Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients

Abstract

Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the ex vivo functionality of neutrophils isolated from blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.

Keywords: allogeneic transplant; chemotaxis; granulocyte colony stimulating factor (G-CSF); hematopoietic stem cell donor; neutrophil.

Figure 1

G-CSF treatment affects neutrophil migration. Stem cell donors before and after treatment were analyzed for neutrophil function. (A) Neutrophil count measured before and after G-CSF-treatment in the stem cell donor group (n = 10). Revealed a significant increase in the number of circulating neutrophils after G-CSF treatment. (B) Distribution of half migration time during chemotaxis within the stem cell donor group before and after G-CSF-treatment. Half migration time was defined as the time required for half-maximal fluorescence signal indicative for migration toward 10 nM fMLP. (C) The distribution of velocity during chemotaxis before and after G-CSF-treatment is indicated. Velocity was measured as Δ %/min at half migration time indicating the speed of the neutrophils when they have reached half maximal fluorescence in the bottom well. Blood cell differentiation analysis (D) before and (E) after purification of neutrophils from stem cell donors before (n = 3) and after (n = 4) GCSF treatment confirmed that the purification enriched mature neutrophils >95% independent of previous G-CSF treatment. Statistical analysis was performed using a Wilcoxon signed-rank test (A–C). ^*p < 0.05, ^**p < 0.01.

Figure 2

Systemic G-CSF treatment impairs chemotaxis in neutrophils from healthy donors. Purified neutrophils from peripheral blood of healthy, untreated donors (n = 8) were incubated with 0, 10, or 50 ng/ml human recombinant G-CSF. Thereafter, a chemotaxis assay was performed using fMLP as chemoattractant and half migration time (A) as well as velocity at half migration time (B) was determined. Neutrophils pre-treated with 0, 10, and 50 ng/ml of G-CSF respectively were stained with a PE-labeled anti-fMLP receptor antibody. The histogram is showing a representative experiment of three similar replications (C). Statistical analysis was performed with a Kruskal-Wallis test for (A) (p = 0.0098) and (B) (p = 0.0093), complemented with Mann-Whitney U-tests showing that samples 0 and 50 ng/ml are significantly different for (A,B) with ^*p < 0.05.

Figure 3

Plasma from G-CSF-treated individuals contains lower amount of IL-8 than untreated controls. G-CSF (A), IL-8 (B), and CRP (D) concentrations in plasma obtained from stem cell donors (n = 10) before and after G-CSF-treatment. Each value was determined from a mean of four technical replicates for G-CSF and IL-8. For IL-8 (B) all the samples after G-CSF treatment were below the range of the assay (0.72 pg/ml). To be able to perform statistical analysis the values were extrapolated to 0.72 pg/ml. (C) Levels of IL-8 secreted by neutrophils in vitro after G-CSF (50 ng/ml) or mock treatment for 30 min and subsequently either stimulated with LPS (1 μg/ml, labeled as LPS +) or left untreated (labeled as LPS -). The levels of IL-8 were measured by ELISA after 6 h stimulation. The CRP values (D) were measured as single measurements for each donor before and after G-CSF treatment. Statistical analysis was performed using a paired students t-test (C) and Mann-Whitney U-test (A,B,D) when data was not normally distributed. ^***p < 0.001 and ^****p < 0.0001.

Figure 4

The chemotactic activity remains impaired in allogeneic transplant recipients. (A) Distribution of half migration time in the stem cell donors before G-CSF treatment (n = 10), serving as controls, and samples from patients undergoing allogeneic stem cell transplantation at a medium of 48.9 days (n = 9), indicated as sample 1, and 80.2 days (n = 12), indicated as sample 2, after transplant, respectively. The half migration time is measured as the time it takes to reach half of max fluorescent and is a measure of neutrophil speed in the process of chemotaxis. (B) The distribution of velocity during chemotaxis comparing stem cell donors before G-CSF with samples from patients undergoing allogeneic stem cell transplantation as described above. The evaluation shows that the velocity at half max migration goes down early after allogeneic stem cell transplantation which confirms that the chemotaxis in neutrophils is slower early after stem cell transplantation. Statistical analysis was performed with a Kruskal–Wallis test for (A) (p = 0.0466) and (B) (p = complemented with Mann–Whitney U-tests showing that samples “before G-CSF treatment” and “allogeneic sample 1” are significantly different for (A,B) with ^*p < 0.05.

Figure 5

ROS production and phagocytosis are not affected by G-CSF in stem cell donors and neither in allogeneic transplant recipients. (A) Production of ROS by neutrophils was measured as area under the curve (AUC) in the stem cell donor group before and after G-CSF-treatment (n = 10). Statistical analysis was performed using an unpaired students t-test. (B) Production of ROS measured as AUC in the samples from patients undergoing allogeneic stem cell transplantation using the samples from the stem cell donors before G-CSF-treatment as a control. Statistical analysis was performed using a Kruskal-Wallis test due to skewness of data. (C,D) Phagocytosis measured as the uptake of pH-sensitive pHrodo beads that triggered phagocytosis by neutrophil isolated from (C) stem cell donor group before and after G-CSF-treatment (n = 10) and from (D) patients undergoing allogeneic stem cell transplantation using the samples from the stem cell donors before G-CSF-treatment as a control. Phagocytosis rate was calculated as percent out of 100 percent control. Statistical analysis was performed using a One-way ANOVA with Tukey's multiple comparison tests. Statistical analyses in (A-D) did not reveal significance p < 0.05.