Exploratory analysis of the neutrophil to lymphocyte ratio in patients with pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare, progressive vascular disease characterized by elevated pulmonary vascular resistance and right heart failure [1]. The pathomechanism of PAH consists of endothelial dysfunction, in-situ thrombosis, occlusive vascular remodeling with loss of small vessels and perivascular inflammation including the development of tertiary lymphoid follicles [2, 3]. The importance of the immune system to regulate pulmonary vascular homeostasis has been demonstrated. In experimental models of pulmonary hypertension (PH), vascular inflammation may even precede vascular remodeling [3‐5]. Accumulation of circulating cytokines such as interleukin (IL) 2, 6, 8, 10 and 12p70 and C-reactive protein (CRP) has been associated with poor overall survival in patients with PAH [6‐8]. However, the inflammatory state found in patients with PAH has not yet been incorporated into the clinical management of the disease.

Parameters of the differential white blood cell (WBC) count have been linked to poor outcome in cardiovascular diseases. In patients with coronary heart disease or chronic left heart failure, increased neutrophils, decreased lymphocytes and particularly an increased neutrophil/lymphocyte ratio have been related to poor outcome [9‐11]. The ratio of the absolute numbers of neutrophils and lymphocytes is a simple and readily assessable measure of the inflammatory state, which may be considered more stable than the individual cell counts and less affected by acute conditions [11]. To date, there is only one study assessing the clinical significance of the neutrophil/lymphocyte ratio in patients with PAH. The study demonstrated that an increased neutrophil/lymphocyte ratio was associated with poor functional class and elevated mortality. The prediction of outcome, however, was not independent of other parameters of disease severity [12].

The present study was conducted to assess the association of total and differential WBC count parameters with functional and hemodynamic parameters as well as transplantation-free survival in patients with PAH.

The present study revealed that increased relative number of neutrophils and particularly increased neutrophil/lymphocyte ratio were associated with more severe disease and poor outcome in patients with PAH. On the other hand, the absolute number of cell count was not found to be associated with disease severity and survival. The prediction of transplantation-free survival of the neutrophil/lymphocyte ratio was found to be independent of functional, hemodynamic parameters, GFR, CRP levels and co-morbidities in the present cohort of patients. This implication on the outcome sustained significant in subsets of patients with incident PAH or in PAH patients without co-morbidities known for their risk for left heart disease, but not in patients with only idiopathic disease. In addition to these findings, we observed that elevated eosinophils were associated with impaired hemodynamics and functional capacity, but not with outcome. Elevated eosinophils were found in patients with lower mPAP and PVR. Although this is, to our knowledge, the first clinical analysis of circulating eosinophils in patients with PAH, these findings challenge the observations derived from experimental PH and need to be validated.

Clinical management of patients with PAH involves the assessment of circulating biomarkers that reveal severity of pulmonary vascular disease by reflecting the impairment of related organs such as right heart or renal failure [1, 16]. Levels of NT-proBNP for instance are elevated following right ventricular impairment and reflect the severity of pulmonary vascular flow obstruction in patients with PAH [18]. Impaired renal function may represent decreased systemic perfusion as a consequence of decreased cardiac output [18]. Among hematological parameters, an increased red blood cell distribution width (RDW) was independently associated with increased mortality in patients with PAH [19, 20]. In a cohort of patients with idiopathic PAH, RDW was even superior to NT-proBNP and 6WMD in predicting mortality [19]. The RDW may be linked to ineffective erythropoiesis, hemolysis, chronic inflammation and/or iron-deficiency. Although, it is now increasingly recognized that the immune system is involved in the pathogenesis of patients with PAH, marker of the inflammatory state has not yet been incorporated into clinical management [1, 3].

Severe disease and higher mortality in PAH patients have been associated with increased circulating CRP levels in PAH [8]. Despite that the neutrophil/lymphocyte ratio correlated with CRP level, which has also been shown previously [21], its prediction of outcome was independent of the level of CRP in our cohort of patients. Thus, the total and differential WBC count, which represents an inexpensive and readily assessable parameter of inflammation, may render additional information on the disease state and patients’ outcome. In cardiovascular diseases, a ratio shifted towards an increased neutrophil/lymphocyte ratio has been observed to be associated with more severe diseases such as acute coronary syndrome or ischemic heart disease [9‐11]. When added to the Framingham risk score in a general population cohort, the neutrophil/lymphocyte ratio reclassified those with a intermediate risk category as having lower or higher probability of cardiovascular mortality [11]. Explanation has here been suggested by the pro-atherogenic and pro-inflammatory effect of neutrophils on one side and the regulatory, quiescent action of lymphocytes (i.e., subsets) on the other side [10, 11, 22]. In addition, neutrophil/lymphocyte ratio seems to be increased among diabetic patients and in these it was also shown to be independently associated with cardiovascular events [23, 24]. The co-occurrence of cardiovascular risk factors has been increasingly observed alongside with a demographical shift towards older age in patients diagnosed with PAH who had entered clinical trials and registries [25]. This has led to the use of terms such as ‘atypical PAH’, which refers to patients who have 3 or more risk factors for left heart disease [25]. In a subsequent subset of patients with no CVRF (including arterial hypertension, diabetes mellitus, coronary heart disease, atrial fibrillation and body mass index ≥ 30 kg/m²) the neutrophil/lymphocyte ratio remained associated with survival in our cohort of PAH patients. Comparable to the results from the international Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) registry [25] the outcome of PAH with 3 or more CVRF in our cohort was similar to those patients with less than 3 CVRF.

Several studies have reported on metabolic remodeling as a feature in the diseased pulmonary vascular in PAH. Central to this is the energy metabolism in mitochondria, which is also implicated in other metabolic diseases such as diabetes. For instance, suppression of mitochondrial function with a shift towards glycolysis by loss of the mitochondrial deacetylase sirtuin 3 or by loss of the proton transporter uncoupling protein 2 (UCP2) led to spontaneous development of PH in mice [26, 27]. Interestingly, insulin resistance is also present in experimental PH of BMPR2 mutated mice, the predominant cause of hereditary PAH in humans [28].

In the pulmonary arteries neutrophils may release cytokines, enzymes and other factors when passing the pulmonary circulation. For instance, neutrophil elastase, an enzyme also produced by pulmonary vascular smooth muscle cells (SMC), can liberate growth factors from the extracellular matrix [29]. Inhibition of elastase has been shown to prevent and also reverse experimental PH by inducing apoptosis of SMC [30]. Interestingly, it has been demonstrated that neutrophils isolated from patients with IPAH showed an increased release of mediators such as elastase or leukotriene B4 compared with neutrophils from healthy control subjects [31]. Leukotriene B4 levels were also found to elevated in patients with PAH and its inhibition was shown to reverse experimental PH [32].

An altered composition of lymphocytes, particularly within the T helper cell compartment has been found in patients with PAH [3]. Deficiency of T cells in an athymic rat led to a particular aggressive type of experimental PH, probably due to the lack of regulatory T cells which are sufficient to self-limit vascular inflammation [4]. On the other hand, activation of the nuclear factor of activated T-cells (NFAT), which in PAH occurs though expression of Moloney murine leukemia virus (Pim-1) not only in T cells but also in pulmonary vascular SMC, was reported to induce a proproliferative and antiapoptotic phenotype of pulmonary vascular cells [33]. Interestingly, circulating level of Pim-1 has been found to be associated with disease severity and outcome in patients with PAH [34]. Here we observed that the number of circulating lymphocytes as retrieved from the WBC count is a less strong prognostic factors than the number of circulating neutrophils or the neutrophil/lymphocyte ratio.

Activation of the Th2 pathway by immunization and prolonged intermittent challenge via the airways induced severe muscularization of small- to medium-sized pulmonary arteries [35]. In addition, it has been shown that the presence of eosinophils is necessary for vascular remodeling in experimental PH induced by allergic inflammation [36]. However, the role of eosinophils and the Th2 pathway in non-allergic experimental PH are not clear. For instance, the presence of eosinophils in cancer is associated with less aggressive tumor biology and better outcome indicating an anti-mitogenic effect [37].

Foremost, limitations are those inherent to the retrospective observational study design. Further studies are now needed to confirm present results and to evaluate the underlying pathophysiological mechanisms. Particularly in the context of an underlying disease associated with PAH such as connective tissue disease, since our results base on a mixed cohort of patients with different form of PAH. Moreover, the WBC count parameters represent a snapshot of the inflammatory state. Longitudinal data of the WBC count were not available for this analysis. There are no established cut-off values for the WBC parameters. However, the cut-off applied in our analyses was consistent with prior findings. For instance studies showed that a neutrophil/lymphocyte ratio > 4.7 and > 4.5 predicted mortality in left heart disease [11, 38]. Unlike previous studies we did not observe a prognostic implication of PVR, CI or RAP in our cohort of patients [16].

Our results indicate that neutrophilic inflammation with high relative number of neutrophils and a high neutrophil/lymphocyte ratio may be associated with functional and hemodynamic deterioration, as well as with elevated mortality or the need for lung transplantation. The observed prediction of outcome may be independent of functional and hemodynamic parameters, co-morbidities and the level of CRP. These findings may further incorporate the inflammatory state found in patients with PAH into the clinical perspective of the disease. The WBC count represents an inexpensive and readily assessable parameter of inflammation, which may be able to add relevant prognostic information to the clinical management of patients with PAH. However, to be considered as parameter in the routine assessment of patients with PAH further studies and validation are required.