BCG immunisation impairs vascular relaxation and enhances atherosclerosis in the chronically hypercholesterolameic rabbit ML Tickner, DJ Lamb, AC Dreux, W El-Sankary, SMO Hourani, L-J Eales-Reynolds, GAA Ferns. School of Biomedical & Life Sciences, University of Surrey, Guildford, GU2 7XH, UK.

Print abstract Search PubMed for: Tickner ML Lamb DJ Dreux AC El-Sankary W Hourani SMO Eales-Reynolds Ferns GAA

We have previously shown that bacillus Calmette-Guerin (BCG) immunisation enhances atherosclerosis in the cholesterol-fed rabbit (Lamb et al., 1999). Recent studies have implicated immune responses to heat shock protein-60/65 (HSP) as a causal factor (Xu, 2002). The aim of this study was to use a BCG immunisation model to investigate the relationship between anti-HSP immune responses, endothelial dysfunction and atherosclerosis in the chronic low-level hypercholesterolaemic rabbit model.

24 male New Zealand white rabbits (weight range 2.6 - 3.5kg) were divided into four experimental groups. Animals were injected intradermally with BCG vaccine (4-13x10⁶ units) (n=12) or saline (n=12), boosted after 2 weeks and after a further 2 weeks fed either a 0.25-1% cholesterol diet or a chow diet for 16 weeks. Blood cholesterol levels were maintained at 10-12mmol/l by altering the dietary cholesterol content. Plasma titres of HSP60/65 specific antibodies were measured by indirect ELISA. Animals were anaesthetised with xylazine and ketamine (s.c.), heparinised and killed with an overdose of pentobarbitone. Carotid artery rings were mounted between two stainless steel stirrups and pre-contracted with cumulative concentrations of phenylephrine (0.01-30µM). Carotid rings were re-contracted with 3µM phenylephrine and relaxed with cumulative concentrations of the endothelium-dependent vasodilator acetylcholine (0.01-3µM) or the endothelium-independent vasodilator, sodium nitroprusside (SNP; 0.01-300µM). Responses were recorded isometrically using force displacement transducers and analysed statistically using one-way ANOVA. Aortae were stained en face with oil red O. The intima and media areas were measured in aortic cross sections and analysed statistically using Mann-Whitney U test.

Plasma levels of anti-mycobacterial antibodies rose following BCG immunisation, but anti-HSP antibodies developed only in the BCG-immunised, cholesterol-fed rabbits. Endothelial function was impaired in the BCG immunised, cholesterol-fed rabbits compared to control animals as measured by acetylcholine-mediated relaxation of isolated carotid artery rings (_pD₂ 7.18±0.16 vs 7.75±0.17. This impairment was positively associated with the level of plasma anti-HSP-60 antibodies (P<0.01). There was no difference in the relaxation to SNP (_pD₂ 5.4±0.2 vs 5.3±0.1). BCG immunised, cholesterol-fed rabbits developed more extensive aortic lesions (72.3±3.5% vs 51.8±8.6%); P<0.05 and thicker intima:media area ratio (61.6±16.2 vs 3.3±2.9); P<0.01 compared to control animals. Aortic endothelium from cholesterol-fed, but not chow-fed, rabbits stained positively for HSP-60, independently of the immunisation protocol.

These results suggest that BCG immunisation impairs endothelial responses and increases atherosclerosis in chronic low-level hypercholesterolaemic rabbits. This is mediated, at least in part, by immune responses against mycobacterial and vascular HSP.

Lamb, D.J., et al (1999). Atherosclerosis 143, 105-113.
Xu, Q. (2002). Arterioscler Throm Vasc Biol 22, 1547-1559.