Received: October 15, 2017
Accepted: January 11, 2018
Mediterr J Hematol Infect Dis 2018, 10(1): e2018017 DOI 10.4084/MJHID.2018.017
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Background and Objectives:
Inflammatory markers that influence bone pain crisis (BPC) and other
complications of sickle cell anaemia (SCA) are numerous and play
various roles. This study determined the plasma levels of tumour
necrosis factor (TNF) - α, interleukin - 8 (IL-8), and endothelin - 1
(ET-1) in adult SCA patients during BPC and in steady state. In
addition, the plasma levels of these cytokines were correlated with the
severity of BPC of the patients.
Pro-inflammatory mediators have been extensively studied in SCA, but there is a paucity of information that relates the cytokines to the severity of bone pain crisis. TNF-α is a pro-inflammatory cytokine that stimulates tumour necrosis via its receptors, TNFR55 and TNFR75. TNF-α is produced mainly by monocytes/macrophages and to a less extent by T-cells, smooth muscle cells, adipocytes, and fibroblasts. TNF-α worsens vaso-occlusion in SCA by enhancing endothelial adhesiveness, activating leucocytes, and coagulation cascade.[10-12] Several studies have shown altered levels of some cytokines in SCA patients.[12-20] Some found a significantly higher level of TNF-α in SCA patients in VOC and/or during steady state than in healthy HbA controls.[16-18] Contrarily, Tavakkoli et al. and Graido-Gonzalez et al. demonstrated insignificantly elevated TNF-α level in SCA patients in VOC compared with patients in the steady-state group and controls.[13,21]
Interleukin-8 (IL-8) is a CXC chemokine that stimulates endothelial cell proliferation and angiogenesis through its receptors (CXCR1 and CXCR2), which are expressed mainly by neutrophils.[20,22] IL-8 is produced by neutrophils, endothelial cells, macrophages, fibroblasts, and platelets. IL-8 activates re-arrangement of the cytoskeleton, changes in intracellular Ca++ levels, integrins, promotion of protein-granule exocytosis, and respiratory burst in neutrophils.[23,24] IL-8 contributes to the initiation and propagation of inflammation by activating neutrophils, which are the first line recruits to the site of vascular injury. Similar to the role of IL-1 and TNF-α, IL-8 increases the endothelial adhesiveness of the sickle red cells leading to impairment of microcirculation and exacerbating painful episodes.[11,15,25] The most potent vasoconstrictor known, endothelins (ET), are a family of 21 amino acid peptides and ET-1 is the most prevalent subtype of the four subtypes characterised. ET-1 constricts large vessels, resistance arterioles and even post-capillary venules, the usual site of vaso-occlusion in SCA. Endothelin is produced by endothelial cells at a steady rate that is increased following endothelial injury or activation. The release of ET-1 is modulated by TNF-α and other inflammatory mediators. Therefore, the ET-1 level is expectedly elevated in SCA patients because of the ongoing endothelial activation and elevated pro-inflammatory cytokines. Endothelins act via two specific G-protein–coupled membrane receptors, ETR-A and ETR-B, which are on vascular smooth muscle cells and the smooth muscle contraction results from inositol-triphosphate–mediated increases in intracellular calcium. Though ET-1 is rapidly internalised and cleared from circulation by the lungs within minutes, its vasoconstrictive effect lasts as long as 1 hour. In in vitro assays, endothelin stimulated monocyte production of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8 etc.), neutrophil production of platelet-activating factor (PAF). ET-1 also enhances monocyte and neutrophil chemotaxis.[30-32] Endothelins upregulate endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, which are adhesion molecules that participate in the recruitment of leukocytes to sites of inflammation. Conversely, neutrophil proteases play a crucial role in cleaving bioactive ET-1 from its precursor molecule, thereby leading to the production of active ET and resulting in a vicious cycle with worsening inflammation.[21,34] We, therefore, hypothesize that pro-inflammatory mediators would increase with the severity of bone pain in SCA patients. The aim of our study was to determine the plasma levels of TNF- α, IL-8 and ET-1 in SCA patients during bone pain crisis compare with those of SCA patients in steady state and to correlate these with the severity of pain.
Materials and Methods
The researcher/attending Physician interviewed all SCA participants, and questionnaires were completed. The survey contained sections on bio-data, past medical history obtained from the patients’ notes, and clinical assessment for the management of the bone pain crisis. University of Ibadan/University College Hospital ethics review committee approved the study (UI/EC/14/0089), and all participants gave written informed consent.
Clinical severity assessment. With the aid of a questionnaire, all SCA patients were assessed for bone pain after clerking for general and organ-specific signs and symptoms. Pain assessment included pain site (ribs, sternum, back, lower or upper limbs), pain duration (up to 4 days; 5-7 days or more than 7 days), and pain intensity based on single dimensional verbal pain numerical rating system (that is the patient’s perception of pain on a scale of 1-10). The grading of pain was essential because its grade guided the type or class of analgesia administered to abate the pain. The pain was managed as mild for a verbal, numerical score 1-4; as moderate for a score of 5-6; and as severe for a score of 7-10.[35,36] However, for this study, the assessment of patient’s pain included the doctor’s perception of the patient’s pain. This aspect had questions on behaviour of patient during painful episode (normal, agitated, very disturbed, or too quiet) and the analgesic used to abort the pain (Paracetamol; Non-Steroidal Anti-inflammatory Drugs [NSAIDs]; Opioids; patient-controlled analgesia [PCA]; or intensive care unit [ICU] care) adapted from WHO analgesic ladder.[25,37]
For ease of comparability of variables in this study, patient’s and doctor’s perceptions were analyzed and summarized as total summary pain score (TSPS). TSPS was adapted for this study and calculated as follows:
TSPS = [patient’s pain score x duration of pain] + [patient’s behaviour] + [analgaesia used]
Each characteristic of patient’s pain was scored as follows: pain intensity was accorded 1, 2 or 3 respectively for verbal, numerical score 1-4, 5-6, 7-10 respectively. Duration of pain prior to patient’s presentation accorded 1, 2 or 3 for a pain that was respectively less than 4 days, or lasted 5-6 days; or lasted more than 7 days. The same was done for the immediate analgesic intervention to abate the pain: accorded 1, 2, or 3 respectively for Paracetamol/NSAIDs; Opioids; or for the pain that necessitated patient-controlled analgesia/intensive care. Patient’s behaviour attracted a maximum of four (4) for a patient who was too quiet, 3 for a very disturbed patient, 2 for an agitated patient and 1 for a patient who appeared normal.
Venous blood was collected from all the participants at the time of presentation to the hospital and dispensed into two EDTA vacutainers. One for analysis of the haematological parameters, the second tube was centrifuged, and plasma was stored in aliquots at -20°C until cytokines were assayed.
Haematological parameters. Complete blood count (CBC) was performed using Sysmex XS -1000i (Sysmex Corporation, Kobe, Japan), a fully automated 5-part counter.
Plasma Cytokine Assays. Plasma TNF-α, IL-8 and ET-1 were quantified using high-sensitivity commercial enzyme-linked immunosorbent assay (ELISA) kits (Span® Biotec Limited, Shenzhen, China) in accordance with the manufacturer’s instructions.
Statistical analysis. Data were analyzed using SPSS version 22.0 (Statistical Package for Social Sciences, Inc., Chicago, Ill.). The descriptive data were presented as means ± standard deviation except otherwise stated. Frequencies were shown in tables and graphs. Kruskal-Wallis test was used to compare means of the independent variables. Significant results were subjected to post hoc analyses for pairwise comparisons. Spearman rho analysis was performed for correlation of the haematological parameters and/or cytokines with the severity of bone pain crisis. Results were considered statistically significant if p<0.05.
|Table 1. Demographic and haematological parameters of all participants..|
Plasma levels of TNF-α, IL-8 and ET-1 in sickle cell anaemia patients and haemoglobin A controls. The plasma levels of TNF-α, IL-8 and ET-1 of the different groups were compared in table 2. The mean plasma TNF-α was significantly higher in the BPC group (373.78±354.95ng/L) than in the steady state group (99.77±92.86 ng/L, p=0.000), and in the HbA controls (82.20±29.32 ng/L, p=0.000). Similarly, the mean plasma IL-8 level was significantly higher in the BPC group (464.11±475.99 ng/L, p=0.005) than in the steady state group (233.57±294.35ng/L, p=0.005) and in the HbA group (183.92±198.58 ng/L, p=0.002). The mean plasma ET-1 was significantly higher in the BPC group (164.90±214.76 ng/L) than in the steady state (56.16±48.34 ng/L, p=0.038) and in the HbA groups (51.18±28.70 ng/L, p=0.042).
|Table 2. Plasma levels of TNF-α, IL-8 and ET-1 levels in sickle cell anaemia patients in bone pain crisis (BPC), steady state (STEADY) and haemoglobin A controls (HbA). n=90.|
Severity of bone pain crisis in the bone pain crisis group. Table 3 summarizes the clinical severity of bone pain crisis in the SCA patients at presentation. Of the 30 patients in the BPC group, a majority 15 (50%) reported a verbal, numerical pain score of 7-10 while two (6.7%) reported a mild pain score of 1-4 and the remaining 13 reported a moderate score of 5-6. Half (15) of the BPC patients reported that they had experienced bone pain for a few hours to 4 days and nine (30%) had experienced pain for 5 to 7 days. Six (20%) of the patients had pain for more than 7 days prior to presentation. From the physician’s perception, five patients (16%) were normal in their behaviour, 15 (50%) were agitated, eight (26.7%) were very disturbed, and only two (6.7%) were too quiet during clinical examination. Most of the patients were relieved of pain following administration of opioids, 22 (73.3%); or paracetamol/NSAIDs, 8 (23.3%). None of the patients required patient-controlled analgesia or intensive care unit to relieve pain.
The perception of the patient and physician were summed up for ease of comparability as the total summary pain score (TSPS), Table 4. Most patients 15 (50%) had a moderate TSPS and 12 (40%) had a mild TSPS while three (10%) had severe TSPS.
|Table 3. Severity scores of sickle cell anaemia patients in a bone pain crisis.|
|Table 4. Total summary pain score (TSPS) of sickle cell anaemia patients in bone pain crisis.|
Site of bone pain crisis in the BPC group. Concerning the site of bone pain, multiple sites were involved. SCA patients in bone pain crisis presented with pain in the various parts of the body: The upper limbs were the most frequently involved parts with right being 13/76 (17%) and Left 17/76 (22%). The other sites involved were right lower limb 9/76 (12%); left lower limb 21/76 (16%); spine 12/76 (16%); and chest (ribs and/or sternum) 09/76 (12%).
Comparing the plasma levels of TNF-α, IL-8 and ET-1 in the bone pain severity groups. Plasma levels of the pro-inflammatory markers (TNF-α, IL-8 and ET-1) were compared among the BPC severity groups and shown in Figures 1A-C. Plasma levels of the pro-inflammatory mediators were highly variable. The severe BPC group had the most elevated mean plasma TNF-α (610.49 ± 352.82 ng/L), IL-8 (691.61 ± 966.20 ng/L), and ET-1 (336.40 ±24.38 ng/L). Mean plasma TNF-α level was significantly higher in the mild, moderate and severe than steady state group (p=0.000, p=0.004 and p=0.001 respectively), figure 1A. The IL-8 level was significantly higher in the mild and moderate groups than the steady state group (p=0.037 and p=0.019), figure 1B. Plasma ET-1 level was significantly higher in the mild and severe groups than the steady state group (p=0.013 and p=0.006), figure 1C. Post hoc (i.e. within the group) comparison of plasma level of TNF-α, IL-8 and ET-1 between mild and moderate; moderate and severe; and mild and severe bone pain severity groups were not statistically significant.
Correlation of TNF- α, IL-8 and ET-1 in sickle cell anaemia patients during bone pain crisis. The correlation was performed for TNF- α and ET-1 in SCA patients during bone pain crisis (n=30), Figure 2. Spearman rho correlation analysis among the SCA patients in the bone pain crisis group revealed a significant positive correlation (r=0.854, p=0.000) for TNF-α and ET-1. IL-8 and ET-1 were not significantly correlated (r=0.017, p=0.927) in the BPC group. Similarly, IL-8 and TNF- α (r=0.004, p=0.985) were not significantly correlated.
|Figure 2. Correlation of plasma TNF-α and ET-1 in SCA patients during BPC. n=30.|
Significantly elevated TNF-α observed in the SCA groups is similar to findings of Lanaro et al. and Goncalves et al. Both groups of researchers found that SCA patients have increased circulating TNF-α and IL-8 levels at steady state and during the crisis.[15,16] Apart from enhancing endothelial adhesiveness, TNF-α activates leucocytes and the coagulation cascade, leading to the elevation of plasma levels of acute-phase plasma proteins such as fibrinogen that aid erythrocyte adhesion to endothelium.[41,42] These contribute to the development of vascular occlusion in SCA patients. Endothelin-1 is a potent vasoconstrictor of arterioles and post-capillary venules that contribute to vaso-occlusion, bone pain crisis, acute chest syndrome and nephropathy. ET-1 upregulates synthesis of adhesion molecules like ICAM -1, VCAM-1, and E-selectin in endothelial cells thereby participating in leucocyte adhesion at sites of inflammation.[21,27,28,43] An elevated level of ET-1 results from low oxygen tension in SCA patients because hypoxia is a potent stimulus for the production and release of ET-1 by vascular endothelial cells.[21,39]
Comparing the verbal pain numerical scoring system with the total severity score system (adopted in this study for ease of comparability of variables), only three (10%) of the bone pain crisis SCA group were in severe pain at presentation. It would then not be surprising that most of the patients 23(76.7%) were managed as outpatients and none had pain that was severe enough to warrant patient-controlled analgesia (PCA) or intensive care unit (ICU) admission. This finding is similar to the outcome of the study by Etienne-Julan et al., in which an episodic pain index was adopted for children with SCD. From those above, it could be inferred that elevated levels of pro-inflammatory mediators contributed to the severity of BPC in the patients. Pain and the immune system influence each other in such a manner that makes it difficult to determine whether blocking nociception or reducing the production of these pro-inflammatory cytokines would results in less severe pain. Pro-inflammatory cytokines stimulate pain via the cyclooxygenase-1/prostaglandin E2 induction at the tissue injury site and the spinal cord thus, increasing neuronal sensitivity to pain stimuli. This mechanism corroborates the observation relating to the location of pain in the BPC group. In this study (data not shown) BPC involving the spinal vertebrae was more frequent among the moderate and the severe pain groups probably because of the proximity of the infarctive injury to the spinal cord.[44,45] IL-8 like other chemokines are responsible primarily for migration of leucocytes to the sites of tissue injury or inflammation as seen in VOC. It also participates in synaptic transmission and formation of secondary messenger systems in neurons and glial cells, hence favouring nociception. Tumour necrosis factor-α and ET-1 were mostly positively correlated with the bone-pain crisis severity groups indicating the role of both peptides in the pathogenesis of the BPC.
Limitation of the study
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