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ORIGINAL ARTICLE
Year : 2023 | Volume
: 22
| Issue : 4 | Page : 446-450
Interleukin-6 and interleukin-8 levels in children with aplastic anemia and its correlation with disease severity and response to immunosuppressive therapy
Anurag Singh, Sharvan Kumar Bhargawa, Geeta Yadav, Rashmi Kushwaha, Shailendra Prasad Verma, Tanya Tripathi, Uma Shankar Singh, Anil Kumar Tripathi
Department of Pathology, King George's Medical University, Lucknow, Uttar Pradesh, India
Correspondence Address:
Rashmi Kushwaha
Department of Pathology, King George's Medical University, Lucknow - 226 003, Uttar Pradesh
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_106_22
Abstract
Background: Aplastic anemia (AA) is an uncommon condition characterized by pancytopenia and hypocellular bone marrow. Interleukin (IL)-6 and IL-8 have been shown to inhibit myelopoiesis and are major mediators of tissue damage. The primary goal of this study was to determine the IL-6 and IL-8 levels in children with AA, as well as their relationship to illness severity and immunosuppressive medication response. Materials and Methods: The IL-6 and IL-8 levels were tested in 50 children aged 3–18 years who had AA. As controls, 50 healthy age and sex matched individuals were used. A sandwich enzyme-linked immunosorbent assay kit (solid-phase) was used to measure IL-6 and IL-8 levels quantitatively. The concentrations of IL-6 and IL-8 in pg/mL were used to represent the results. Immunosuppressive medication was given to the patients in accordance with the British Committee for Standards in Haematology Guidelines 2009. Results: The patients' average age was 11.3 ± 3.7 years. Patients with AA had significantly higher IL-6 and IL-8 levels than controls (278.88 ± 216.03 vs. 4.51 ± 3.26; P < 0.001) and (120.28 ± 94.98 vs. 1.79 ± 0.78; P < 0.001), respectively. The IL-6 and IL-8 levels were also investigated with respect to AA severity, with statistically significant differences (P < 0.01) between different grading strata. Patients with very severe AA (VSAA) had the highest IL-6 levels (499.52 ± 66.19), followed by severe AA (SAA) (201.28 ± 157.77) and non-SAA (NSAA) (22.62 ± 14.63). For IL-8 levels, a similar trend (P < 0.01) was detected, with values of 209.81 ± 38.85, 92.12 ± 78.0, and 9.29 ± 10.68 for VSAA, SAA, and NSAA, respectively. After 6 months of immunosuppressive treatment (IST), mean levels of IL-6 and IL-8 in responders and nonresponders were again assessed. The mean IL-6 level in the responders' group (46.50 ± 45.41) was significantly lower, when compared to the nonresponders' group (145.76 ± 116.32) (P < 0.001). Similarly, the mean IL-8 level in the responder's group (33.57 ± 27.14) was significantly lower, compared to the nonresponder's group (97.49 ± 69.00) (P < 0.001). Conclusions: Children with AA had higher IL-6 and IL-8 levels than normal age- and sex-matched controls. Increased levels were linked to the severity of the condition, suggesting that IL may have a role in AA. IL levels can be monitored in AA patients during IST, which can assist in predicting response to IST.
Résumé
Contexte: L'anémie aplastique (AA) est une affection peu fréquente caractérisée par une pancytopénie et une moelle osseuse hypocellulaire. Il a été démontré que l'interleukine (IL)-6 et l'IL-8 inhibent la myélopoïèse et sont des médiateurs majeurs des lésions tissulaires. L'objectif principal de cette étude était de déterminer les niveaux d'IL-6 et d'IL-8 chez les enfants atteints d'AA, ainsi que leur relation avec la gravité de la maladie et la réponse aux médicaments immunosuppresseurs. Matériel et méthodes: Les niveaux d'IL-6 et d'IL-8 ont été testés chez 50 enfants âgés de 3 à 18 ans atteints d'AA. 50 témoins sains appariés par l'âge et le sexe ont été utilisés. Un kit de dosage immuno-enzymatique en sandwich (phase solide) a été utilisé pour mesurer quantitativement les niveaux d'IL-6 et d'IL-8. de manière quantitative. Les concentrations d'IL-6 et d'IL-8 en pg/mL ont été utilisées pour représenter les résultats. Des médicaments immunosuppresseurs ont été administrés aux patients conformément aux directives 2009 du British Committee for Standards in Haematology. Résultats: L'âge moyen des patients était de 11,3 ± 3,7 ans. Les patients atteints d'AA présentaient des taux d'IL-6 et d'IL-8 significativement plus élevés que les témoins (278,88 ± 216,03 contre 4,51 ± 3,26 ; P < 0,001) et (120,28 ± 94,98 contre 1,79 ± 0,78 ; P < 0,001), respectivement. Les taux d'IL-6 et d'IL-8 ont également été étudiés en fonction de la gravité de l'AA, avec des différences statistiquement significatives (P < 0,01) entre les différentes strates de classement. Les patients présentant une AA très sévère (VSAA) avaient les taux d'IL-6 les plus élevés (499,52 ± 66,19), suivis par les patients atteints d'AA sévère (SAA) (201,28 ± 157,77) et les patients non-SAA (NSAA) (22,62 ± 14,63). Pour les niveaux d'IL-8, une tendance similaire (P < 0,01) a été détectée, avec des valeurs de 209,81 ± 38,85, 92,12 ± 78,0, et 9,29 ± 10,68 pour les VSAA, SAA et NSAA, respectivement. Après 6 mois de traitement immunosuppresseur traitement immunosuppresseur (IST), les niveaux moyens d'IL-6 et d'IL-8 chez les répondeurs et les non-répondeurs ont été à nouveau évalués. Le taux moyen d'IL-6 dans le groupe des répondeurs (46,50 ± 45,41) était significativement plus faible, par rapport au groupe des non-répondeurs (145,76 ± 116,32) (P < 0,001). De même, le niveau moyen d'IL-8 dans le groupe des répondeurs (33,57 ± 27,14) était significativement plus faible que dans le groupe des non-répondeurs (97,49 ± 69,00) (P < 0,001). Conclusions: Les enfants atteints d'AA présentaient des niveaux d'IL-6 et d'IL-8 plus élevés que les témoins normaux appariés selon l'âge et le sexe. L'augmentation des taux était liée à la gravité de l'affection, suggérant que l'IL pourrait jouer un rôle dans l'AA. Les niveaux d'IL peuvent être surveillés chez les patients atteints d'AA pendant l'IST, ce qui peut aider à prédire la réponse à l'IST.
Mots-clés: Anémie aplastique, traitement immunosuppresseur, interleukine-6, interleukine-8
Keywords: Aplastic anemia, immunosuppressive treatment, interleukin-6, interleukin-8
Introduction 
Aplastic anemia (AA) is an uncommon condition characterized by pancytopenia and hypocellular bone marrow (BM). A continuous deficiency of hematopoietic stem cells and their granulocytic, erythroid, and megakaryocytic progenitors in the marrow is the fundamental abnormality in AA. The cause of AA is unknown; however, investigations have shown that immunologic abnormalities have a role.[1] Marrow stromal cells, regulatory cells, and stem cells generate a variety of growth factors, cytokines, and/or chemokines, which are key components of the marrow microenvironment and control hematopoiesis in an autocrine/paracrine way.[2] Interleukin-6 (IL-6) is a multifunctional cytokine that regulates B-cell terminal differentiation, T-cell differentiation, and macrophage growth and activation. The balance between interleukin-17 (IL-17)-producing CD4+ T-cells (T-helper 17 [Th17] cells) and regulatory T-cells (Tregs) can be altered by an increased level of IL-6.[3] In the pathogenesis of AA, as well as in many other autoimmune illnesses, the Th17/Tregs imbalance is significant.[4],[5] Increased Th17 cells and reduced Tregs may lead to overproduction of several negative regulators of hematopoiesis, such as IL-8 and tumor necrosis factor (TNF), leading to the development of AA.[6],[7],[8] IL-8 is a chemokine that inhibits myelopoiesis and is produced by a variety of cells, including macrophages, fibroblasts, neutrophils, and lymphoid T-cells. IL-6 and IL-8 have been shown to inhibit myelopoiesis and are major mediators of tissue damage during acute inflammation.[9] As a result, it is possible that continuous overproduction of IL-6 and IL-8 plays a role in AA. Early detection and treatment of AA in children can reduce the need for numerous blood transfusions and pretreatment infections, improving overall survival.
The primary goal of the present research was to determine the IL-6 and IL-8 levels in children with AA, as well as their relationship to illness severity and immunosuppressive medication response.
Materials and Methods For 2 years, this prospective descriptive research was undertaken at the department of pathology and clinical hematology at King George's Medical University in Lucknow, a tertiary care hospital in Uttar Pradesh, India. Patients with AA aged 3–18 years were consecutively enrolled in this research. Each patient's sociodemographic data were documented. The study population was divided into three socioeconomic groups on the basis of the modified Kuppuswamy socioeconomic scale. A peripheral blood count, absolute reticulocyte count (ARC), BM aspiration smear, and trephine biopsy study were conducted in all of the cases. AA was diagnosed using widely accepted criteria,[10],[11] which included hypocellular BM (cellularity <25%) on trephine biopsy in the absence of fibrosis or neoplastic infiltration in peripheral blood with at least two of the following: hemoglobin <10 g/dL, platelet count <50 × 109/L, and absolute neutrophil count (ANC) 1.5 × 109/L. Severe AA (SAA) is defined as ANC <0.5 × 109/L, and platelets <20 × 109/L with ARC of <20 × 109/L. Whereas the diagnosis of very SAA (VSAA) is rendered when ANC is <0.2 × 109/L. The remaining individuals were diagnosed with non-SAA (NSAA). Patients with aplasia of marrow caused by chemotherapy and hereditary BM suppression/failure syndrome were ruled out based on the detailed clinical history, family history, a thorough physical examination, and a chromosomal fragility test with mitomycin C. Patients who were eligible for BM transplantation or absconded to follow-up during the study period were also excluded from the study. Patients were given immunosuppressive medication such as anti-thymocyte globulin and cyclosporine, according to the Guidelines of the British Committee for Standards in Haematology 2009.[12] This study also included 50 age- and sex-matched healthy controls, from whom 2 ml of peripheral blood was obtained in a plain vial under rigorous aseptic conditions, for measurement of levels of IL-6 and IL-8. The University's Ethics Committee gave its approval to this research. Clinical records and all samples were collected after the informed consent of guardian and parents. Blood was drawn from the research participants' peripheral veins in a sterile container under strict aseptic circumstances. The serum was isolated and kept in separate vials at −60°C for subsequent analysis after centrifuging the sample of blood at 4000 rpm for 10 min. A enzyme-linked immunosorbent assay (ELISA) kit (sandwich solid phase) was used to measure IL-6 and IL-8, according to the manufacturer's instructions (Elabscience ELISA kit). IL-6 and IL-8 values in pg/mL were used to display the data.
Statistical analysis
The statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS) software for windows version 21.0 (Armonk, NY: IBM Corp). The proportions and percentages of discrete/categorical data, as well as the mean and standard deviation (SD), were used to summarize the data (SD). The Chi-square test was used to compare all of the categorical data. Significant was defined as a P < 0.05.
Results The study comprised a total of 50 patients who met the inclusion criteria (29 males and 21 females). The age group ranged from 3 to 18 years old (median age, 11 years). [Table 1] shows the clinic demographic characteristics of the patients, with the majority of them belonging to a lower socioeconomic class. Patients with aplastic anemia had significantly higher lL-6 and IL-8 levels than controls (278.88 ± 216.03 vs. 4.51 ± 3.26; P < 0.001) and (120.28 ± 94.98 vs. 1.79 ± 0.78; P < 0.001), respectively. The IL-6 and IL-8 levels were investigated in connection to AA severity, and statistically significant differences (P < 0.01) were found between different grading strata. Patients with VSAA had the highest IL-6 levels (499.52 ± 66.19), followed by SAA (201.28 ± 157.77) and NSAA (22.62 ± 14.63). For IL-8 levels, a similar trend (P < 0.01) was detected, with values of 209.81 ± 38.85, 92.12 ± 78.0, and 9.29 ± 10.68 for VSAA, SAA, and NSAA, respectively [Table 2]. After 6 months of immunosuppressive treatment (IST), 39 of the 50 cases achieved a partial or complete response (responders group). Eleven patients did not respond to the IST treatment (nonresponders group). There were no significant differences in age, gender, or disease severity between the responders and nonresponders groups. The difference in hematological profile at diagnosis between the two groups, which included white blood cell (P = 0.43), ANC (P = 0.52), absolute lymphocyte count (ALC) (P = 0.32), hemoglobin (P = 0.37), red blood cell (P = 0.52), ARC (P = 0.11), and platelet count (P = 0.59) was not statistically significant.
Table 2: Comparisons of interleukin-6 and interleukin-8 in between nonsevere, severe, and very severe cases of aplastic anemia at diagnosisAt the time of initial diagnosis, responders had 288.21 ± 226.03 IL-6 levels, whereas nonresponders had 262.29 ± 202.22 (P = 0.68). Similarly, mean IL-8 levels were 120.01 ± 98.93 in responders and 120.75 ± 90.30 in nonresponders at the time of initial diagnosis (P = 0.979). There was no statistically significant difference in the IL-6 and IL-8 levels at diagnosis between the responders and nonresponders groups [Table 3].
Table 3: Comparisons of interleukin-6 and interleukin-8 in between responders and nonresponders at diagnosis and after 6 monthsAfter 6 months of IST, the mean levels of IL-6 and IL-8 in responders and nonresponders were again assessed. The IL-6 mean level in the responders group was less 46.50 ± 45.41 when compared to nonresponders' group 145.76 ± 116.32, and this difference was statistically significant (P < 0.001). Similarly, 'the level of mean IL-8 in responders was 33.57 ± 27.14, compared to nonresponders 97.49 ± 69.00 (P < 0.001) [Table 3]. In responders, the mean IL-6 and IL-8 levels was reduced, although they were not similar to controls [Table 3].
Discussion Because the primary etiology of AA is currently thought to be mostly immune-related diseases, as well as a variety of other variables such as BM mesenchymal stem cells and defective hematopoietic stem/progenitor cells, only a proportion of patients respond effectively to IST. A great number of researches have been conducted to identify a biomarker for predicting IST response. The ARC and ALC may be a straightforward predictor of response to IST, according to a retrospective examination of 316 individuals.[13] However, in terms of ARC and ALC, the current study found no statistically significant differences between responders and nonresponders. After a thorough search of the literature, only a few studies on the role of immune factors such as IL6 and IL8 were found. ILs, particularly IL-6, IL-10, and IL-8, have been examined in a variety of diseases, with high levels seen in pulmonary diseases, systemic sclerosis, and infections.[14],[15],[16] Only a few researches have explored the role of IL-6 and IL-8 in patients of AA, and only a couple of studies have looked into the role of IL-6 and IL-8 in AA of children.
When comparing patients to controls, significantly increased IL-6 and IL-8 levels were found in the current study. IL level was also linked to varied disease severity levels, with the most elevated levels found in patients of VSAA. Increased IL-8 was also identified in both peripheral blood and BM in a study done earlier in adult patients.[17] In cases of very severe and severe disease, levels of IL-6 and IL-8 were significantly higher, followed by nonsevere disease. A study of adult patients with AA found that IL-17 caused macrophages to synthesize more IL-6, IL-8, and TNF-alfa.[7] A study done by Tang et al. also showed elevated IL-8 levels in the peripheral blood of patients having AA.[18] The level of IL-8 also showed a significant increase as disease severity increased.[18] IL-6 and IL-8 were also found to be released by stromal cells in the BM in a few mouse investigations. The integrity of the BM microenvironment may be harmed by IL-6.[19] According to the findings, IL-8 suppresses myelopoiesis by limiting the myeloid progenitors proliferation through the transmission of inhibitory signals through a receptor-ligand-mediated pathway. Increased levels of this cytokine in the blood may cause neutrophil activation-mediated death in the peripheral circulation.[20]
The present research found no significant correlation between responders and nonresponders in terms of levels of IL-6 and IL-8 at the initial diagnosis. Similar findings were found in a study conducted by Gupta et al.[21] However, Lu et al. found that elevated IL-6 and IL-8 levels at the time of diagnosis are a predictor of favorable response to IST.[22] The disparity between this study and the work of Lu et al. could be attributable to geographic differences in the environment and the severity of the illness. After 6 months of IST, IL-6 and IL-8 levels were assessed in connection to the response to IST. When compared to patients who did not respond to treatment, those who responded had low levels of IL-6 and IL-8, and the difference was statistically significant. IL levels had dropped in responders, although they were still greater than in controls. These findings indicate that IL-6 and IL-8 play a significant role in AA cases that responded to IST and were linked to immunologic abnormalities. Extensive literature searches turned up only one study in children with AA, by Gupta et al., that also correlated IL levels after 6 months of IST in between responders and nonresponders. The findings of this study were similar to those of Gupta et al.[21]
Th17/Tregs imbalance is particularly critical in the early stages of AA development. Treg dysfunction sets the stage for Th17 cell growth. Early treatment with anti-IL-7 antibodies can help to lessen the severity of BM failure.[5],[6] After the early stage, the Th17/Treg imbalance can lead to the release of various negative hematopoiesis regulators, as well as the traditional Th1/Th2 imbalance, which can be corrected only after AA treatment.[23],[24],[25]
There were certain limitations in our research, due to the small sample size and the lack of measurement of Th17 cells and Tregs, we can only assume that elevated levels of IL-6 and IL-8 may predict response rate through the Th17/Tregs balance. More research with a larger sample size along with measurement of Th17 cells and Tregs is needed to determine the impact of IL-6 and IL-8 in predicting IST response.
Conclusions The current study findings revealed that children with AA have elevated blood IL-6 and IL-8 levels. Increased levels were linked to the severity of the disease, and so seem to play an essential role in AA. The initial levels of IL-6 and IL-8 at diagnosis had no significant correlation between responders and nonresponders. IL levels can be monitored in AA patients during IST, which can assist in predicting response to IST.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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