CD66b overexpression and homotypic aggregation of human peripheral blood neutrophils after activation by a gram-positive stimulus

Abstract

Neutrophils represent the main component of innate immunity in the clearance of bacterial infections. To pass the tissue and to localize and reach the site of infection, the peripheral blood neutrophils have to pass through a complex receptor-mediated interaction with the endothelial layer. Under pathophysiological conditions, such as severe sepsis, this process is impaired and often characterized by neutrophil aggregation. In this study, we examined the impact of three different Staphylococcus aureus strains on the activation status of human peripheral blood neutrophils by coincubation of bacterial culture supernatant with whole blood. This complex interaction of a gram-positive stimulus with blood components leads to a special neutrophil activation phenotype, which is characterized by an overexpression of the cell-surface molecule CD66b. The process is accompanied by a strong increase of homotypic aggregates and seems to be initialized by a massive activation impulse caused by the interplay of plasma components. This maximum activation of neutrophils prior to the complex and highly regulated activation required for transmigration might play a key role in the neutrophil dysfunction in gram-positive sepsis.

Mechanotransduction in neutrophil activation and deactivation

“…Neutrophil functions that are amenable to in vitro and ex vivo biochemical assessment include calcium mobilization (29), F-actin assembly (30), adhesion (31), aggregation (13), degranulation (19), phagocytosis (21) and reactive oxygen species (ROS) production (32). Those which can be quantified in vivo include formation of neutrophil extracellular traps or NETs (33), recruitment and migration (34).…”

“…In fact, nuclear morphology is used in clinical settings as I/T ratio, distinguishing immature (I) PMNs from the total neutrophil count (T) (10,11). Neutrophils can also be identified based on specific surface markers such as CD66a (12) and CD66b (12,13). The nuclei of mature neutrophils stay multi-lobulated and remain intact (1), as neutrophils emerge from the bone marrow where they are born/generated/constituted, into circulating blood and into tissues where they undertake anti-microbial, pro-inflammatory and other functions.…”

Soluble CEACAM8 Interacts with CEACAM1 Inhibiting TLR2-Triggered Immune Responses

“…The carcino-embryonic antigen-related cell adhesion molecule (CEACAM)8, often better known as CD66b, encodes a glycosylphosphatidylinositol (GPI)-linked glycoprotein, which is exclusively expressed by human granulocytes [4]–[6]. CEACAM8 belongs to the carcinoembryonic antigen (CEA) family of the immunoglobulin superfamily.…”

Comparison of capillary and venous blood in the analysis of concentration and function of leucocyte sub-populations

“…Cells were identified with anti-human monoclonal antibodies as helper T lymphocytes (expressing CD3 + CD4 + ), cytotoxic T lymphocytes (CD3 + CD8 + ), B lymphocytes (CD3 − CD19 + ), natural killer (NK) cells (CD3 − CD56 + ) and monocytes (CD14 + ). Granulocytes were labelled with antibodies against specific antigenic structures that relate to neutrophil developmental stage and distinct neutrophilic functions (i.e., chemotaxis, adhesion) (CD66b, CD16b, CD11b and CD18) (Schmidt et al 2012;Lund-Johansen and Terstappen 1993;Kuijpers et al 1991;Elghetany 2002;Wang et al 2013). Whole blood (capillary site 25 μL; venous site 100 μL) was aliquoted into two 5 mL tubes (BD Falcon™ Tubes) containing previously aliquoted monoclonal antibodies with concentrations as per manufacturer's recommendation for venous tube and adjusted proportionally for capillary tubes (Table 1).…”

“…Some of these antigenic structures are also markers of specific cell functions and may be up-regulated or down-regulated as necessary. Owing to the functional relevance of these granulocyte surface antigens (Schmidt et al 2012;Lund-Johansen and Terstappen 1993;Kuijpers et al 1991;Elghetany 2002;Wang et al 2013), the level of expression of these markers was also assessed. Profiling such surface markers may elucidate developmental distribution of neutrophils as well as possible alterations in functional capabilities according to vascular location.…”