White Blood Cells and Blood Pressure

Supplemental Digital Content is available in the text.


Blood pressure measurement procedure in the UK Biobank study
The participant was collected from the waiting area and was seated in a curtained office. The participant took the first part of the interview questionnaire. After completing this, the staff member informed the participant that they would now take the first of two measurements of blood pressure. The participant was asked to sit with their feet parallel to each other, toes pointing forward and soles of feet flat on the floor. There should be no restrictive clothing to impede the circulation to their left upper arm: the participant was asked to loosen or remove any restrictive clothing. The right arm was only used if the left was not practical: e.g. amputee, shunt, mastectomy, axiliary clearance). Since resting blood pressure was measured, the staff member took care not to engage the participant in conversation. A Seca tape measure was used to determine the circumference of midpoint of upper arm, and the appropriate size of blood pressure cuff was selected. The appropriate sized cuff was put on the upper arm of participant (it might, depending on size of upper arm, encircle the arm several times). The cuff was rotated so that the green marker tab indicating the centre of the cuff bladder lied over the brachial artery, which was located at the inner part of front of the elbow. The rubber inflation tubing sit over the brachial artery in line with the participant's middle finger, and the bottom of cuff sit 1-2 cm above the elbow joint. When the cuff was correctly positioned it was secured with Velcro fasteners. The participant was asked to place their arm on the desktop, so that the cuff was at about same level as their heart, and to breathe in and out slowly five times in a relaxed fashion. The rubber inflation tubing was connected to the Omron blood pressure monitor and, "Start" on the monitor was pressed. When the blood pressure result was displayed on monitor, the "Retrieve" button on the computer was selected to transfer results from the monitor and then "Accept" to complete the first measurement. If there was a problem with the measurement, "Reject" was selected and the measurement repeated. If the largest cuff size was too small for the participant, or if the electronic blood pressure monitor failed to produce a reading, a sphygmomanometer with an inflatable cuff was used in conjunction with a stethoscope. The IT system prompted the staff member to scan the sphygmomanometer by using the barcode reader before taking the measurement. After completing the first measurement, the rubber inflation tubing was disconnected from the Omron monitor, with the cuff left in place. The participant was asked to gently shake their arm, and open and close their hand a few times. A timer in the computer ensured that the second blood pressure reading could not be taken until at least 1 minute had elapsed. This rest period was used to complete the second part of the interview. The inflation tubing was reconnected to the Omron blood pressure monitor and a second reading was made using the same procedure as before. Then the cuff was removed.

Supplemental Tables
Supplemental Table 1: Spearman's rho rank correlation coefficients for pairs of white blood cell counts in the UK Biobank study (n=384,721 subjects used in this study)  Median-oriented estimates are given in mmHg units relative to the 3 rd quintile of certain cell type count. Analyses were adjusted for sex, age, age squared, BMI, smoking status and alcohol intake frequency. P values significant after Bonferroni correction for multiple testing (150 tests i.e. 5 types of blood cell counts x 3 BP indices x 10 between-quintile differences and 15 tests for continuously defined white blood cell counts) are depicted in bold.
Cont. = estimates concerning continuously defined count of particular white blood cell count 6  NF-kappa-B inhibitor alpha (also known as IκBα) sequesters stress-induced transcription factor NF-κB in an inactive state in the cytoplasm; NF-κB plays crucial role in lymphocyte development/function and thus in adoptive immunity 18,19 endothelium-specific IκBα overexpression results in milder renal damage and reduced T cell infiltration, but has no effect on blood pressure level in hypertension as compared to wild type controls 20 ; pharmacological inhibition of NF-κB signaling reduces cardiac hypertrophy in spontaneously hypertensive rats (SHRs) without effects on BP level 21 rs9913156 intergenic ARRB2 DBP Arrestin beta 2 is a regulator of G-protein coupled receptor (GPCR) signaling 22 no significant difference in AngII-induced SBP raise in Apoe -/-Arrb2 -/mice as compared to Apoe -/-Arrb2 +/+ 23 ; angiotensin II type 1 receptor (AGTR1)/Arrb2/ERK1/2 signaling axis regulates renal fibrosis 24 rs60515486 intron AGBL2 SBP, DBP AGBL2 gene encodes cytoplasmic carboxypeptidase 2 (CCP2) that is responsible for deglutamylation of targeted protein such as α-tubulin or myosin light chain kinase (MYLK) 25 -rs76382185 intergenic S1PR1 DBP S1P receptor type 1 and its ligand, Sphingosine-1-Phosphate are crucial for the egress of lymphocytes from lymph nodes and for regulation of eNOS dependent vasodilation 26,27 EC-specific S1pr1 knockout mice have increased basal blood pressure level, as well exacerbated hypertension in response to AngII infusion 26 ; S1pr1 agonist, SEW2871, administration decreases BP of hypertensive mice 28 rs3745621 intron SAE1 DBP SAE1 activates SUMO (1-3) proteins, which covalently bind to target proteins introducing posttranslational modification called SUMOylation 29,30 AngII induces upregulation of SUMO-1 and its target, activating transcription factor 3 (ATF3) in the mice aorta. ATF3 SUMOylation is involved in endothelial cell dysfunction and inflammation via increasing ATF3 protein stability 31 ; SUMO-1 overexpression protects against pressure overload-induced left ventricular hypertrophy 32 ; SUMOylation of RORγt transcription factor by SUMO 2 is crucial for the TH17 lymphocyte differentiation and thymocyte development 33

Supplemental table 4: MR analyses testing effects of counts of white blood cells subpopulations on blood pressure-related traits
Ubiquitin-associated and SH3 domain-containing protein B (UBASH3B, also termed STS-1 or TULA-2) negatively regulates signaling through T cell receptor (TCR) 34 T cells isolated from UBASH3B and UBASH3A double knockout mice are hyper-responsive to TCR stimulation and exhibit enhanced cytokine production 34 ; single knockouts also exhibit exacerbated inflammation 35 rs12598529 intron ADCY9 DBP Adenylate cyclase 9 (AC9) catalyses the formation of cyclic AMP (cAMP) in response to beta-adrenergic signaling activation 36 ; cAMP abundantly produced by AC9 in T reg cells is transferred via gap junction to responder T cells which in turn become suppressed 37,38 Loss of ADCY9 results in an improved endothelial function in femoral arteries of healthy, as well as in atherosclerotic mice; loss of ADCY9 protects mice from atherosclerosis due to reduced macrophage accumulation/proliferation in aortic wall 39  Presented SNPs were previously associated with lymphocyte count at p<5x10 -8 1 and with SBP and/or DBP 2 in the same direction at Bonferronicorrected p value threshold given 121 SNPs tested (p<4.1x10 -4 ) in Mendelian Randomization analysis on lymphocyte count and BP indices in the current study.

Supplemental figure 4: Level of medication-adjusted, blood pressure indices in relation to quintiles of five counts of white blood cell types after additional adjustment for salt intake
Estimated marginal means of BP indices, from GLM analysis adjusted for sex, age, age squared, BMI, smoking status, salt intake and alcohol intake frequency, are presented according to quintiles of counts of white blood cell subpopulations. All ANOVA tests, assessing global between-quintiles differences in BP indices were significant at p<10 -11 . Post-hoc tests revealed that all comparisons, except for eosinophil count and DBP, between the 1 st and the 5 th quintile of any cell type count with respect to any BP index were significant at Bonferroni corrected p<0.05, given 150 tests (5 types of blood cell counts x 3 BP indices x 10 between-quintile differences) performed.

Supplemental figure 5: Partial residual plots derived from GAM analyses testing continuous level of five white blood cell types in relation to medication-adjusted blood pressure indices in the UK Biobank
Red ticks correspond to between-quintile boundaries.
X axes were limited in order to exclude 0.5% subjects with the highest level of particular white blood cell count.

Supplemental figure 6: Scatter plots of SNPs used as IVs for lymphocyte (A,C) or eosinophil (B,D) count with SBP (A,B) or DBP (C,D).
SNPs rs3184504 (A,C) and rs653178 (B,D) are depicted in red.

Supplemental figure 7: Leave-one-out plots presenting IVW causal estimates testing effect of lymphocyte or eosinophil count on SBP/DBP level
Top 25 SNPs that inflate or deflate IVW causal estimates the most are depicted. UACR SNP-specific estimates were derived from GWAS of the CKD Gen consortium (A) 5 or meta-analysis of UK Biobank and CKD Gen consortium (B) 4

Supplemental figure 11: Leave-one-out plots presenting IVW causal estimates testing effect of lymphocyte count on UACR level
Top 25 SNPs that inflate or deflate IVW causal estimates the most are depicted.