Human neutrophil Fc gamma receptors: different buttons for different responses.

Neutrophils are fundamental cells in host defense. These leukocytes are quickly recruited from the blood to sites of infection or tissue damage. At these sites, neutrophils initiate several innate immune responses, including phagocytosis, production of reactive oxygen species, degranulation to release proteases and other antimicrobial compounds, production of inflammatory mediators, and formation of neutrophil extracellular traps. In addition to their role in innate immunity, neutrophils are now recognized as cells that also regulate adaptive immunity, via interaction with dendritic cells and lymphocytes. Neutrophils also respond to adaptive immunity by interacting with antibody molecules. Indeed, antibody molecules allow neutrophils to have antigen-specific responses. Neutrophils express different receptors for antibodies. The receptors for immunoglobulin G molecules are known as Fcγ receptors. Upon Fcγ receptor aggregation on the cell membrane, these receptors trigger distinct signal transduction cascades that activate particular cellular responses. In this review, we describe the major Fcγ receptors expressed on human neutrophils and discuss how each Fcγ receptor activates a choice of signaling pathways to stimulate particular neutrophil responses.

Low-Density Neutrophils in Healthy Individuals Display a Mature Primed Phenotype.

Neutrophils are the most abundant leukocytes in human peripheral blood, comprising about 70% of all leukocytes. They are regarded as the first line of defense of the innate immune system, but neutrophils have also the ability of regulating the adaptive immune response. Recently, However, multiple phenotypes and functional states of neutrophils have been reported, particularly in inflammation, autoimmunity, and cancer. One possible subtype of neutrophils, the so-called low-density neutrophils (LDN) is found among mononuclear cells (MNC), monocytes and lymphocytes, after separating the leukocytes from blood by density gradient centrifugation. LDN increase in numbers during several pathological conditions. However, LDN present in healthy conditions have not been investigated further. Therefore, in order to confirm the presence of LDN in blood of healthy individuals and to explore some of their cellular functions, neutrophils and MNC were isolated by density gradient centrifugation. Purified neutrophils were further characterized by multicolor flow cytometry (FACS) and then, using the same FACS parameters cells in the MNC fraction were analyzed. Within the MNC, LDN were consistently found. These LDN had a normal mature neutrophil morphology and displayed a CD10+, CD11b+, CD14low, CD15high, CD16bhigh, CD62L+, CD66b+, and CXCR4+ phenotype. These LDN had an enhanced reactive oxygen species (ROS) production and increased phagocytic capacity and were able to produce neutrophil extracellular traps (NET) similarly to neutrophils. These data confirm the presence of a small number of LDN is blood of healthy individuals and suggest that these LDN represent mature cells with a primed phenotype.

The Antibody Receptor Fc Gamma Receptor IIIb Induces Calcium Entry via Transient Receptor Potential Melastatin 2 in Human Neutrophils.

Human neutrophils express two unique antibody receptors for IgG, the FcγRIIa and the FcγRIIIb. FcγRIIa contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence within its cytoplasmic tail, which is important for initiating signaling. In contrast, FcγRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor with no cytoplasmic tail. Although, the initial signaling mechanism for FcγRIIIb remains unknown, it is clear that both receptors are capable of initiating distinct neutrophil cellular functions. For example, FcγRIIa is known to induce an increase in L-selectin expression and efficient phagocytosis, while FcγRIIIb does not promote these responses. In contrast, FcγRIIIb has been reported to induce actin polymerization, activation of ß1 integrins, and formation of neutrophils extracellular traps (NET) much more efficiently than FcγRIIa. Another function where these receptors seem to act differently is the increase of cytoplasmic calcium concentration. It has been known for a long time that FcγRIIa induces production of inositol triphosphate (IP3) to release calcium from intracellular stores, while FcγRIIIb does not use this phospholipid. Thus, the mechanism for FcγRIIIb-mediated calcium rise remains unknown. Transient Receptor Potential Melastatin 2 (TRPM2) is a calcium permeable channel expressed in many cell types including vascular smooth cells, endothelial cells and leukocytes. TRPM2 can be activated by protein kinase C (PKC) and by oxidative stress. Because we previously found that FcγRIIIb stimulation leading to NET formation involves PKC activation and reactive oxygen species (ROS) production, in this report we explored whether TRPM2 is activated via FcγRIIIb and mediates calcium rise in human neutrophils. Calcium rise was monitored after Fcγ receptors were stimulated by specific monoclonal antibodies in Fura-2-loaded neutrophils. The bacterial peptide fMLF and FcγRIIa induced a calcium rise coming initially from internal pools. In contrast, FcγRIIIb caused a calcium rise by inducing calcium entry from the extracellular medium. In addition, in the presence of 2-aminoethoxydiphenyl borate (2-APB) or of clotrimazole, two inhibitors of TRPM2, FcγRIIIb-induced calcium rise was blocked. fMLF- or FcγRIIa-induced calcium rise was not affected by these inhibitors. These data suggest for the first time that FcγRIIIb aggregation activates TRPM2, to induce an increase in cytoplasmic calcium concentration through calcium internalization in human neutrophils.

Transforming Growth Factor-ß-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-ß-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-ß-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.