COPD is a highly prevalent yet underdiagnosed disease, with increasing morbidity and mortality rates. Complex underlying mechanisms that are reflected by diverse clinical presentation are making this disease challenging for specific diagnosis and therapy. Ideally, due to COPD heterogeneity, a particular endotype and phenotype should be recognised for each patient to personalise its treatment. It is now recognised that inflammation is present in COPD not just at the local level i.e. in lungs and airways but also at the whole-body level, with persistent systemic inflammome being demonstrated in some patients (30). Blood is a very accessible sample obtained by quite non-invasive way. Therefore, searching for a good peripheral blood diagnostic, prognostic, predictive biomarker and/or biomarker of disease severity in any disease is recommendable, and especially in complex and heterogeneous diseases. In this study, we assessed concentration of Hsp70 in peripheral blood of patients with stable phase of COPD and its association with disease characteristics defined by spirometry and clinical presentation. We found significantly elevated eHsp70 in overall COPD patients compared to healthy subjects. This increase was related to degree of airflow limitations as well as symptoms burden and history of exacerbation, and eHsp70 concentrations were the highest in patients with GOLD 4 stage and GOLD D group. It is also important to emphasize that eHsp70 was elevated even in patients classified as GOLD A and GOLD 2 (which is in clinical practice often the lowest GOLD stage) compared to overall control group. To the best of our knowledge, this is the first study that assessed eHsp70 concentrations in COPD patients subdivided by GOLD ABCD classification, and the first one to show GOLD stages dependent differences in eHsp70 levels. In addition, when participants of the control group were subdivided according to their smoking status, patients belonging to GOLD 2 and GOLD A subgroups had higher eHsp70 levels than never smoking individuals, but similar eHsp70 levels as smokers with normal lung function. Therefore, it could be suggested that so-called healthy smokers might be more susceptible to altered inflammatory responses provoked by eHsp70 being a danger signal to immune system, and some of them might even develop COPD in time to come. This assumption should be tested in the future studies. In addition, as only 20% of smokers develop COPD, a specific individual genetic makeup seems to be also decisive.
By searching the literature, we found only three studies that assessed Hsp70 concentration in the blood of COPD patients so far (4, 16, 27). However, in addition to being performed on significantly lower number of participants compared to our study, there are some concerns regarding their sample and ELISA kit selection. Reported concentrations on eHsp70 are very dependent on the matrix in which it was measured. Whitham and Fortes showed that eHsp70 concentrations were the highest in EDTA plasma, its values in heparinized plasma were somewhat lower, while the lowest eHsp70 levels were measured in serum, and they hypothesized that this was due to the binding of eHsp70 to aggregated clotting proteins in serum. Therefore, they recommended EDTA plasma as a sample of choice in the future investigations (8). This is especially important at least for the studies with healthy participants at rest as their eHsp70 values tend to be very low as well as for the studies with elderly subjects as their eHsp70 concentrations are significantly lower than in young individuals (25). Still, in previous researches assessing eHsp70 in COPD patients that were mostly older subjects, serum (4, 27) or heparinized plasma (16) were used. In addition, there are several concerns regarding ELISA kit applied in previous studies. Ünver et al. used an adopted ELISA kit that is not entirely appropriate for blood as a matrix, and this could be the reason for extremely high eHsp70 values they obtained in the study although they used serum as the sample (4). Hacker et al. also used an adopted ELISA kit that was specific for intracellular Hsp70 determination in cell lysates (27). It is important to recognise limitations of those assays, as they are not optimised for biological fluids such as blood. On the other hand, Cui et al. selected a proper ELISA kit validated for serum and plasma with EDTA used as an anticoagulant, but not with citrate and heparin. However, they used heparinized plasma as the sample for eHsp70 measurement, and this could be the reason for higher eHsp70 values they obtained (16). In our study, we used EDTA plasma and ELISA kit that was more sensitive and could detect lower eHsp70 concentrations compared to other ELISA kit also validated for EDTA plasma and serum (31). With this choice of sample and kit we were able to detect eHsp70 in each study participant (controls and patients), and, as already mentioned, we were also able to distinguish patients by their disease severity. Dong et al. reported that the expression of intracellular Hsp70 was closely related with COPD disease severity (17). However, an association of extracellular Hsp70 with disease severity was not shown for COPD yet, but was demonstrated for some other diseases including asthma, chronic heart failure and rheumatoid arthritis (3, 32, 33).
In this study, we obtained positive associations between eHsp70 and multicomponent COPD indices (BODCAT, BODEx, CODEx, DOSE) that reflect airflow obstruction, smoking status, symptoms and history of exacerbations, which are all important in assessing patients’ overall condition. We also obtained significant negative associations between eHsp70 and lung function parameters, and this was only reported for the expression of intracellular Hsp70 in COPD patients (17). Finally, eHsp70 showed to have a good predictive characteristic with its OR of 7.63 (95% CI = 3.68–15.82).
Although we presented some novel and interesting results, our study has some limitations. It did not include COPD patients in GOLD C group or in GOLD 1 stage. However, in clinical practice COPD patients belonging to GOLD 1 group rarely contact their physician due to very mild symptoms, and GOLD C category of patients is also very rare as the patients that do not have many symptoms are not usually frequent exacerbators. In addition, larger number of participants should be recruited in the further studies and longitudinal study design should be considered.