|Year : 2014 | Volume
| Issue : 2 | Page : 137-142
Study of complement activation, C3 and interleukin-6 levels in burn patients and their role as prognostic markers
S Modi1, M Rashid2, A Malik3, M Shahid3
1 Department of Microbiology, ICARE Institute of Medical Sciences & Research and Dr. B. C. Roy Hospital, Haldia, West Bengal, India
2 Department of Microbiology, SMS and R, Sharda University, Greater Noida, Uttar Pradesh, India
3 Department of Microbiology, J. N. Medical College, AMU, Aligarh, Uttar Pradesh, India
|Date of Submission||25-Feb-2013|
|Date of Acceptance||28-Oct-2013|
|Date of Web Publication||2-Apr-2014|
Department of Microbiology, ICARE Institute of Medical Sciences & Research and Dr. B. C. Roy Hospital, Haldia, West Bengal
Source of Support: Indian Council of Medical Research, New Delhi, Conflict of Interest: None
Purpose: The management of burn patients is always challenging for the clinician due to high risk of bacterial sepsis, multi-organ failure and death. Our objective was to study complement activation, C3 and interleukin-6 (IL-6) levels in burn patients and evaluate their role as prognostic markers. Materials and Methods: A total of 63 burn patients and 60 healthy controls were included in this study. Blood was collected from patients within 24 h and at 7 th day of injury. Complement activation was determined by crossed electrophoresis and counter-current immunoelectrophoresis. C3 levels were measured using a single radial immunodiffusion. IL-6 was detected by ELISA. Results: All patients showed initial complement activation. Mean C3 levels showed an inverse correlation with the severity of burn. Patients with ≥20% burns had lower C3 than the controls (P < 0.001) and those with <20% burns (P < 0.001). Patients with ≥40% burns had activated complement and low C3 in 2 nd week; they subsequently developed infection. Complement was inactive and C3 levels recovered in patients with <40% burns. The non-survivors showed significantly lower C3 than the survivors (P < 0.05) in 2 nd samples. Patients who developed infection had C3 significantly lower than those who remained free of infection (P < 0.05). All patients showed initial elevation in IL-6 levels. Patients with ≥60% burns had significantly higher IL-6 than controls (P < 0.001) and those with <60% burns (P < 0.001). Non-survivors had higher IL-6 than survivors in both samples (P < 0.001). Patients who developed infection showed significantly higher IL-6 in 2 nd samples than those without infection (P < 0.001). Conclusions: Complement activation, C3 and IL-6 levels correlated well with the severity of injury and development of infection in burn patients. These parameters can be used to predict the onset of infection, septicaemia and mortality in burn patients.
Keywords: Complement activation, C3, crossed electrophoresis, interleukin-6, single radial immunodiffusion
|How to cite this article:|
Modi S, Rashid M, Malik A, Shahid M. Study of complement activation, C3 and interleukin-6 levels in burn patients and their role as prognostic markers. Indian J Med Microbiol 2014;32:137-42
|How to cite this URL:|
Modi S, Rashid M, Malik A, Shahid M. Study of complement activation, C3 and interleukin-6 levels in burn patients and their role as prognostic markers. Indian J Med Microbiol [serial online] 2014 [cited 2019 Dec 15];32:137-42. Available from: http://www.ijmm.org/text.asp?2014/32/2/137/129793
| ~ Introduction|| |
Immunosuppression following burn injury has generated a lot of interest in recent times because of its observed influence on the prognosis of the burn patients. Inhibition of phagocytosis and suppression of humoral and cell mediated immunity are well recognised consequences of burn injury. Many of these alterations have been shown to correlate with an increased rate of sepsis in the post injury period. ,,
Tissue injury leads to complement activation in both humans and animals, with subsequent utilisation and depletion of the complement components, mainly C3, C4 and C5. ,,, Chances of bacterial infections increase in the presence of persistently reduced complement levels. , Failure of C3 levels to return to normal within specified time have been reported to invariably indicate septic complications. ,,
Recently, it has become clear that interleukin-6 (IL-6), earlier known as hybridoma growth factor, is involved in systemic changes associated with tissue injury and infection.  IL-6 is released by a variety of lymphoid and non-lymphoid cells such as T cells, B cells, monocytes, fibroblasts, keratinocytes, endothelial cells, mesangium cells and several tumour cells. IL-6 is released by the damaged tissues and it induces the synthesis of proteins in the liver (acute phase proteins) that protect the host against inflammatory reactions. 
However, most aspects of the effects of burn injury on complement and cytokine functions are still under investigation. Further studies are needed to determine the role of complement activation and cytokine stimulation following burn injury.
In this study, we analysed serum complement activation, C3 levels and IL-6 levels in relation to the severity of injury as well as clinical outcome in burn patients.
| ~ Materials and Methods|| |
This case-control study, approved by our Institutional Ethics Committee, was conducted in the Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India, during the period August 2006 to December 2007. Informed consent was obtained from the patients or their relatives for participation in the study. Treatment given to the patients remained unaffected by the outcome of the study.
A total of 63 recently burned patients and 60 healthy controls were included in this study. Patients with various categories of burns (electrical, thermal and chemical) were included and assessed for the severity of burn injury by calculating the percentage of total burnt body surface area (% TBSA).  The following types of cases were excluded from the study group: Patients with obvious infections at other sites, patients with a recent history of diseases such as malaria/typhoid/viral hepatitis/other infectious diseases, and patients with chronic diseases such as diabetes mellitus/tuberculosis/chronic obstructive pulmonary disease/malignancies etc.
A detailed clinical history was taken at admission and physical examination was carried out to record body temperature, blood pressure, pulse rate, respiratory rate, body weight and urinary output, in addition to the extent of burn injury.
A total volume of 5 ml blood was collected by venepuncture from each patient within 24 h of injury and a second sample on the 7 th day following burn injury. Blood was also collected from the members of the control group. Serum was separated and stored at −20°C until further tested. Complement activation was studied using counter-current immunoelectrophoresis  using anti-C3 antiserum (Sigma-Aldrich Ltd., USA). 3 mm wells were punched 1 cm apart on agarose overlay gel. Anti-C3 antiserum was added to the wells on the anodic side, while those on the cathodic side contained the serum samples. Electrophoresis was performed at 50 V for 45 min. Presence of two lines in between the antigen-antibody wells was considered to indicate activated complement and the two lines were taken to be of C3 and C3b split product [Figure 1]. The results were confirmed by crossed electrophoresis,  wherein on a pre-set gel plate, wells were punched on the cathodic side. Serum samples were added to the wells and electrophoresis was performed at 50 V for 90 min. A narrow strip of the gel containing the separated components of the serum was cut and placed on another plate which was overlayed with 1% agarose containing anti-C3 antiserum. The separated components were electrophoresed again at right angles at 50 V for 1 h. The presence of two peaks, corresponding to C3 and C3b complement fraction, indicated complement activation [Figure 2]. Presence of a single line in counter current immunoelectrophoresis and a single peak in crossed electrophoresis indicated inactive complement. C3 levels in the sera of patients and controls were measured using single radial immunodiffusion test  using anti-C3 antiserum (Sigma-Aldrich Ltd., USA). 2% molten agarose and anti-C3 antiserum (in 1:16 dilution) were mixed in equal quantities and the solution was overlayed on a glass slide. Wells were punched in the gel and serum samples were introduced in them. The slide was incubated at 37°C for 1-2 h in a humid chamber and then at 4°C for 72 h. The slides were examined for rings of precipitation [Figure 3]. The squares of the diameters of the precipitin rings around the wells were measured and plotted on a graph against standard C3 levels to get a quantitative estimation of serum C3 levels. Detection and quantitative measurement of IL-6 was done by microwell ELISA technique using AviBion Human IL-6 ELISA kit (Orgenium Laboratories, Finland).
|Figure 1: Counter-current Immunoelectrophoresis to detect complement activation; ← = 2 lines indicating activated complement ← = Single line indicating inactive complement|
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|Figure 2: Crossed electrophoresis to detect complement activation. Presence of 2 peaks of C3 and C3b indicates activated complement|
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All the patients were monitored in the post-burn period for the following clinical signs and symptoms of septicaemia, noting only their presence or absence without quantifying them: Fever, wound infection, chest infection, subphrenic or pelvic abscess, renal failure, mental disorientation, bleeding diathesis and features of shock. Blood culture was performed whenever required to confirm septicaemia. Wound swab culture was done to isolate the organism causing wound infection.
Results are expressed as complement status: Activated/inactive; C3 levels − mg/dl ± standard deviation; IL-6 levels − pg/ml ± standard deviation.
Statistical analysis was performed manually using Chi-square and unpaired t-tests. P < 0.05 was considered to be significant.
| ~ Results|| |
The age groups found most prone to burn injury were 30-40 years (n = 18, 28.6%), 10-20 years (n = 12, 19.0%) and 60-70 years (n = 12, 19.0%). TBSA was 60-80% in 24 (38.1%) patients, ≥80% in 18 (28.6%) and 40-60% in 12 (19.0%) patients.
The mean C3 levels of patients were found to decrease with the increase in the severity of burn injury. Patients with ≥20% burns had mean C3 levels in 1 st samples significantly lower than controls (P < 0.001) as well as those with <20% burns (P < 0.001). Complement was found to be activated in the 1 st samples of all patients while it was inactive in the controls. In the 2 nd serum samples, complement was active and C3 levels continued to be low only in patients with ≥40% burns. Patients with 40-60% burns had mean C3 level of 90.1 ± 40.8 mg/dl, those with 60-80% burns had mean C3 level of 62.9 ± 26.3 mg/dl while those with ≥80% burns had mean C3 level of 59.4 ± 23.9 mg/dl in their 2 nd samples. All these values were significantly lower than controls (P < 0.05, P < 0.001 and P < 0.001 respectively). These patients subsequently developed septic complications and expired during the course of treatment. Patients with <40% burns showed inactive complement as well as recovery of C3 levels in their 2 nd samples [Table 1] and Graph 1].
All burn patients showed initial elevation in IL-6 levels. Mean IL-6 levels were found to increase with the increase in the percentage of burn injury. Significant elevation in mean IL-6 levels in 1 st samples was found in patients with ≥20% burns when compared with control mean IL-6 levels (P < 0.001). In patients with ≥60% burns, very high mean IL-6 levels were encountered in the 2 nd samples as well. The 1 st sample mean IL-6 levels of patients with 20-40%, 40-60% and 60-80% burns were similar, but in patients with ≥80% TBSA, the 1 st sample mean IL-6 levels were significantly higher than those with <80% burns (P < 0.05). In the 2 nd samples, patients with ≥60% TBSA had higher mean IL-6 levels than controls (P < 0.001) as well as patients with <60% burns (P < 0.0001) [Table 1] and Graph 2].
A total of 42 patients who did not survive had mean C3 levels of 28.1 ± 11.1 mg/dl and 60.9 ± 29.0 mg/dl in 1 st and 2 nd samples respectively, both being significantly lower than controls (P < 0.001). Their 1 st and 2 nd sample mean IL-6 levels were 440.5 ± 661.3 pg/ml and 1992.7 ± 1089.7 pg/ml, respectively, this increase was also statistically significant (P < 0.001). All these cases had a longer mean duration of hospital stay than patients who survived. 6 patients who had septicaemia, but survived had significantly lower mean C3 levels and higher mean IL-6 levels than controls. A total of 12 survivors with local infection had mean C3 levels of 28.9 ± 13.6 mg/dl and 136.2 ± 14.9 mg/dl in 1 st and 2 nd samples respectively, while their 1 st and 2 nd sample mean IL-6 levels were 191.8 ± 12.2 pg/ml and 311.4 ± 70.2 pg/ml, the difference in both mean C3 and mean IL-6 levels in comparison with controls being significant (P < 0.001) [Table 2].
|Table 2: Mean C3 and IL-6 levels in cases in relation to clinical outcome|
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Overall, the 1 st sample mean C3 levels of survivors were not significantly different from that of the non-survivors. However, the 2 nd sample mean C3 levels of the non-survivors were significantly lower than the survivors (P < 0.05). The mean IL-6 of 1 st as well as 2 nd samples in non-survivors was significantly higher than survivors (P < 0.001). Furthermore, the patients who developed infection were found to have significantly lower mean C3 levels and higher IL-6 levels in the 2 nd samples than patients who did not develop any infection.
The mean duration of hospital stay was the lowest among the survivors without infection while the non-survivors with septicaemia had a comparatively higher duration of hospital stay, as depicted in Graphs 3 and 4.
Bacterial infection was found to occur in most of the burn patients. 48 (76.2%) patients had positive blood culture as well as wound swab culture. 12 (19.0%) patients were positive only for wound swab culture whereas blood culture was negative in these patients.
The most common isolate in the 1 st week following burn injury was Staphylococcus aureus while Pseudomonas species was the predominant isolate from the 2 nd week onwards. Overall, 41.7% isolates were of Pseudomonas species followed by S. aureus (33.3%), Escherichia coli (13.9%), Streptococcus pyogenes (5.6%) and Enterococcus faecalis (5.6%) [Table 3].
| ~ Discussion|| |
The general immune competence of a patient with severe burns is important in determining the final outcome. The complement system is one of the principal effectors of the humoral arm of the immune system and is important in defence against microbial invasion. ,
Complement activation is generally beneficial to the host but in patients who develop infections following injury, profound and persistent complement activation leads to consumption and depletion of essential components of complement and the necessary functions of chemotaxis, opsonisation and microbial cell lysis are hampered. This is called consumptive opsinopathy which occurs in severe bacterial infections such as those which occur in the immediate post-burn period. 
An established parameter in this post-traumatic immunosuppression could be of great value to the clinician in predicting the outcome of injury in the traumatised patients with a reasonable degree of accuracy especially since immunosuppression is reported to correlate well with the occurrence of septic complications in these patients. The relationship in humans of acute injury to complement activation (as measured by a decrease in circulating activity) or the appearance in blood of activation products has been reported.  Extensive amounts of non-viable tissue have been found to be associated with significant depletion of C3, C4 and C5 within 24 h after injury. 
Serum C3 levels are found to exhibit an inverse relation with the extent of burn injury. A minor trauma produces a less pronounced depletion of C3 compared to patients with extensive amounts of non-viable tissue. ,
In this study, we observed complement activation and a fall in serum C3 levels within hours of burn injury. All patients showed initial complement activation, whereas complement was found inactive in controls. Mean C3 levels correlated with the severity of burn injury. Patients with ≥20% burns had significantly lower mean C3 than the controls (P < 0.001) and those with <20% burns (P < 0.001). Patients with ≥40% burns continued to have activated complement and low C3 levels in their 2 nd samples, and they subsequently developed a bacterial infection, whereas complement was inactive and C3 levels recovered in patients with <40% burns. The non-survivors showed significantly lower C3 levels than the survivors in their 2 nd samples (P < 0.05). Patients who developed infection also had mean C3 levels significantly lower than those who remained free of infection (P < 0.05).
Cytokine stimulation has been observed within hours of injury. High levels of IL-6 are seen in various diseases, in patients with multiple injuries, burns, septicaemia etc., The important role of IL-6 in inflammatory reactions led to study of its role in multi-organ failure and sepsis. 
Increased levels of IL-6 within hours of thermal trauma has been reported by several workers. , Persistence of high IL-6 levels in relation to sepsis has also been observed , Several workers have observed a positive correlation between circulating IL-6 levels and the magnitude of burn injury. ,,
We also found raised IL-6 levels within hours of burn injury. All the burn patients showed initial elevation in IL-6 levels. Patients with ≥60% burns had significantly higher IL-6 levels than controls (P < 0.001) and those with < 60% burns (P < 0.001). Non-survivors had higher mean IL-6 levels than survivors in both samples (P < 0.001). Furthermore, patients who developed infection showed significantly higher IL-6 levels in 2 nd samples than those without infection (P < 0.001).
| ~ Conclusions|| |
Complement activation, C3 and IL-6 levels correlated well with the severity of injury and development of infection in burn patients. Thus, these parameters can be used to predict the development of infection, septicaemia and mortality in burn patients.
| ~ Acknowledgments|| |
The authors wish to thank the Indian Council of Medical Research (ICMR), New Delhi, India, for providing financial assistance for this study and Prof. Aziz Khan (retd.), Department of Community Medicine, J.N. Medical College, AMU, Aligarh, for carrying out manual statistical analyses of the data.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]