There is compelling evidence that systemic inflammation plays a role in the development and progression of coronary heart disease (CHD) [1
]. White blood cell (WBC) counts are elevated in patients presenting with acute coronary syndromes. Early studies have shown that patients with elevated white blood cell (WBC) counts were at higher risk of mortality and recurrent AMI [2
]. More recent studies have confirmed the association between an elevated WBC and clinical outcomes in AMI patients and furthermore suggested that WBC could serve as a simple and inexpensive tool for risk stratification in acute coronary syndromes [4
The pro-inflammatory cytokines IL-1 and IL-6 have also been implicated in several mechanisms that contribute to the development of CHD and increased levels of IL-6 are associated with higher cardiovascular mortality [6
]. Pro-inflammatory cytokines stimulate the liver to synthesize acute phase proteins, which in turn are associated with a higher risk of cardiovascular events [6
]. For example, C-reactive protein (CRP) whose concentration in serum is increased in response to inflammatory stimuli is also elevated in ACS compared to stable angina or vasospastic angina [7
]. Elevated CRP is associated with increased cardiac morbidity [10
]. Increased levels of fibrinogen, another acute phase protein are a strong predictor of a poor outcome in unstable CAD, and are also reported in patients with asymptomatic atherosclerosis [13
]. Similarly, plasma vWF levels are strongly associated with ischaemic heart and cerebrovascular disease [16
]. Thus, a variety of inflammation markers are risk factors for myocardial infarction and other acute coronary syndromes [17
]. Inflammatory proteins may not only be markers of disease but may contribute to its pathogenesis. For example, there is evidence that CRP and fibrinogen promote vascular inflammation and thrombosis [18
In spite of the mounting evidence implicating a group of inflammation-related markers and coronary disease, the mechanisms are still poorly understood. While inflammation in the vessel wall may be a consequence of environmental factors (infection, tissue injury), it is also possible that genetic factors contribute to an abnormal or exaggerated inflammatory response. Moreover, interactions between inflammatory markers and variants of thrombotic genes may contribute to the coronary thrombosis that underlies ACS. We sought to determine whether the elevated white cell count in ACS is influenced by pro-inflammatory genetic variants.
The IL1 chromosomal cluster of genes contains an extended haplotype spanning the IL1beta and IL1RN genes [21
]. This haplotype is represented by the IL1RN intronic repeat *2 allele [22
]. Carriers of *2 appears to be over-represented in some diseases associated with inflammation [21
]. IL1 Receptor Antagonist levels are elevated in acute MI, but it is unclear whether the *2 allele carriers have an altered risk of cardiovascular disease [26
]. The role of the IL1 cluster haplotype in modulating WBC levels in coronary patients has not been investigated, and we wished to determine if cardiovascular patients carrying *2 had an elevated WBC. The IL6 promoter variant -174G allele is associated with higher levels of acute phase IL6, with heterozygotes intermediate between the two homozygotes [29
]. The G allele is associated with both arthritis and susceptibility to Kaposi sarcoma and has been associated with atherosclerosis [30
]. We wished to evaluate whether there the IL6-174GG genotype was associated with higher WBC than the -174CC genotype.
The hypothesis we wished to test was whether or not the elevated WBC seen in patients with an acute event is elevated in carriers of pro-inflammatory genetic variants. In the absence of careful studies of WBC before and after an acute event, the pathophysiological causation of elevated WBC in patients that presented with MI is incompletely understood. The impact of myocardial damage, and inflammatory states prior to the MI, are likely to be two important factors, although extent of ischaemia was not shown to be strongly associated with WBC in another study [5
]. It is unclear if inflammatory responses associated with IL1 and IL6 cytokines are key to modulating either of these processes. From a clinical perspective, identifying any association between pro-inflammatory variants and WBC (resulting from either process, and regardless of cause) could provide a perspective on the anti-inflammatory mechanisms that may be relevant in improving patient prognosis. Additionally, we examined the relationship of the pro-inflammatory variants with CRP, another marker of inflammation, and with variants in thrombotic genes previously implicated in coronary thrombosis.
In order to test this hypothesis, baseline white cell counts around the time of presentation were investigated from two clinical trial studies (sample sizes of patients with white cell counts at follow-up were not sufficient to permit a powerful test of such effects at later time points). The first study was the multinational OPUS-TIMI 16 study [33
]. Patients had to have either acute myocardial infarction or unstable angina, with an associated high-risk feature, and be enrolled within 72 hours from the onset of their acute coronary syndrome. For comparison purposes, a group of patients with acute myocardial infarction or unstable angina from the EXCITE trial (who were recruited at the time of a percutaneous intervention, and therefore are mainly outside the time period of an acute event) and a group of patients presenting with stable angina (also from the EXCITE study) were similarly examined [34