Multidrug resistance-associated protein-1 (MRP1) genetic variants, MRP1 protein levels and severity of COPD

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with an influx of neutrophils, macrophages and CD8⁺ T-lymphocytes in the airways and lung tissue[1]. Smoking generates oxidative stress resulting from an oxidant - antioxidant imbalance, and oxidative stress markers are increased in airspaces, blood and urine of smokers and COPD patients[2]. Oxidative stress can be reduced by members of the ATP-binding cassette (ABC) superfamily of transporters. One such a transporter is multidrug resistance-associated protein-1, MRP1, (official name ABCC1, ABC subfamily C, member 1) that plays an important role in normal lung physiology by protecting against toxic xenobiotics and endogenous metabolites[3].

MRP1 was first detected in small cell lung cancer. It has been shown to be highly expressed in the normal human lung [4, 5] and particularly at the basolateral side of human bronchial epithelial cells[6]. Interestingly, we have previously shown that MRP1 is less expressed in bronchial epithelium of COPD patients compared to healthy subjects[7]. Mrp1/Mdr1a/1b triple knock-out mice had a poor ability for smoke-induced IL-8 production compared with wild type mice, which associated with almost complete absence of inflammatory cells in response to cigarette smoke[8]. An additional study demonstrated that cigarette smoke extract inhibits MRP1 activity in bronchial epithelial cells in vitro[9]. Thus there is a clear role for MRP1 in COPD.

A total of 51 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) > 5% are required to tag the entire MRP1 gene in Caucasians[10]. We have shown that two SNPs in MRP1 significantly associate with a lower or higher level of FEV₁ in two independent population-based cohorts. Two additional SNPs had a significant effect of the same, negative magnitude on the level or decline of FEV₁. One SNP was a significant predictor of COPD in the general population [11].

So far, no study has focused on the relation between MRP1 polymorphisms and the level of lung function, inflammatory markers and MRP1 protein in lung tissue of individuals with established COPD. We had the unique opportunity to do so in a recently finished, two center trial in COPD that amongst others studies inflammatory markers in bronchial biopsies and induced sputum[12]. Furthermore, we assessed whether MRP1 protein levels in bronchial biopsies of COPD patients are associated with MRP1 SNPs.

The clinical characteristics of COPD patients and controls are presented in Table 1.

DNA was available from 110 out of 114 COPD patients and from 37 controls. All 5 MRP1 SNPs were in Hardy Weinberg Equilibrium (HWE, p > 0.05) and were not highly correlated with each other (the highest r² in our population is 0.34) (See figure S1 in Additional file 2). There were no significant differences in genotype distributions between the COPD patients and the general population-based control cohort (Additional file 1). Likewise, there were no significant differences in genotype distributions between the COPD patients and controls (Table 2).

Table 3 shows the number and the percentage of inflammatory cells in bronchial biopsies and induced sputum from the COPD patients.

This is the first study linking MRP1 SNPs with the severity of COPD and additionally with the intensity of MRP1 staining in bronchial biopsies. Our results suggest a role of MRP1 in COPD severity, as indicated by the associations of rs212093 genotypes with a higher level of FEV₁ and less inflammatory cells in bronchial biopsies. Additionally, the SNPs rs504348 and rs4781699 were associated with less airway wall inflammation and rs4148382 with a lower FEV₁ level and increased sputum cell numbers. Moreover, the before mentioned SNPs rs4148382 and rs4781699 were associated with respectively higher and lower levels of MRP1 protein in bronchial biopsies of COPD patients (see summary of the results in Figure 5).

Since first described in 1992 [4], a fair amount of data on the structure, substrate, function, and regulation of this transporter has been gathered. MRP1 is a member of the human ATP-binding cassette superfamily of transporters which regulates the traffic of molecules across cell membranes. The MRP1 pump confers resistance to several chemotherapeutic agents including vincristine, daunorubicin and methotrexate[20, 21]. In addition to protecting cells within the body against drugs, environmental toxins and heavy metals, MRP1 has been suggested to be involved in the cellular oxidative defence system and inflammation [22, 23], both being important in COPD development and progression.

We showed that the MRP1 polymorphism rs212093 was significantly associated with a higher FEV₁ level. In line with this, rs212093 SNP was associated with lower numbers of plasma cells, macrophages, neutrophils and mast cells in bronchial biopsies, cells implicated in COPD previously. Increased numbers of neutrophils have been reported in bronchial biopsies of smokers with airflow limitation, an increase that was associated with a lower FEV₁[24]. Neutrophils and macrophages release proteolytic enzymes and generate oxidants, which cause tissue damage as well as cytokines and chemokines that can potentiate inflammation and trigger an immune response. We previously reported a larger number of B lymphocytes in bronchial biopsies of patients with COPD than in controls without airflow limitation[25]. Furthermore, epithelial cells of smokers with COPD contain higher macrophage and mast cell numbers than smokers without COPD[26]. In a triple knock-out mouse model, we previously demonstrated that the inflammatory response to inhalation of cigarette smoke is reduced when MRP1 is absent[8]. Linking previously reported increased airway wall inflammation in COPD with genetic variants of MRP1 we found rs212093 to be associated with lower numbers of inflammatory cells in bronchial biopsies, therefore, this SNP might play a protective role in COPD. This SNP located in 3'region is known to be in complete linkage disequilibrium with rs129081 located in the 3' untranslated region [10] and therefore this polymorphism might be involved in the regulation of MRP1 mRNA stability[11].

One could raise the issue of multiple testing and that we should have adjusted for this in our analyses, but we feel that applying a sequential (classical) Bonferroni correction is not appropriate in the current dataset for a number of reasons[27]. Firstly, our choice for the current study was explicitly driven by our previous findings, suggesting that there might be associations between MRP1 SNPs and COPD severity. Thus, we explicitly hypothesized on the main outcome variables on forehand. Secondly, a Bonferroni correction would not take into account the potential clustering of outcome variables, which might occur jointly at high or low levels, e.g. a Pearson's correlation coefficient r = 0.79 for macrophages and lymphocytes in induced sputum, or are defined as each others ratios[27]. This suggests one might preferentially test a cluster of outcome variables as "one outcome variable" rather than test all variables separately.

It has been shown previously that higher neutrophil percentages in induced sputum correlate with lower FEV₁ levels [28], therefore it is of interest that rs4148382, located in 3'region of MRP1, is associated significantly with higher total cells counts and neutrophils in induced sputum and lower FEV₁ level. The association with total cell counts might be driven by the neutrophils which represent 72% of the total cells in induced sputum. The functional consequence of this particular SNP is not known so far and it is not known whether any functional polymorphism is in linkage disequilibrium with it. This polymorphism is located closely to the 5'end of the MRP6 which maps also on chromosome 16. However, MRP6 mRNA is moderately present in human lung extracts [29] and highly expressed in the liver and kidney [6], which might suggest indeed that the effect of this particular SNP is within MRP1 and not MRP6. How this SNP functionally contributes to COPD severity has to be further unravelled in future studies.

The observed effects in the current study appear to be opposite to previous findings in the same general population as described by Siedlinski et al[11]. In the current study, which extends the previous findings, we observed that rs4148382 associated with a lower FEV₁ level in COPD patients, whereas in the general population from the Doetinchem study rs4148382 associated with a higher FEV₁ level [11]. With respect to these findings it is worth mentioning that the present study was not designed to compare the direction or magnitude of effect estimates between the COPD patients and general population with respect to FEV₁ and genetic factors. The opposite effects are likely due to the fact that we selected a COPD subset of the Doetinchem general population for the current study by matching on the clinical characteristics age, number of packyears and FEV₁/FVC<70%. Although both groups had almost the same number of packyears (median 25^th - 75^th percentile) (40 (34.1 - 48.7) vs. 41.8 (31.2 -54.7)), the matched COPD subset in the general population had a higher lung function (mean FEV₁ % predicted (SD) = 79.7 (13.4)) than our current COPD patients (49.5 (8.8)). This suggests that the COPD subset of subjects from the Doetinchem study who, fulfilled the GOLD criteria of COPD, might be less susceptible to cigarette smoke and COPD development. Therefore, the patients included in the current study with established COPD were probably not comparable with the heavy smokers from the general population based control cohort (Doetinchem).

Additionally, we have calculated the haplotypes of MRP1 and assessed the effects of these haplotypes on FEV₁ level and inflammatory cells in bronchial biopsies and induced sputum. We observed that the effects of MRP1 haplotypes are due to the specific SNP constituting these haplotypes, and therefore didn't add new information. Details on the MRP1 haplotypes are presented in the Additional file 1.

Decreased or increased functional MRP1 expression may have a high impact on development and/or progression of lung diseases and protection against air pollution and inhaled toxic compounds such as present in cigarette smoke[6, 7, 30]. One of our earlier studies showed that the MRP1 intensity in bronchial biopsies of COPD patients was lower compared to healthy individuals[7]. How can we reconcile this with our current findings of MRP1 staining in COPD patients and controls? One option is that this might be due to differences in staining between paraffin and frozen biopsies[31]. More important, it might be due to underlying differences of MRP1 genotyping distribution in the two populations. It appeared that the previous low intensity of MRP1 staining was driven by wild type individuals [7] and if we would have known this at that time, it might have had a different impact on the interpretation of the results. MRP1 is an essential pump for glutathione (GSH) - conjugates such as the inflammatory mediator leukotriene C4 (LTC4) as well as substrates in the presence of GSH (i.e. glutathione disulphide, GSSG) [32], thereby decreasing intracellular concentrations of toxic compounds. Given the rarity of homozygote mutant (AA) individuals for rs4148382 all the conclusions about this SNP are drawn based on the heterozygote (GA) individuals in COPD patients. It might be that in particular individuals who are heterozygote for rs4148382 SNP can have a locally high MRP1 protein level which therefore might lead to more severe inflammation at that site. Clearly, further research needs to investigate this approach in a larger sample of subjects with or without COPD.

In conclusion, our study is the first to demonstrate that MRP1 plays a role in COPD severity, given the association of polymorphisms in MRP1 with airway wall inflammation, the level of lung function and moreover MRP1 protein levels in subjects with established COPD. This is an important step forward linking MRP1 polymorphisms with the pathophysiology of COPD.