Nitric oxide inhalation and glucocorticoids as combined treatment in human experimental endotoxemia: It takes not always two to tango.

To the Editor: We read with interest the article by Hållström et al (1) about the effects on the innate immune response of inhaled nitric oxide (iNO) during experimental endotoxemia. All 15 volunteers received hydrocortisone 30 mins after the endotoxin challenge. The study design was crossover, randomized, double-blind, and placebocontrolled regarding iNO. At least 3 to 4 wks elapsed between the experiments. This is the first study in healthy volunteers in which two potential immune modulating therapies are combined in the human endotoxin model and in our view several issues concerning this complex study design and its subsequent influence on the results should be addressed. We recently performed a comparable trial in which we induced experimental human endotoxemia twice in 12 healthy male volunteers, in the presence or absence of a pharmacological intervention or placebo. Similarly, this study was also performed in a crossover design, 10 to 18 days apart (mean, 14 days). After deblinding of the study, however, we discovered an important tolerance effect of the administration of endotoxin, with a lower inflammatory response during the second visit regardless of the pharmacological intervention (active or placebo). We would like to know whether such effects were also encountered during the study by Hållström et al. Previous crossover studies with the human endotoxin model generally choose a washout period of 2 wks on average (2) or a washout period of 6 wks on average (3) to avoid the potential effects of lipopolysaccharide (LPS)antibody formation. Why did they choose a washout period of 3 to 4 wks? Inhaled nitric oxide may modulate the acute neutrophilic inflammation of the lung parenchyma and dysfunction of the alveolar-capillary membrane that characterizes acute respiratory distress syndrome at several levels. Given the fact that inhaled nitric oxide is inactivated within seconds (by hemoglobin in blood, by haptoglobin-hemoglobin complexes in plasma, and by a reaction with heme ferrous iron and ferric iron that forms nitrosyl-hemoglobin), the question is if the very low concentrations of iNO can attenuate the (parenterally administrated) LPS-mediated cytokine production outside of the lung. There is no human data that 30 ppm iNO in fact can attenuate the LPS-mediated cytokine production in the lung, which could be examined in humans with use of LPS inhalation (4). In addition, it is unclear to us why hydrocortisone was administrated to all of the volunteers. Why examine the possible effect of iNO during a suppressed innate immune response if the effect of iNO alone is questionable? It now remains possible that iNO in the absence of steroid treatment may influence the LPSinduced response. Furthermore, in this protocol hydrocortisone was injected 30 mins after endotoxin infusion as a bolus (2 mg/kg), while other studies that investigate the immune modulating effects of glucocorticoids in the human endotoxin model use pretreatment with continuous infusion of hydrocortisone during a period of at least 6 hrs before endotoxin infusion (5). It is unclear if and to what extent hydrocortisone administration 30 mins after the induction of endotoxemia will influence the innate immune response, given that no control group for the steroid treatment was tested. In our view, time control experiments in which LPS is administrated twice to volunteers without another pharmacological intervention are needed to demonstrate the presence of absence of time-related effects on the innate immune response of LPS. In addition, we feel it is preferable to study one pharmacological intervention at the time or to use four study groups (both iNO and cortisone randomized) if combined effects need to be established. The authors have not disclosed any potential conflicts of interest.