Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

Signal Inhibitory Receptor on Leukocytes-1 is highly expressed on lung monocytes, but absent on mononuclear phagocytes in skin and colon.

Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is expressed on human blood monocytes and granulocytes and inhibits myeloid effector functions. On monocytes, but not granulocytes, SIRL-1 expression is low or absent in individuals with the single nucleotide polymorphism (SNP) rs612529C. The expression of SIRL-1 in tissue and the influence of rs612529 hereon is currently unknown. Here, we used flow cytometry to determine SIRL-1 expression on immune cells in human blood and three barrier tissues; skin, colon and lung. SIRL-1 was expressed by virtually all neutrophils and eosinophils in these tissues. In contrast, SIRL-1 was not expressed by monocyte-derived cells in skin and colon, whereas it was highly expressed by lung classical monocytes. Lung monocytes from individuals with a rs612529C allele had decreased SIRL-1 expression, consistent with the genotype association in blood. Within the different monocyte subsets in blood and lung, SIRL-1 expression was highest in classical monocytes and lowest in nonclassical monocytes. SIRL-1 was not expressed by dendritic cells in blood and barrier tissues. Together, these results indicate that SIRL-1 is differentially expressed on phagocyte subsets in blood and barrier tissues, and that its expression on monocytes is genotype- and tissue-specific. Immune regulation of monocytes by SIRL-1 may be of particular importance in the lung.

Secretion of hepatoma-derived growth factor is regulated by N-terminal processing.

Hepatoma-derived growth factor (HDGF) was first purified as a growth factor secreted by hepatoma cells. It promotes angiogenesis and has been related to tumorigenesis. To date, little is known about the molecular mechanisms of HDGF functions and especially its routes or regulation of secretion. Here we show that secretion of HDGF requires the N-terminal 10 amino acids and that this peptide can mediate secretion of other proteins, such as enhanced green fluorescent protein, if fused to their N-terminus. Our results further demonstrate that cysteine residues at positions 12 and 108 are linked via an intramolecular disulfide bridge. Surprisingly, phosphorylation of serine 165 in the C-terminal part of HDGF plays a critical role in the secretion process. If this serine is replaced by alanine, the N-terminus is truncated, the intramolecular disulfide bridge is not formed and the protein is not secreted. In summary, these observations provide a model of how phosphorylation, a disulfide bridge and proteolytic cleavage are involved in HDGF secretion.