Elsevier

Cytokine

Volume 80, April 2016, Pages 18-25
Cytokine

Plasma levels of TGF-β1 in homeostasis of the inflammation in sickle cell disease

https://doi.org/10.1016/j.cyto.2016.02.012Get rights and content

Highlights

Abstract

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-β), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-β1, and also checked the influence of the presence of −509C/T polymorphism in TGFB1 gene on TGF-β1 plasma values. The plasma levels of TGF-β1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sβ-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The −509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-β1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-β1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-β1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-β1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.

Introduction

Sickle cell disease (SCD) comprehends the group of hemolytic anemia caused by the presence of hemoglobin S (Hb S), either in homozygosis (sickle cell anemia), or in association with other hemoglobin variants or thalassemias [1]. Hb S originates from a point mutation in the beta globin gene (HBB:c.20A > T; rs334) resulting in replacement of glutamic acid for valine in the polypeptide chain, causing structural and biochemical modifications in the hemoglobin molecule [1], [2], [3]. Sickle cell anemia is the most common form of the disease, as well as the most severe. The association of Hb S with other variants such as Hb C (HBB:c.19G > A; rs33930165) and Hb D-Los Angeles (HBB:c.364G > C; rs33946267) are less frequent and less severe, while its association with beta thalassemia results in moderate to severe clinical types, depending on the beta-thalassemia mutation inherited [1], [4].

Complications of SCD start with the polymerization of Hb S, erythrocyte sickling, exposure of membrane proteins, hemolytic anemia and ischemia-reperfusion cycles. These recurrent cycles result from vascular occlusion and represent the main stimulus for the inflammatory process, due to endothelial dysfunction, increased vascular inflammation, coagulation activation and oxidative stress created during the restoration of blood flow [5]. Although it plays a protective role in the infection control and promotes tissue repair, the exaggerated inflammatory response can also cause tissue damage. In SCD, inflammation may occur in acute and chronic forms, due to polymerization of Hb S, which not only results in erythrocyte sickling and intravascular hemolysis, but also a series of cellular interactions mediated by inflammatory cytokines [6].

The transforming growth factor beta (TGF-β) is a pleiotropic cytokine that affects cell proliferation, survival and migration and might act as both positive and negative regulator during gene transcription, depending on the target genes and cellular context. It exists in three isoforms: TGF-β1, 2 and 3, whereas TGF-β1 is the most abundant [7]. TGF-β plays an important role in inflammation resolution, since it is associated with the inhibition of immune cells proliferation and the activity suppression of immune system precursor cells. It also acts as a potential inhibitor on T cells differentiation and apoptosis inducer in B cells, besides participating in the chemotaxis and polarization of macrophages and neutrophils at the inflammation site. On the other hand, it is involved in the releasing of proinflammatory cytokines from neutrophils and in stimulating pro-inflammatory Th17 cells lineage differentiation [8], [9].

The TGF-β production can be controlled by single nucleotide polymorphisms (SNP) in its gene, such as the −509C/T on TGFB1 (rs1800469), wherein the mutant allele T is associated with high circulating levels of TGF-β1 [10]. The elevation in the TGF-β1 levels has been described in sickle cell anemia [11], [12] and genetic polymorphisms in TGFB, its receptors and members of its activation pathway were related to subphenotypes of the disease, including clinical complications such as myocardial infarction, osteonecrosis, priapism, leg ulcer and pulmonary hypertension [13].

In the present study we built on the observation of the inflammatory condition in SCD patients – genotypes SS, SD-Los Angeles (SD), S/beta-thalassemia (Sβ-thal) and SC – based on the association of TGF-β1 plasma levels to the total amount of white blood cells (WBC), neutrophils and platelets in the peripheral blood. In addition, we evaluated the relation between the −509C/T polymorphism in the TGFB1 gene and the TGF-β1 plasma values.

Section snippets

Subjects

The study consisted of 115 SCD patients from the Arthur de Siqueira Cavalcanti Institute of Hematology (Hemorio) in Rio de Janeiro, RJ, Brazil. In order to minimize biases in the analysis, all patients were selected according to exclusion criteria, namely: anti-inflammatory prescription for three weeks prior to sample collection, use of hydroxyurea for up to six months preceding the collection date and blood transfusions carried out in <60 days (or Hb A > 10.0%) [14]. In addition, only individuals

Characterization of the study groups

After characterization of the 115 samples obtained from patients with SCD, 67 (58.3%) were from patients with Hb SS, 30 (26.1%) with Hb SC, 10 (8.7%) with Hb Sβ-thal and eight (6.9%) with Hb SD. All 58 control subjects were confirmed with Hb AA profile. The hemoglobin profile presented by the five study groups are detailed in Table 2.

−509C/T polymorphism frequency and its influence on circulating levels of TGF-β1

The −509C/T polymorphism was investigated in all samples. In the case group we found 39.1% of the CT genotype and 9.6% of TT homozygotes. In the control group, the

Discussion

We describe here the differences between SCD genotypes based on inference of the inflammatory condition, by the dosage of TGF-β1 linked to WBC, neutrophils and platelets counts. The evaluated study groups were the four most common genotypes of SCD: Hb SS, Hb SD, Hb Sβ-thal and Hb SC. A control group with Hb AA was used as reference for TGF-β1 dosages and for the effect of −509C/T polymorphism in circulating protein levels.

Although the literature presents studies related to the inflammatory

Conclusion

In summary, the results presented in this study indicate that the plasma levels of TGF-β1 are elevated in SCD, especially sickle cell anemia, the most clinically severe condition. Their levels are related to WBC, evidencing the chronic inflammatory condition triggered by the disease, and associated to the count of circulating platelets, suggesting that increased plasma levels of TGF-β1 can be indirectly inferred by increased platelet count in the blood. Because of the strong anti-inflammatory

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

The authors thank the FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for financial support. Process no. 2012/19653-1. In addition, we thank Gabriel Gandolphi for English text review.

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