Erythrocyte sialoglycoproteins engage Siglec-9 on neutrophils to suppress activation

Abstract

Healthy blood neutrophils are functionally quiescent in the bloodstream, have a short lifespan, and exit the circulation to carry out innate immune functions, or undergo rapid apoptosis and macrophage-mediated clearance to mitigate host tissue damage. Limitation of unnecessary intravascular neutrophil activation is also important to prevent serious inflammatory pathologies. Because neutrophils become easily activated after purification, we carried out ex vivo comparisons with neutrophils maintained in whole blood. We found a difference in activation state, with purified neutrophils showing signs of increased reactivity: shedding of l-selectin, CD11b upregulation, increased oxidative burst, and faster progression to apoptosis. We discovered that erythrocytes suppressed neutrophil activation ex vivo and in vitro, including reduced l-selectin shedding, oxidative burst, chemotaxis, neutrophil extracellular trap formation, bacterial killing, and induction of apoptosis. Selective and specific modification of sialic acid side chains on erythrocyte surfaces with mild sodium metaperiodate oxidation followed by aldehyde quenching with 4-methyl-3-thiosemicarbazide reduced neutrophil binding to erythrocytes and restored neutrophil activation. By enzyme-linked immunosorbent assay and immunofluorescence, we found that glycophorin A, the most abundant sialoglycoprotein on erythrocytes, engaged neutrophil Siglec-9, a sialic acid-recognizing receptor known to dampen innate immune cell activation. These studies demonstrate a previously unsuspected role for erythrocytes in suppressing neutrophils ex vivo and in vitro and help explain why neutrophils become easily activated after separation from whole blood. We propose that a sialic acid-based "self-associated molecular pattern" on erythrocytes also helps maintain neutrophil quiescence in the bloodstream. Our findings may be relevant to some prior experimental and clinical studies of neutrophils.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

“…31 Membrane effects vary even among epitopes within the same target protein. Surface proteins may have important intrinsic roles that could be disrupted by ligand binding, such as GPA interaction with neutrophils to promote quiescence 32 or ion transport by band 3. 33 It is critical to examine untoward effects to identify optimal RBC targets.…”

Tamm–Horsfall glycoprotein engages human Siglec‐9 to modulate neutrophil activation in the urinary tract

“…To stop the reaction, NaIO 4 was removed by transferring sample to a 30kDa Microcon column (EMD Millipore), centrifuged for 15 min at 14,000 × g at 4°C, and washed 3× with ice cold 1× DPBS. Sialic acid aldehydes were then quenched with 4-methyl-3-thiosemicarbazide (MTSC, Sigma Aldrich) as performed previously 32 . Subsequent treatment with FITC-thiosemicarbazide verified all aldehydes were fully quenched under assay conditions (data not shown).…”

Role of negative regulation of immune signaling pathways in neutrophil function

“…Clec12 is also a negative regulator of PMN ROS production and it has been demonstrated that monosodium urate crystals (MSU)-induced p40 phox phosphorylation and subsequent ROS generation is increased in the absence of Clec12 signaling [64]. Glycophorin A, a sialylated erythrocyte membrane glycoprotein, engages PMN via the ITIM-containing inhibitory receptor Siglec 9 to suppress PMN activation and down-regulate the oxidative response [65]. Similarly, murine PMN lacking Siglec E have an enhanced oxidative burst following stimulation compared to WT PMN [66].…”

“…As such, increased PMN surface expression of CD47 is associated with a delay in apoptosis and poor prognosis in non-small cell lung cancer patients [78]. Finally, recent studies have demonstrated that sialylated erythrocyte surface glycans engage the inhibitory immunoreceptor Siglec 9 on circulating PMN to suppress activation and apoptosis [65]. …”

“…As discussed above, PMN activation in the circulation is suppressed by erythrocyte sialoglycoproteins interacting with Siglec 9 [65]. In addition, increased concentrations of erythrocytes significantly reduced extracellular DNA release by PMN in response to PMA.…”