Effects of IL-6 and IL-6 blockade on neutrophil function in vitro and in vivo

Abstract

Reports on the regulation of neutrophil function by IL-6 are often conflicting. Therapeutic inhibition of IL-6 in RA is associated with occasional neutropenia, but the mechanisms underlying this observation are poorly understood. This study investigated interactions between IL-6, the anti-IL-6 receptor agent tocilizumab (TCZ) and neutrophils in vitro and in vivo. Neutrophils were isolated from healthy controls and incubated in vitro with pharmacologically relevant concentrations of IL-6 or TCZ. Neutrophils were also isolated from RA patients, including a cohort following TCZ therapy. Apoptosis was measured by annexin V/propidium iodide (PI) flow cytometry; phagocytosis was measured by incubating apoptotic neutrophils with THP-1-derived macrophages; chemotaxis was measured using cell migration through hanging-cell inserts towards IL-8 and cell surface proteins, including adhesion molecules CD11b (αMβ2 integrin) and CD62L (L-selectin) were measured by flow cytometry. IL-6 (10-100 ng/ml) did not affect the rate of neutrophil apoptosis, priming of the respiratory burst or adhesion molecule expression nor act as a neutrophil chemoattractant. However, IL-6 enhanced signal transducer and activator of transcription 3 (STAT3) activation and neutrophil migration towards IL-8. TCZ in vitro did not induce apoptosis or phagocytosis of neutrophils, nor did it have a significant effect upon apoptosis or cell surface molecule expression. Neutrophil functions in ex vivo neutrophils from RA patients receiving TCZ treatment were unaffected. Therapeutic blockade of IL-6, while inducing a transient neutropenia, does not directly affect neutrophil functions associated with host defence. TCZ-associated neutropenia cannot be explained by direct induction of apoptosis by TCZ, induction of apoptosis following depletion of IL-6, nor increased phagocytosis of neutrophils.