TNFSF/TNFRSF cytokine gene expression in sickle cell anemia: Up-regulated TNF-like cytokine 1A (TL1A) and its decoy receptor (DcR3) in peripheral blood mononuclear cells and plasma.

BACKGROUND: Sickle cell anemia (SCA), a disorder with an important inflammatory component, where vasoocclusion is major contributor to the disease pathophysiology. Pro-inflammatory cytokines play an important regulatory role in the process of inflammation. We investigated the expression TL1A/DR3/DcR3 cytokine signaling pathway in peripheral blood mononuclear cells (PBMC) and their corresponding plasma levels in SCA subjects who presented with acute painful episodes. MATERIALS AND METHODS: PBMC were isolated from the blood of SCA subjects and normal healthy controls. RNA isolated from PBMC was used for real time gene expression of TL1A/DR3/DcR3. Gene expression was compared in subgroups within SCA subjects with co-inherited fetal hemoglobin (HbF) or alpha-globin gene deletions. Plasma prepared from blood was used for determination of TL1A/DR3/DcR3 proteins by ELISA assays. RESULTS: In the PBMC of SCA subjects, expression of TL1A and DcR3 is elevated, while DR3 expression is lowered in comparison to normal control PBMC. In SCA subjects with HbF > 10%, TL1A/DcR3 expression is lower, while HbF < 10% is associated with increased TL1A/DcR3 expression. Moreover, subjects with HbF > 10% appear to have significantly fewer pain episodes in comparison to those with HbF < 10%. Deletion of alpha-globin genes appears to have no significant effect on TL1A/DR3/DcR3 expression. Circulating levels of TL1A, DR3 and DcR3 in plasma were significantly elevated in SCA subjects. CONCLUSIONS: Elevated TL1A and DcR3 expression in PBMC of SCA subjects during painful vasoocclusive crisis, suggest an altered TL1A expression may contribute to the pathophysiology of vasoocclusive crisis in SCA. HbF > 10% appears to moderate TL1A elevation, while HbF < 10% exacerbates TL1A/DcR3 responses. Furthermore, subjects with HbF > 10% have significantly lower pain episodes reported as compared to subjects with HbF < 10%.

Fetal hemoglobin in sickle cell anemia: genetic studies of the Arab-Indian haplotype.

Sickle cell anemia is common in the Middle East and India where the HbS gene is sometimes associated with the Arab-Indian (AI) ß-globin gene (HBB) cluster haplotype. In this haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are 3-4 fold higher than those found in patients with HbS haplotypes of African origin. Little is known about the genetic elements that modulate HbF in AI haplotype patients. We therefore studied Saudi HbS homozygotes with the AI haplotype (mean HbF 19.2±7.0%, range 3.6 to 39.6%) and employed targeted genotyping of polymorphic sites to explore cis- and trans- acting elements associated with high HbF expression. We also described sequences which appear to be unique to the AI haplotype for which future functional studies are needed to further define their role in HbF modulation. All cases, regardless of HbF concentration, were homozygous for AI haplotype-specific elements cis to HBB. SNPs in BCL11A and HBS1L-MYB that were associated with HbF in other populations explained only 8.8% of the variation in HbF. KLF1 polymorphisms associated previously with high HbF were not present in the 44 patients tested. More than 90% of the HbF variance in sickle cell patients with the AI haplotype remains unexplained by the genetic loci that we studied. The dispersion of HbF levels among AI haplotype patients suggests that other genetic elements modulate the effects of the known cis- and trans-acting regulators. These regulatory elements, which remain to be discovered, might be specific in the Saudi and some other populations where HbF levels are especially high.

Monocytes from sickle cell disease patients induce differential pulmonary endothelial gene expression via activation of NF-κB signaling pathway.

Monocyte-endothelial interactions play an important role in inflammatory diseases and may modulate vasculopathy in sickle cell disease, a disorder with an important inflammatory component. We co-incubated normal and sickle monocytes, lymphocytes and TNF-α with pulmonary microvascular and arterial endothelial cells and compared the expression of genes coding for adhesion molecules and cytokines that might contribute to sickle vasoocclusion. Monocyte-endothelial cell co-incubation resulted in up-regulation of L-selectin, E-selectin, VCAM-1, ICAM-1, MCP-1, MMP-1, TNF-α, IL-6 and IL-1ß and down-regulation of eNOS. Lymphocyte-endothelial cell co-incubations, induced similar effects restricted to pulmonary artery endothelial cells. TNF-α had similar effects on the endothelial cells as monocytes did, however monocyte induced gene expression in endothelial cells was not TNF-α dependent but was regulated through the NF-κB pathway. Sickle monocytes lead to altered expression of L-selectin, MCP-1 and MMP-1 in pulmonary vascular endothelium when compared with normal monocytes. The gene expression changes we observed could reflect pathological events of sickle vasoocclusion.

Effect of sodium butyrate on lung vascular TNFSF15 (TL1A) expression: differential expression patterns in pulmonary artery and microvascular endothelial cells.

Vascular endothelial growth inhibitor TNFSF15 (TL1A), a ligand for TNFRSF25 (DR3) and decoy receptor TNFRSF6B (DcR3), is expressed in human pulmonary arterial (HPAEC) and lung microvascular (HMVEC) endothelial cells where it might modulate inflammation and sickle vasculopathy. Pulmonary disease, endothelial abnormalities and inflammation are prominent features of sickle cell disease (SCD). Butyrate has opposing effects on endogenous TNFSF15 expression in pulmonary endothelium, acting as an inhibitor in HPAEC and an inducer in HMVEC. Similar effects were observed with a known cytokine TNF-alpha in these two cell types. Furthermore the TNFSF15 promoter utilized different combinations of cis-elements for its expression in these two cell types. AP1-like and G-rich sequence elements were critical for promoter activity in large vessel HPAEC while AP1-like and NF-kappaB consensus sequence elements were required in small vessel HMVEC. The requirement of an NF-kappaB sequence element by the TNFSF15 promoter in HMVEC but not in HPAEC supported the notion that HMVEC might be a target of inflammation and vasoocclusion in SCD. The dual effects of butyrate-dependant TNFSF15 regulation in lung endothelium may help in identify inflammatory pathways and understand the role of HMVEC in pathogenesis of vasoocclusion in SCD.

Differential gene expression in pulmonary artery endothelial cells exposed to sickle cell plasma.

Clinical variability in sickle cell disease (SCD) suggests a role for extra-erythrocytic factors in the pathogenesis of vasoocclusion. We hypothesized that endothelial cell (EC) dysfunction, one possible modifier of disease variability, results from induction of phenotypic changes by circulating factors. Accordingly, we analyzed gene expression in cultured human pulmonary artery ECs (HPAEC) exposed to plasma from 1) sickle acute chest syndrome (ACS) patients, 2) SCD patients at steady state, 3) normal volunteers, and 4) serum-free media, using whole genome microarrays (U133A-B GeneChip, Affymetrix). Data were analyzed by Bayesian analysis of differential gene expression (BADGE). Differential expression was defined by the probability of >1.5 fold change in signal intensity greater than 0.999 and a predicted score of 70-100, measured by cross-validation. Compared with normal plasma, plasma from SCD patients (steady state) resulted in differential expression of 50 genes in HPAEC. Of these genes, molecules involved in cholesterol biosynthesis and lipid transport, the cellular stress response, and extracellular matrix proteins were most prominent. Another 58 genes were differentially expressed in HPAEC exposed to plasma from ACS patients. The pattern of altered gene expression suggests that plasma from SCD patients induces an EC phenotype which is anti-apoptotic and favors cholesterol biosynthesis. An altered EC phenotype elicited by SCD plasma may contribute to the pathogenesis of sickle vasoocclusion.