Neutrophils in chronic lymphocytic leukemia are permanently activated and have functional defects

Abstract

A growing body of studies highlights involvement of neutrophils in cancer development and progression. Our aim was to assess the phenotypic and functional properties of circulating neutrophils from patients with chronic lymphocytic leukemia (CLL). The percentage of CD54+ and CD64+ neutrophils as well as CD54 expression on these cells were higher in CLL patients than in age-matched healthy controls. Neutrophils from CLL produced more reactive oxygen species (ROS) compared to controls in both resting and activated conditions. Lipopolysaccharide-induced production of IL-1β and TNF-a as well as reduced TLR2 expression in neutrophils from CLL than in neutrophils from controls suggesting their tolerant state. Finally, phenotypic alterations of neutrophils, particularly elevation of CD64 and CD54 markers, correlated with disease activity and treatment, and low percentage of neutrophils. Taken together, the alterations in percentage and functional characteristics of neutrophils reflect the clinical course of CLL. Our data provide first evidence that neutrophils in CLL are permanently primed and have functional defects.

Keywords: chronic lymphocytic leukemia; disease activity; enhanced ROS production; flow cytometry; neutrophils.

Figure 1. Distribution of percentage of surface markers (CD64, CD54, CD62L, CD11b) on circulating neutrophils from healthy controls and patients with CLL

Group means are indicated by horizontal bars, error bars indicate 95%CI; P values for differences between two groups are stated.

Figure 2. Distribution of CD64, CD54 and CD62L positive neutrophils in CLL subgroups

A. non-active vs active disease, B. untreated vs treated disease, C. mutated vs unmutated IGHV gene status, D. CLL patients without infection vs with ongoing infection. Group means are indicated by horizontal bars, error bars indicate 95%CI; P values for differences between two groups are stated.

Figure 3. Percentage of circulating neutrophils and surface markers (CD64, CD54, CD62L, CD11b) in CLL subgroups

Comparison of studied parameters (medians) between A. non-active (light red columns) vs active (dark red columns) disease, B. untreated (light blue columns) vs treated (dark blue columns) disease, C. mutated (light violet columns) vs unmutated (dark violet columns) IGHV gene status. The arrows indicate the increase/decrease in studied parameters in non-active/untreated/mutated CLL subgroups compared to active/treated/unmutated subgroups, respectively.

Figure 4. Comparison of percentage of circulating neutrophils and surface markers (CD64, CD54, CD62L, CD11b) in CLL patients and healthy controls

CLL is coloured yellow, control subjects green. Discrimination between CLL and controls using combination of two parameters: A. CD54 (%) - neutrophils (%) and B. CD64 (%) - neutrophils (%). The dots represent the percentage of CD54/CD64 (y-axis) and neutrophils (x-axis) in individual subjects. C. Comparison of studied parameters (medians) between CLL (yellow columns) and healthy groups (green columns). The arrows indicate the increase/decrease in studied parameters in CLL patients compared to healthy controls.

Figure 5. Multidimensional clustering of CLL subgroups

Discriminant 3-D models with combination of CD64 (%) - CD54 (%) - neutrophils (%) show the separation of following CLL subgroups: A. non-active (light red clouds) vs active (dark red clouds) disease, B. untreated (light blue clouds) vs treated (dark blue clouds) disease, C. mutated (light violet clouds) vs unmutated (dark violet clouds) IGHV gene status. The dots represent the percentage of CD54 (x-axis), CD64 (y-axis) and neutrophils (z-axis) of individual subjects. The colour bulks represent the probability intervals: the more saturated color the higher probability (intervals: > 90, 90-80, 80-70, 70-60, and 60-50%) of correct classification.

Figure 6. Functional analyses of circulating neutrophils

A. Basal (rest) and fMLP- and PMA-induced ROS production by neutrophils from the blood of CLL patients (CLL, n = 18) and healthy controls (control, n = 17). Error bars represent means ± SEM. Induced (*P < 0.05, **P < 0.01, ***P < 0.001) vs resting ROS production; (^#P < 0.05, ^##P < 0.01) in CLL vs control group. B. IL-1β and C.TNF-a release by neutrophils isolated from the blood of CLL patients (CLL, n = 16) and healthy controls (control, n = 7) after priming with LPS. Error bars are means ± SEM (*P < 0.05, **P < 0.01) vs media; (^#P < 0.05, ^##P < 0.01) vs control group.