanced Immunodiagnostic Training and Research Centre and National Facility For Tribal and Herbal Medicine (NFTHM) Centre for financial support. Funding We are thankful to UGC Advanced Immunodiagnostic Training and Research Centre and National Facility For Tribal And Herbal Medicine (NFTHM) Centre for financial support. Competing Interests : No Reference 1. International Diabetes Federation; IDF Diabetes Atlas; 6th edn.; International Diabetes Federation; 2013; http://www.idf.org/diabetesatlas ISBN: 2-930229-853. 2. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation. Diabet Med. 1998; 15:539–553. 3. Maahs DM, West NA, Lawrence JM and Mayer-Davis EJ. Epidemiology of type 1 diabetes. Endocrinol Metab Clin North Am. 2010; 39: 481–97. 4. International Diabetes Federation. IDF Diabetes Atlas. 6th ed. International Diabetes Federation. 2013. 5. Graves PM, Norris JM, Pallansch MA, Gerling IC, Rewers M. The role of enteroviral infections in the development of IDDM: limitations of current approaches. Diabetes. 1997; 46:161–168. 6. Vreugdenhil GR, Schloot NC, Hoorens A et al. Acute onset of type I diabetes mellitus after severe echovirus 9 infection: putative pathogenic pathways. Clin Infect Dis. 2000; 31: 1025– 1031. 7. Yoon JW, Austin M, Onodera T, Notkins AL. Virusinduced diabetes mellitus. N Engl J Med. 1979; 300:1173–1179. 8. Jun HS, Yoon JW. The role of viruses in type I diabetes: two distinct cellular and molecular pathogenic mechanisms of virus-induced diabetes in animals. Diabetologia.2001; 44:271–285. 9. Imagawa A, Hanafusa T, Makino H, Miyagawa J I and Juto P. High titres of IgA antibodies to enterovirus in fulminant type-1 Diabetes. Diabetologia. 2005; 48: 290–293. A-59 AABS; 3(1): 2016 Annals of Applied Bio-Sciences, Vol. 3; Issue 1: 2016 e-ISSN: 2349-6991; p-ISSN: 2455-0396 10. Deresinski S. Infections in the diabetic patient: Strategies for the clinician. Infect. Dis. Rep., 1995; 1: 1-12. 11. Gillani S W, Sulaiman SAS, Sundram S, Sari YO, Baig M, and Iqbal MS. Factors associated with infections in diabetic population. African Journal of Pharmacy and Pharmacology, 2011; 5(11): 1414-1421. 12. Geerlings SE, Hoepelman AI. Immune dysfunction in patients with diabetes mellitus (DM). FEMS Immunol Med Microbiol. 1999; 26:259-65. 13. Moutschen MP, Scheen AJ, Lefebvre PJ. Impaired immune responses in diabetes mellitus: analysis of the factors and mechanisms involved. Relevance to the increased susceptibility of diabetic patients to specific infections. Diabete Metab. 1992;18: 187-201. 14. Dunkelberger JR, Song WC. Complement and its role in innate and adaptive immune responses. Cell Res. 2010; 20(1):34-50. 15. Ballanti E, Perricone C, Greco E, Ballanti M, Di Muzio G, Chimenti MS, Et Al. Complement And Autoimmunity. Immunol Res. 2013; 56(2-3):477-91. 16. Mcmillan DE. Elevation Of Complement Components In Diabetes Mellitus. Diabete Metab. 1980; 6(4):265-70. 17. Barnett AH, Mijovic C, Fletcher J, Chesner I, Kulkuska-Langlands BM, Holder R et al. Low plasma C4 concentrations: association with microangiopathy ill insulin-dependent diabetes. Br Med J. 1984; 289 : 943-945. 18. Bertrams J, Hintzen U, Schlicht V, Schoeps S. C41 another marker for type I diabetes. Lancet. 1982; I : 41. 19. Cooper ME, Duff R, Buchanan R, McPherson J, Jerums G. Low serum C4 concentrations and microangiopathy in type I and type II diabetes. Br Med J. 1986; 292 : 801. 20. Chiarelli F, Verrotti A, La Penna G, Morgese G. Low serum C4 concentrations in type-1 diabetes mellitus. Eur J Pediatr. 1988; 147(2):197-8. 21. Flyvbjerg, Allan. Diabetic angiopathy, the complement system and the tumor necrosis factor superfamily. Nature Reviews Endocrinology. 2010; 6: 94-101. 22. Gorus FK, Vandewalle CL, Winnock F, Lebleu F, Keymeulen BV, et al. Increased prevalence of abnormal immunoglobulin M, G, and A concentrations at clinical onset of insulin-dependent diabetes mellitus: A registry-based study. The Belgian Diabetes Registry. Pancreas. 1998; 16:50–9. 23. Cser A, Ambrus M, Bajtai G, Heim T, Varga F. Juvenile diabetes mellitus and selective IgA deficiency. Ovr Heti. 1974; 1115:803-806. 24. Penny R, Thompson RG, Polmar SM, Schultz RB. Pancreatic malabsorption and IgA deficiency in a child with diabetes. Pediatrics.1971; 78:512-516 25. Silver HKB, Shuster J, Gold P, Hawkins D, Freedman SO. Endocrinopathy and IgA deficiency. Clin Immunol Immunopath. 1973; 1 : 212-219. 26. Svensson J, Eising S, Mortensen HB, Christiansen M, Laursen I, Lernmark A et al. High levels of immunoglobulin E and a continuous increase in immunoglobulin G and immunoglobulin M by age in children with newly diagnosed type 1 diabetes. 2012; 73(1):17–25. 27. Eguchi K, Yagame M, Suzuki D, et al. Significance of high levels of serum IgA and IgA-class circulating immune complexes (IgA-CIC) in patients with noninsulindependent diabetes mellitus. J DiabetComplications. 1995; 9: 42–48. 28. Rodriguez-Segade S, Camina MF, Carnero A, et al. High serum IgA concentrations in patients with diabetes mellitus: agewise distribution and relation to chronic complications. Clin Chem. 1996; 42: 1064– 1067. 29. Islam Laila N, Mahmud Hossain and M. Shamim H. Zahid. Complement mediated bactericidal activity and humoral immune response in type 2 diabetes mellitus. Int J Diabetes Metab. 2006; 14: 92-97. 30. Peleg AY, Weerarathna T, McCarthy JS, Davis TM. Common infections in diabetes: pathogenesis, management and relationship to glycaemic control. Diabetes Metab Res Rev. 2007; 23:3-13.