Our study suggests that prolonged inhalation of helium does not affect the ability of the innate and early adaptive immune system to respond to immune stimuli LPS, LTA or anti-CD3/anti-CD28 ex vivo. TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 levels did not differ at various time points before and after helium inhalation compared to air inhalation. These results are of interest for a broad field, as the use of helium-oxygen mixtures for respiratory disease or with the purpose of cell protection against ischaemia/reperfusion injury in the critical care unit and the operating theatre might expand.
Two in vivo studies using a forearm model of ischaemia-reperfusion injury investigated the protective effect of helium inhalation on endothelial function and additionally looked at systemic immune parameters
[9, 20]. Our results are in line with results found in the first study, in which similar concentrations of helium/oxygen were used
. This study showed that application of three 5-minute cycles of helium (79%He/21%O2) interspersed with 5 min of air breathing before the ischemic episode induces preconditioning in human endothelium
. To investigate the influence on the innate immune system, venous blood was collected at the non-injured arm for analysis of systemic levels of adhesion molecules sVCAM-1, sICAM-1, E-selectin, and proinflammatory cytokines IL-1β and IL-8 at 10 minutes and 3 hours after reperfusion. No effects of forearm ischaemia/reperfusion or helium conditioning could be found on all of these parameters, suggesting no net effect of helium on the investigated immune parameters
In contrast, Lucchinetti and colleagues found immunomodulatory effects after 35 minutes of helium breathing in a concentration of 50%He/50%O2. Venous blood was taken from the injured arm at baseline, and after 5, 10 and 30 minutes of reperfusion to investigate proinflammatory markers on leukocytes. A decrease of CD11b on monocytes at 10 and 30 minutes of reperfusion and a decrease of ICAM-1 on monocytes at 5 minutes of reperfusion was found under helium inhalation in comparison to control. Although no effects on other markers were found, these findings implicated a net negative effect of helium inhalation on systemic immune parameters
. However, differences in duration and concentration of the inhaled helium/oxygen mixtures make a direct comparison between the aforementioned studies difficult. Another difference is the proximity of the induced tissue damage (forearm ischemia-reperfusion) and the point of blood sampling for analysis of immune parameters. In the latter study, blood collection took place from the injured arm – closer to the site of injury - in contrast to the first study in which blood collection took place from the non-injured arm
[9, 20]. It might be possible that the immunomodulatory effect of helium can only be found locally or that helium exerts anti-inflammatory effects only when tissue damage is present. An example is the anti-inflammatory effect of helium in comparison to nitrox breathing that was found in a pig model of acute lung injury
. Anti-inflammatory effects were shown in lung tissue as a reduction of pro-inflammatory cytokine IL-8 and myeloperoxidase, a measure for neutrophil activity. The big difference with the current study is the lack of tissue damage at the time of helium breathing: stimulation of the immune system is done after helium breathing and blood sampling. This provides an objective way of assessing the immunomodulatory characteristics of this noble gas per se.
The main rationale behind the present study was to investigate whether the use of helium gas against organ ischemia/reperfusion injury may have detrimental effects on the immune response. Ischemic and pharmacologic conditioning protocols described in the literature normally do not exceed a total of 30 minutes of intervention
[22–24]. Therefore, 30 minutes of helium inhalation resembles a clinically relevant time frame of gas application. Investigation of the innate immune response after 60 min of helium and air inhalation was done in an additional group, to rule out that prolonged inhalation did exert effects. Furthermore, a concentration of 79% helium is the maximum from a clinical point of view, although a variety of lower helium concentrations have also been used
. Higher percentages of helium lead inevitably to hypoxic gas mixtures. It is highly unlikely that lower concentrations have detrimental effects on the immune system, when a higher dose does not.
In this study, we mainly focused on pro-inflammatory components of the immune system. However, in some cases it is not so clear whether cytokines exhibit purely pro- or anti-inflammatory actions, such as IL-2. This cytokine has pro-inflammatory effects, but might play an anti-inflammatory role in diabetes
. The finding that no effects on pro-inflammatory components were found suggests that no net-effect of helium inhalation on the immune response exists. It has to be noted that clinical outcome of infections is the result of a balance in quantity and time course of pro- and anti-inflammatory components. Therefore, several other anti-inflammatory components of the immune system, as well as other constituents of the innate and early adaptive immune system still need further investigation. In our study we did not investigate cytokine production in fractionated blood leukocytes. Instead, we investigated total amounts of cytokines in whole blood, as this model represents a condition in which many of the physiologically present cellular interactions remain intact. Given the fact that leukocyte counts did not differ between groups at the different time points either, we consider the lack of difference in cytokine levels between groups a good reflection of the unaltered immune status after helium inhalation.
Whole blood stimulation is a widely known model used for various goals. In a recent study it was shown that ex vivo stimulation of whole blood with pathogenic Leptospira induced a cytokine response
. The whole blood stimulation model was also used in another study to show that erythromycin infusion in healthy volunteers reduces IL-8 production after ex vivo stimulation with Streptococcus pneumonia
. Stress-related suppression of cytokine production after whole blood incubation with LPS was shown in a study in which 20 male, healthy volunteers were exposed to bungee jumping
. In this study it was shown that bungee jumping was associated with higher epinephrine, norepinephrine and cortisol levels, but also with increased leukocytes. Nevertheless, the amount of TNF-α and IL-8 levels after ex vivo stimulation with LPS was decreased
Despite the evidence for the applicability of our used model, a limitation of the study is the difficulty of proving absence of an effect while 3 significant differences between helium and air inhalation were found. However, with a total of 168 tests being performed in total, 8 can be significant by chance alone. Furthermore, significant findings at solely one time point of one cytokine after one stimulation type are not likely to reflect a clinically relevant effect of the intervention. As can be seen in the figures, lines of helium and air inhalation intersect at random time points, suggesting that even when an effect seems to be there it does not persist over time.
A second limitation of the study concerns the investigation of healthy, male volunteers only. The target population for helium-induced organ protection often suffers from comorbidity, which might be of influence on the innate immune response to ex vivo stimulation. We have chosen to use a model in which possible confounders by comorbidities were excluded. From literature it is known that sex differences exist in immune defense capacity and cytokine production
. To minimize the influence of this possible confounder we have chosen to investigate males only.