|Year : 2019 | Volume
| Issue : 3 | Page : 163-167
The role of human monocyte-expressing markers (CD163 and MR/CD206) in pediatric sepsis
Eman NasrEldin MD 1, Khalid Ahmed2
1 Clinical Pathology Department, Faculty of Medicine, Assiut University, Asyut, Egypt
2 Pediatric Department, Asyut, Egypt
|Date of Submission||20-Jun-2019|
|Date of Acceptance||18-Sep-2019|
|Date of Web Publication||05-Dec-2019|
Department of Clinical Pathology, Faculty of Medicine, Assiut University, Asyut, 7111
Source of Support: None, Conflict of Interest: None
Background The diagnosis of pediatric sepsis requires sensitive biomarkers to start effective prompt treatment. We evaluated the significance of the haptoglobin–hemoglobin receptor (CD163) and the mannose receptor (MR/CD206) expression and their soluble serum levels as early diagnostic markers for pediatric sepsis.
Patients and methods This prospective study investigated 50 pediatric patients suspected of having sepsis and 50 age-matched healthy control children. Detection of monocyte expression of CD163 and MR was analyzed by flow cytometric technique, and the soluble serum levels of CD163 and MR were determined by enzyme-linked immunosorbent assay. The diagnostic values of the monocytes’ related markers were assessed using the area under the receiver operating characteristic (AUROC) curve.
Results The monocyte expression of CD163 and MR/CD206 and their serum levels were significantly higher in the septic patients than in the controls. We show that the sensitivity of sMR to predict sepsis exceeded that of either C-reactive protein or procalcitonin (area under the curve= 0.98, 0.86, and 0.82, respectively). sMR had higher area under the curve than soluble macrophage-related protein 163 [0.93; 95% confidence interval (CI): 0.79–0.98] followed by monocyte-bound CD163 expression (0.77; 95% CI: 0.61–0.93) and then MR/CD206 (0.68; 95% CI: 0.56–0.90).
Conclusion The study supports the important significance of the monocyte/macrophage-related markers for prediction of sepsis in children and highlights the potential diagnostic use of their soluble forms as new pediatric sepsis biomarkers.
Keywords: monocyte markers, pediatric, sepsis
|How to cite this article:|
NasrEldin E, Ahmed K. The role of human monocyte-expressing markers (CD163 and MR/CD206) in pediatric sepsis. Egypt J Haematol 2019;44:163-7
|How to cite this URL:|
NasrEldin E, Ahmed K. The role of human monocyte-expressing markers (CD163 and MR/CD206) in pediatric sepsis. Egypt J Haematol [serial online] 2019 [cited 2020 Sep 25];44:163-7. Available from: http://www.ehj.eg.net/text.asp?2019/44/3/163/272371
| Introduction|| |
Pediatric sepsis is a life-threatening and a global health condition due to the delay in the culture result, which may contribute significantly to the adverse outcome of sepsis . Early treatment is mandatory and requires the choice of the sensitive and specific biomarker for diagnosis of infection .
A new hopeful method is to analyze the monocyte and macrophage-derived surface markers’ level .
Both haptoglobin–hemoglobin receptor (CD163) and the mannose receptor (MR/206) are markers for macrophages’ or monocytes’ activation . Those scavenger receptors are limited to macrophages, monocytes and dendritic cells . Upon activation during inflammatory diseases and infection, the soluble form of CD163 (sCD163) and soluble MR (sCD206) are shed into the blood, and their serum levels are increased ,. Several biomarkers used for the early diagnosis of sepsis-like C-reactive protein (CRP) may also be elevated in other conditions ,.
Our aim was to investigate the value of monocyte expression of both CD163 and CD206 and their soluble serum levels as an early marker for diagnosis of pediatric sepsis.
| Patients and methods|| |
Study design and patients
This prospective study was conducted according to the principles of the Declaration of Helsinki and was ethically approved by the Medical Ethical Committee, Faculty of Medicine, Assiut University.
This study included 50 ICU pediatric patients diagnosed as having sepsis according to the criteria given by the International Pediatric Sepsis Consensus Conference . Written informed consent was obtained from guardians before enrollment in the study. Exclusion criteria were as follows: Neutrophil count less than 0.5 ×109/l; death during the first day of ICU admission; and congenital or acquired immunodeficiency syndrome.
Cases suspected of having infection underwent diagnostic workup including routine blood test, total and differential leukocytic count, erythrocyte sedimentation rate (ESR), CRP and procalcitonin (PCT) levels, and aerobic and anaerobic bacteriological culture. The diagnosis of sepsis was confirmed by the result of the blood culture. Empirical antibiotic therapy was started until the culture and sensitivity results were obtained. Fifty healthy age-matched and sex-matched children served as controls.
Processing of blood cultures
After enrichment in blood culture bottles by immediately placing up to 2 ml of blood taken by sterile venipuncture into each pediatric-sized blood culture bottle (Peds Plus; Becton-Dickinson Diagnostic Systems, Cockeysville, Maryland, USA), the bottles were incubated at 37°C under both aerobic and anaerobic conditions using the BACTEC 9050 automatic blood culture detection system (Becton-Dickinson Diagnostic Systems). Bottles placed in the instruments were processed and tested as per the manufacturer’s instructions (the bottles were incubated at 37°C with rocking agitation for a total of 5 days. The 9050 U tested each bottle every 10 min.
Bacteremia was identified by the development of microbial growth in one blood culture bottle. Bottles flagged as positive were removed from the data units and subcultured on blood agar, chocolate agar, and MacConkey agar media, according to the results of the Gram stains using a standard protocol. Isolates were identified using the MicroScan Walk-Away 96 system (Beckman Coulter, Inc., NY, USA) according to the manufacturer’s instructions using MicroScan dried overnight Gram-positive panels and MicroScan Dried overnight Gram-negative panels for determining antimicrobial susceptibility and/or identification of the species level.
Analysis of CD163 and CD206
Samples were obtained from each septic patient at admission and from healthy controls for the determination of CD163 and CD206 expression, and their blood levels were drawn in EDTA tubes. One tube was used immediately for flow cytometric analysis, and the other tube was centrifuged for 10 min at 3000 rpm. The plasma, after collection, was aliquoted and stored at −80°C until being used for the enzyme-linked immunosorbent assay (ELISA) analysis.
Monocyte expression of CD163 and CD206
The expression of monocyte-bound CD163 and CD206 were analyzed by flow cytometry. Flow cytometric analysis was performed with 50 µl EDTA-anticoagulated whole blood on Becton Dickinson FACSCalibur flow cytometer (USA). The following monoclonal antibodies at optimal concentration were used: anti-CD14E-PE-Cy7A, anti-CD16-APC-Cy7, anti-CD163-APC, and anti-CD206-FITC (BD Biosciences, San Jose, California, USA). FITC mouse IgG1 was added as an isotype control. Whole blood was stained with a cocktail of optimized quantities of the monoclonal antibody and incubated for 15 min at room temperature in the dark. Red blood cell lysis was carried out by using 2 ml ammonium–chloride-based lysing buffer. Cells were washed with PBS containing 0.5% bovine serum albumin (Sigma Diagnostics Inc., Livonia, U.S.) and reconstituted in 0.5 ml of 1% formaldehyde–PBS.
Flow cytometric analysis was carried out within one hour on at least 50 000 events for each sample to ensure an adequate number of monocytes for analysis. Monocytes (using CD14 as a marker) were identified on the basis of their forward scatter and side scatter. The monocyte expression of CD163 and CD206 was determined by quantifying the median fluorescence intensity emitted by the cell population, and the data were analyzed with the CellQuest Software (Becton Dickinson, Franklin Lakes, New York, USA) ,.
Determination of sCD163 and sCD206 plasma levels
Determination was carried out by sandwich enzyme-linked immunosorbent assay using The Thermo Scientific Pierce Human CD163 (M130) ELISA kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) for the sCD163, and LSBios Human Mannose Receptor/CD206 ELISA kit Life Span Biosciences Inc. (USA) for the sCD206 analysis according to the manufacture instruction
Statistical differences between groups were performed using statistical package for the social sciences version 19 (SPSS Inc., Chicago, Illinois, USA). P value less than 0.05 was considered significant. Comparisons between each group were carried out using the Mann–Whitney, Kruskal–Wallis and Fisher exact test. The receiver operating characteristic curve was used to calculate the area under the curve (AUC), which was used to assess the diagnostic values of each studied marker.
| Results|| |
Laboratory data for the patients enrolled in the study are presented in [Table 1]. Septic patients had a significantly higher total WBC count and absolute monocytic count than the healthy controls.
There were statistically significant differences with respect to the levels of both CRP and PCT between the septic patients and the controls.
Blood cultures drawn for all patients were positive in 80% of the patients. The respiratory tract was the primary site of infection (64%) followed by the gastrointestinal tract (GIT) and then the urinary tract (30, and 6%, respectively).
Staphylococcus aureus and Klebsiella pneumonia were the most common detected organisms (35 and 30%, respectively).
Monocyte-bound CD163 and MR/CD206 expression
Septic Patients had a higher CD14+ monocyte fraction than the healthy controls. The monocyte expression of CD163 and MR/CD206 in the septic patients was significantly higher compared with the healthy controls (P≤0.001 for each comparison; [Table 2]).
sCD163 and sMR/CD206 serum levels were significantly higher in the septic patients than in the controls, and the increase was much more in the level of sMR/CD206 (P≤0.001; [Table 2]).
Receiver operating characteristic curve analysis was performed for both monocyte-bound CD163 and MR/CD206 and also for the serum level of sCD163 and sMR/206 to examine the ability of each marker to discriminate patients in sepsis from the other predictors of sepsis, CRP and PCT ([Figure 1]).
|Figure 1 Receiver operating characteristic (ROC) curves for CD163 expression, CD206 expression, sCD163), sMR, CRP, and PCT for predicting pediatric sepsis.|
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No difference was observed between sCD163 and sMR/206, while both soluble markers performed better than the monocyte-bound markers CD163 and MR/CD206.
We show that the sensitivity of sMR to predict sepsis exceeded that of either CRP or PCT (AUC=0.98, 0.86, and 0.82, respectively). sMR had higher AUC then sCD163 [0.93; 95% confidence interval (CI): 0.79–0.98] followed by monocyte-bound CD163 expression (0.77; 95% CI: 0.61–0.93) and then MR/CD206 (0.68; 95% CI: 0.56–0.90) ([Table 3]).
|Table 3 Area under the receiver operating characteristic curve for predicting sepsis among the studied patients|
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There was no significant difference in the AUC between either CRP or PCT and sCD163 and also monocyte-bound CD163 or MR/CD206.
| Discussion|| |
Sepsis is a very rapid systemic host inflammatory response to infection associated with the activation of many cells, release of mediators, and activation of the coagulation system. Patients with sepsis are usually handled in the ICU ; however, mortality related to sepsis remains high throughout the world . In addition, no efficient prognostic biomarkers for sepsis were found to be used in clinical practice . The child’s innate and adaptive immune response is different from that in the adult , making an early prediction of sepsis in children very helpful to ensure timely use of the proper effective treatments and prevent death . The main cause of pediatric sepsis-associated mortality is the dysregulated inflammatory response of the host including the concurrent occurrence of both proinflammatory and anti-inflammatory immune response .
The main disadvantage of standard bacteriologic cultures is the lack of sensitivity and the delay in obtaining results. Plasma inflammatory biomarkers like CRP and PCT can be useful for the rapid diagnosis of critically ill septic patients and have shown to be helpful for predicting mortality in those patients; however, CRP can be elevated in the other noninfectious conditions ,.
The ability to detect easy suitable inexpensive sepsis biomarkers for high-risk pediatric patients rather than CRP, PCT and other cytokines such as IL-6 and IL-10, which lack sufficient specificity, is a challenge for many researchers, to permit proper prognostic evaluation, which is also the aim of our study.
CD163 is a scavenger transmembrane glycoprotein receptor restricted to the monocyte/macrophage, which plays the main role in the endocytosis and macrophage clearance of the hemoglobin–haptoglobin complexes and the so-called “hemoglobin scavenger receptor” . CD163 receptors are involved in the anti-inflammatory response and regulated by many inflammatory mediators ,.
The sCD163 is a widely recognized macrophage activation marker used as a predictor marker for bacteremia ,. The serum CD163 and monocyte-bound CD163 receptor expression were found to be elevated in sepsis ,; moreover, their levels could determine the clinical outcomes ,.
The macrophage MR (C type I, CD206), a member of the MR family of proteins, is also a highly restricted endocytotic receptor on monocytes, macrophages, and dendritic cell surfaces. It can bind to numerous types of ligands and the MR receptor involved in endocytosis and degradation of micro-organisms and endogenous glycoproteins ,. Serum sMR is a soluble form of the MR found recently in humans as a new biomarker released upon its proteolytic cleavage and detected in high levels among ICU patients with several diseases including sepsis ,.
In this study, we found that the sCD163 and sMR were valuable markers for predicting sepsis in children; their levels were significantly higher among septic patients than normal controls. There was no difference between both sCD163 and sMR to recognize the septic from the nonseptic patients. However, sMR showed a higher AUC (0.98) than those of CRP, PCT, and sCD163, with a better predictive value, supporting that sMR is a useful diagnostic marker for sepsis.Our findings are consistent with those presented by Kjærgaard and colleagues. Similarly, previous studies in critically ill patients reported the higher concentration of sCD163 (1.6 times) compared with that of healthy controls ,.
The monocyte CD163 expression has the ability to discriminate septic patients ,. Similarly to the previous studies ,,, the monocyte expression of CD163 in our study was found to be higher in the pediatric septic patients compared with that in normal controls. We reported the same finding with regard to the monocyte expression of MR/CD206; this finding is not consistent with other research by Kjærgaard and colleagues, who did not find any monocyte expression of MR and support the speculation that MR/CD206 is not expressed on the monocyte surfaces . Other studies have found weak MR/CD206 expression in sepsis ,, while others showed a dramatic increase in the expression of both CD163 and CD206 in septic patients, and higher levels of monocyte CD206 expression were observed among the nonsurvivor patients .
Both sCD163 and sMR soluble markers were superior to the monocyte expression of CD163 and MR/CD206 in differentiating septic patients in children.
In this study, we did not analyze the association between the CD163 and CD206 monocyte expression and clinical outcomes; future studies are needed to address this issue. Moreover, further functional studies of the circulating monocytes together with the cell surface expression are needed to determine the different monocyte phenotypes in pediatric sepsis.
| Conclusion|| |
The study supports the important significance of the monocyte/macrophage-related proteins (CD163 and MR/CD206) in their soluble and cell-bound forms for prediction of sepsis in children and highlights the potential diagnostic use of their soluble forms, especially sMR, as new pediatric sepsis biomarkers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Wiens MO, Kumbakumba E, Kissoon N, Ansermino JM, Ndamira A, Larson CP. Pediatric sepsis in the developing world: challenges in defining sepsis and issues in post-discharge mortality. Clin Epidemiol
Schuetz P, Maurer P, Punjabi V, Desai A, Amin DN, Gluck E. Procalcitonin decrease over 72 hours in US critical care units predicts fatal outcome in sepsis patients. Crit Care
Pierrakos C, Vincent JL. Sepsis biomarkers: a review. Crit Care
Kjærgaard AG, Rødgaard-Hansen S, Dige A, Krog J, Møller HJ et al.
Monocyte expression and soluble levels of the haemoglobin receptor (CD163/sCD163) and the mannose receptor (MR/sMR) in septic and critically ill non-septic ICU patients. PLoS One
Dultz G, Gerber L, Farnik H, Berger A, Vermehren J, Pleli T et al.
Soluble CD163 is an indicator of liver inflammation and fibrosis in patients chronically infected with the hepatitis B virus. J Viral Hepat
Feng L, Zhou X, Su LX, Feng D, Jia YH et al.
Clinical significance of soluble hemoglobin scavenger receptor CD163 (sCD163) in sepsis, a prospective study. PLoS One
Rodgaard-Hansen S, Rafique A, Christensen PA, Maniecki MB, Sandahl TD et al.
A soluble form of the macrophage-related mannose receptor (MR/CD206) is present in human serum and elevated in critical illness. Clin Chem Lab Med
Prashant A, Vishwanath P, Kulkarni P, Sathya Narayana P, Gowdara V, Nataraj SM et al.
Comparative assessment of cytokines and other inflammatory markers for the early diagnosis of neonatal sepsis − a case–control study. PLoS One
Wacker C, Prkno A, Brunkhorst FM, Schlattmann P. Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis. Lancet Infect Dis
Goldstein B, Giroir B, Randolph A, InternationalConsensus Conference on Pediatric Sepsis. International pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med
Wong KL, Yeap WH, Tai JJ, Ong SM, Dang TM et al.
The three human monocyte subsets: implications for health and disease. Immunol Res
Zhu T, Liao X, Feng T, Wu Q, Zhang J, Cao X, Li H. Plasma monocyte chemoattractant protein 1 as a predictive marker for sepsis prognosis: a prospective cohort study. Tohoku J Exp Med
Gaieski DF, Edwards JM, Kallan MJ, Carr BG. Benchmarking the incidence and mortality of severe sepsis in the United States. Crit Care Med
Carlyn CJ, Andersen NJ, Baltch AL, Smith R, Reilly AA, Lawrence DA. Analysis of septic biomarker patterns: prognostic value in predicting septic state. Diagn Microbiol Infect Dis
Ygberg S, Nilsson A. The developing immune system − from foetus to toddler. Acta Paediatr
Hotchkiss RS, Monneret G, Payen D. Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy. Nat Rev Immunol
Liu D, Su L, Han G, Yan P, Xie L. Prognostic valueof procalcitonin in adult patients with sepsis: a systematic review and meta-analysis. PLoS One
Zhang J, She D, Feng D, Jia Y, Xie L. Dynamic changes of serum soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) reflect sepsis severity and can predict prognosis: a prospective study. BMC Infect Dis
Davis BH, Zarev PV. Human monocyte CD163 expression inversely correlates with soluble CD163 plasma levels. Cytometry B Clin Cytom
Moller HJ, Aerts H, Gronbaek H, Peterslund NA, Hyltoft Petersen P, Hornung N et al.
Soluble CD163: a marker molecule for monocyte/macrophage activity in disease. Scand J Clin Lab Invest Suppl
Brunialti MK, Santos MC, Rigato O, Machado FR, Silva E, Salomao R. Increased percentages of T helper cells producing IL-17 and monocytes expressing markers of alternative activation in patients with sepsis. PLoS One
Groselj-Grenc M, Ihan A, Derganc M. Neutrophil and monocyte CD64and CD163 expression in critically ill neonates and children with sepsis: comparison of fluorescence intensities and calculated indexes. Mediators Inflamm
Gaïni S, Pedersen SS, Koldkaer OG, Pedersen C, Moestrup SK et al.
New immunological serum markers in bacteraemia: anti-inflammatory soluble CD163, but not proinflammatory high mobility group-box 1 protein, is related to prognosis. Clin Exp Immunol
Martinez-Pomares L. The mannose receptor. J Leukoc Biol
Kaku Y, Imaoka H, Morimatsu Y, Komohara Y, Ohnishi K, Oda H et al.
Overexpression of CD163, CD204 and CD206 on alveolar macrophages in the lungs of patients with severe chronic obstructive pulmonary disease. PLoS One
Ingels C, Moller HJ, Hansen TK, Wouters PJ, Vanhorebeek I et al.
Circulating levels of the shed scavenger receptor sCD163 and association with outcome of critically ill patients. J Clin Immunol
Smeekens SP, van de Veerdonk FL, Joosten LA, Jacobs L, Jansen T et al.
The classical CD14(+)(+) CD16(−) monocytes, but not the patrolling CD14(+)CD16(+) monocytes, promote Th17 responses to Candida albicans
. Eur J Immunol
[Table 1], [Table 2], [Table 3]