Severe sickle cell anemia is associated with increased plasma levels of TNF-R1 and VCAM-1.

Exposure to TNF-α activates endothelial cells, leading to up-regulation and enhanced cell-surface expression of multiple proteins including VCAM-1 and intra-cellular adhesion molecule-1 (ICAM-1).5 During acute infections, these proteins facilitate the attachment of leukocytes to the vessel wall, ultimately resulting in transmigration into infected tissue.6 In SCA, variable TNF-α signaling might alter disease severity by activating systemic and pulmonary endothelium and by modulating rates of sickle vasoocclusion secondary to altered leukocyte-endothelial cell adhesion.7, 8 Associations between some individual clinical complications of SCA and plasma levels of VCAM-1 and ICAM-1, but not TNF-R1, have been described previously. 9, 10 Here, we report associations between increase plasma levels of VCAM-1 and TNF-R1 and severe SCA, as measured by the SCA severity score, a validated metric which incorporates information about steady-state laboratory data and medical history into a Bayesian network in order to estimate a subject’s probability of dying within the next five years due to complications of SCA.11, 12 In a population of 52 adult subjects with SCA recruited for a study of pulmonary hypertension, we used the severity score to identify the 12 subjects with the most mild clinical presentations (probability of death within five years = 7.5 ± 2.6%, mean ± sd.) and the 12 subjects with the most severe presentations (probability = 80.3 ± 18%). No significant differences were observed between the two groups in terms of age or hydroxyurea treatment (p-values 0.35 and 0.68, respectively), while non-significant trends toward being female and having lower glomerular filtration rates (GFR) were observed in the severe group (p-values 0.1, 0.1). Subjects in the severe group had lower levels of hemoglobin than subjects in the mild group (median 8.3 vs. 9.45 g/dL, p-value = 0.046), though the difference was not significant after adjusting for multiple testing. To look for associations between SCA severity and TNF-α axis activity and endothelial cell activation, steady-state plasma samples from these two groups were tested for levels of E-selectin, ICAM-1, nitric oxide (NO), P-selectin, TNF-α, TNF-R1, and VCAM-1. Only TNF-R1 and VCAM-1 showed significant differences between the two groups after correcting for multiple testing; both biomarkers were significantly higher in severe cases of SCA (p-value = 0.0015 and p-value = 0.0019, respectively, Table 1). Plasma levels of TNF-R1 and VCAM-1 showed a significant correlation after log transformation (Figure 1, p-value = 0.036). Figure 1 Comparison of Plasma VCAM-1 and TNF-R1 TABLE 1 Summary of plasma biomarkers tested for potential association with SCA severity Renal disease, a common occurrence in SCA, can lead to increased plasma levels of TNF-R1 secondary to decreased renal clearance. 13, 14 To evaluate this and other possible confounding factors, we tested for associations between TNF-R1 or VCAM-1 levels and GFR, gender, age, and hydroxyurea treatment status. We also looked for potential association with plasma nitric oxide (NO), since endothelial cell-surface expression of leukocyte adhesion proteins is altered by NO bioavailibility.15, 16, 17 TNF-R1 levels were found to be associated with gender (p-value = 0.048), but were not significantly associated with GFR, hydroxyurea use, NO levels, or with age (p-value > 0.05 in all cases). VCAM-1 levels were not significantly associated with any of these factors (p-value > 0.05). The relationship between TNF-R1 and SCA severity remained significant in a linear regression model involving both TNF-R1 and gender (p-value = 4.6 × 10−03), indicating that TNF-R1 as a plasma biomarker is significantly related to increased SCA severity beyond its association with gender. In contrast to our results, multiple groups have shown that hydroxyurea therapy was associated with decreased levels of multiple inflammatory biomarkers, including VCAM-1; this may be due to our small sample size, or to differences in efficacy of hydroxyurea treatment. 9, 18, 19 We identified 155 SNPs in 16 candidate genes which either code for one of the six biomarkers, or are involved in their release into the plasma. SNP data was obtained from a previous GWAS, which examined 1265 subjects with SCA from the Cooperative Study of Sickle Cell Disease (CSSCD) using the severity score as a phenotype. While none of the 155 SNPs met stringent criteria for genome-wide significance, several of these SNPs did show significant association with SCA severity at moderate levels (BF > 3; Table 2).4 TABLE II Genetic associations in biomarker and related genes Of the 47 SNPs examined in the six candidate genes coding for the plasma biomarkers (ICAM1, SELE, SELP, TNF, TNFRSF1A, and VCAM1), only one (rs1041163), located in the promoter of VCAM1, was significantly associated with SCA severity (BF = 3.5). This same SNP was previously reported to be associated with small vessel stroke in the CSSCD.20 We also examined 108 SNPs in 10 genes involved in the release of these biomarkers or in the generation of NO (ADAM17, ARFGEF2, ARF1, ARF3, ERAP-1, IL1B, NOS1, NOS2A, NOS3, and NUCB2).21–25 Of these SNPs, the top association was with rs2273102, located in intron 12 of ARFGEF2, a gene known to be involved in endothelial release of TNF-R1 into circulation in an ADAM17-independent manner (BF = 70 under a dominant model).24 Three of the other 15 SNPs in ARFGEF2 (rs6019548, rs1115535, and rs6019566) also showed significant associations with SCA severity (BF > 3). Using SNP set enrichment (SSE) analysis, which summarizes genetic association across multiple SNPs within a single gene, ARFGEF2 as a whole was found to be significantly related to SCA severity ( p-value = 5.5 × 10−05).4 Significant association with SCA severity was also observed with rs1143634, located in the gene IL1B (BF > 30 under a recessive model). IL1B codes for the cytokine IL-1B, which induces ADAM17-dependent release of TNF-R1 into circulation, among other roles.21 None of the other 15 genes were significantly related to SCA severity by SSE analysis. Our data showing severity-related variability in plasma levels of biomarkers of TNF-α signaling, together with genetic association between SCA severity and genes involved in biomarker generation and release, supports the hypothesis that genetic variability along the TNF-α signaling pathway may modulate the severity of SCA. Further investigation, including the confirmation of both the genetic and biomarker associations with SCA severity in a single population, is necessary to develop a more complete understanding of the ways in which genetic variation in TNF-α signaling might affect the clinical manifestations of this disease. With such confirmation, the associations shown here between SCA severity and variability along the TNF-α signaling pathway might be used to further prospectively stratify subjects with SCA into different levels of risk, and, ultimately, may lead to the ability to more rationally designed therapeutic plans for subjects with different clinical presentations of SCA.