Tolerance to repeat exposure of inhaled endotoxin: an observation in healthy humans

To the Editors: 

We read with interest the articles on endotoxin research in the May issue of the European Respiratory Journal . The editorial by Bals 1 aptly raised the yet unanswered questions concerning the timing (acute versus chronic) and doses of inhaled endotoxin relevant to health and disease, and the questions of whether the outcome of such exposure is always detrimental.

To this end, we wish to add our own preliminary observation of the possibility of tolerance to repeat exposure of inhaled lipopolysaccharide (LPS) in healthy nonatopic humans at 4 weeks. In a double-blind, crossover study, eight healthy human subjects were randomised to receiving either a single inhaled dose of 50 µg salmeterol or placebo prior to being challenged with a 15-µg dose of Escherichia …


Tolerance to repeat exposure of inhaled endotoxin: an observation in healthy humans
To the Editors: We read with interest the articles on endotoxin research in the May issue of the European Respiratory Journal. The editorial by BALS [1] aptly raised the yet unanswered questions concerning the timing (acute versus chronic) and doses of inhaled endotoxin relevant to health and disease, and the questions of whether the outcome of such exposure is always detrimental.
To this end, we wish to add our own preliminary observation of the possibility of tolerance to repeat exposure of inhaled lipopolysaccharide (LPS) in healthy nonatopic humans at 4 weeks. In a double-blind, crossover study, eight healthy human subjects were randomised to receiving either a single inhaled dose of 50 mg salmeterol or placebo prior to being challenged with a 15-mg dose of Escherichia coli serotype 026:B6 (Sigma, Poole, UK), in two visits separated by 4 weeks. Using 1 week prior as a baseline, sputum induced at the 6th h after LPS challenge showed no significant differences in the increase of total cell counts in the two treatment periods (mean difference (95% confidence interval) salmeterol versus placebo: 10.6610 6 cells?mL -1 (-9.71-30.9); p50.25) or neutrophils (11.7610 6 cells?mL -1 (-8.33-31.92); p50.20; unpublished data). The assertion that salmeterol does not protect against airway neutrophilic inflammation was subsequently supported in a more robust study, where subjects were randomised to receiving either daily salmeterol for 3 weeks or placebo, prior to inhaled LPS challenge, in a crossover study [2].
Retrospective power analysis of our results first alerted us to the possibility of intrinsic biological phenomena in a study design of sequential inhaled LPS challenges. Data were then re-analysed with the purpose of looking into the reproducibility of sputum neutrophilia between the two inhaled challenges, treating the effects of the single-dose salmeterol as no more than placebo [2]. Our findings showed that following the first LPS challenge, the mean total sputum cell counts increased by 31.23610 6 cells?mL -1 (95% CI: 13.27-49.20) and the mean sputum neutrophil counts rose by 30.3610 6 cells?mL -1 (12.59-48.11). However, following the second LPS challenge, the mean total sputum cell counts only increased by 11.3610 6 cells?mL -1 (2.14-24.89) and mean sputum neutrophil counts by 10.9610 6 cells?mL -1 (1.02-22.9). The difference between the means was statistically significant (p50.01; mean difference: 19.8610 6 cells?mL -1 (6.16-33.56) for total sputum cell counts; 19.4610 6 cells?mL -1 (4.73-34.08) for sputum neutrophil counts; fig. 1).
Using such a human experimental model of airway neutrophilia to understand the inhaled effects of endotoxin [3], and to examine for potential anti-inflammatory properties of therapeutic agents [2] appears to be a validated approach. MICHEL et al. LPS on two occasions separated by o3 weeks. However, none of these studies had observed tolerance towards subsequent LPS challenge(s) in their healthy human subjects at doses of LPS described that were higher than ours. It is possible that tolerance in healthy nonatopic human subjects only occurs in exposure to lower doses of inhaled endoxin. In fact, existing literature indicates that exposure of 30-40 mg inhaled LPS is probably the clinical threshold to induce symptoms and lung function changes for healthy subjects [4].
More research is required to validate our preliminary observation.

L.C. Loh
Dept of Medicine, Clinical School, International Medical University, Jalan Rasah, Malaysia.