BACKGROUND: Neutropenia is a major risk factor for infectious complications in hematopoietic cell transplantation (HCT) recipients. However, much less is understood about neutrophil function in the posttransplant period. Cathelicidins are multifunctional host defense peptides. The sole human homolog, Cathelicidin Antimicrobial Peptide (CAMP, OMIM * 600474) is secreted by invading leukocytes, especially neutrophils and plays an important and pleiotropic role in host defense. It acts both as a direct bactericidal agent, but also affects the activity of many immune cells, including prolonging the lifespan of neutrophils. Another critical step in the microbicidal function of neutrophils is the generation of reactive oxygen species. OBJECTIVE: To characterize neutrophil function and CAMP expression over time in children following HCT. METHODS: A prospective study of children undergoing HCT was conducted. Blood samples were collected at 3 months, 6 mo., and 1 y post-HCT timepoints. White blood cells were purified using a PolymorphPrep gradient (Axis-Shield Diagnostics Ltd., Scotland). CAMP expression was measured using Clone H7 antibody (Santa Cruz Biotechnology Inc., USA) in fixed and permeabilized cells. Neutrophil activation was measured as reduction in membrane potential following stimulation with phorbol 12-myristate 13-acetate (PMA) using fluorescence staining with 3,3'-dipentyloxacarbocyanine iodide (DiOC5). Neutrophil hydrogen peroxide generation was measured by oxidation, and resultant increased staining of the probe 2',7' dichlorohydrofluorescein diacetate (DCFH-DA) following PMA stimulation. STATISTICS: Categorical variables were summarized by frequency and percent, and continuous variables were summarized by median and inter-quartile range (IQR). Distributions of continuous variables were examined and were log-transformed if skewed. Linear mixed-effects model was used to evaluate temporal trends and correlations between pairs of variables across time. points. The Bayesian Information Criterion was used to identify the optimal model that balanced model goodness-of-fit and complexity. RESULTS: Sixty-three patients were enrolled, median age 6.1 y (0.4 - 20.5 y). A majority of transplants (69%) were allogeneic. Patient and transplant characteristics are described in Table 1. CAMP was expressed in 95.8% of white blood cells (IQR 80.3 - 99.0%) (Figure 1). CAMP expression in neutrophils, measured as fold-increase above background staining, showed a median value of 474 (IQR 355 - 623). CAMP expression was inversely corelated with absolute neutrophil counts, with 37 folds increase (95% CI 11 to 63 folds, p = 0.006; Figure 2) for every 106 per milliliter reduction in neutrophil counts. Neutrophil activation measured as reduction in membrane potential was expressed as % decrease in DiOC5 staining following PMA stimulation with a median value of 41% (IQR 31 - 56%). Peroxide generation expressed as % increase in DCFH-DA staining following PMA stimulation showed a median value of 304% (IQR 170 - 630%). The reduction in membrane potential was correlated with peroxide generation. For every 1% reduction in membrane potential, hydrogen peroxide production increased by 2% (95% CI 1 to 3%, Figure 3). CAMP expression in neutrophils was not associated with neutrophil function measured by change in membrane potential or peroxide generation. None of the covariates examined (age, transplant type, and timepoints posttransplant) was found to be associated with CAMP expression or measures of neutrophil function. CONCLUSIONS: We have demonstrated that neutrophils, as characterized by changes in membrane potential and reactive oxygen species generation in response to PMA stimulation, are functional in children as early as 3 months following HCT. Of the parameters assessed in this study the increase in CAMP expression in neutrophils in patients with lower numbers of neutrophils in circulation after HCT stands out and this finding may potentially be of importance. Further studies will be necessary to determine if this is specific to HCT setting or if this can also be observed in normal individuals. It will also be important to study if this is reflected in the serum levels of the CAMP-derived active peptides.
No relevant conflicts of interest to declare.
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