Biochemical and functional characterization of the leucocyte tyrosine phosphatase CD4S (CD4SRO, 180 kD) from human neutrophils. In vivo upregulation of CD45RO plasma membrane expression on patients undergoing haemodialysis

Abstract

SUMMARYThe biochemical and functional characterization, and the regulation of plasma membrane expression of the leucocyte tyrosine phosphatase CD45. have been investigated in neutrophils from healthy donors and patients undergoing haemodialysis. CD45 proteins of 180 kD and 130-150 kD were precipitated from neutrophils from both healthy subjects and haemodialysed patients. Prolonged storing, as well as trypsin treatment of satnples containing the 180-kD CD45 protein, generated the 130-150-kD polypeptides. The 130-150-kD CD45 polypeptides carried extracellular CD45 epitopes. including the sialic acid-related UCHLI epitope (CD45RO). Furthermore, these trypsin-generated CD45 polypeptides did not possess phosphatase activity, which could be detected on the 180-kD protein. A remarkable quantitative increase o^ cell surface expression of the neutrophil CD45 components was detected both afler in vitro neutrophil activation and after dialysis treatment with neutropenic membranes. The CD45 biochemical pattern did not qualitatively ehange upon either in vitro or in vivo dialysis-induced neutrophil activation. The upreguiated expression of CD45 on neutrophils from dialysed patients correlated with the neutropenic effect induced by the different dialyser membranes. Maximal upregulation ofC045 expression was observed alter ISminofdialysis with neutropenic membranes, and norrnal expression levels were restored after 1 h. By contrast, increase of CD45 plasma mernbrane expression induced in vitro by treatment of normal neutrophils with thedegranulatory agents fMLPor Ca-^ ionophore was maintained. These results demonstrate that neutrophil cell surface expression ofthe 180-kD CD45 protein is upregulated during the in vivo haemodialysis process, and suggest that a proteotytic activity could regulate the enzymatic activity of CD45 by degranulation of its cytoplasmic phosphatase domains.

Human Polymorphonuclear Neutrophil Phenotypes Generated in vitro

“…As is known, CD4 is co-receptor for helper T cells and primary receptor for HIV virus [21]. CD45-RO molecule, a characteristic marker of memory and effector T cells, is a tyrosine phosphatase that regulates lymphocyte activation [22] and its expression in PMN neutrophils has been described in 96.7% ± 2.6 % on cell surface [32]. CD45 has also been reported to modulate respiratory burst activation and its engagement synergizes with formylated peptide stimulation of respiratory burst [58].…”

“…In this work, HLA-DR molecule expression is demonstrated, in addition to costimulatory molecules B7 expression and CD4 and CD45RO unconventional molecules in NETs. Our assumption that it was feasible to find this set of molecules in NETs related to described PMN neutrophils phenotypes (PMN neutrophil-APC and PMN neutrophil-CD4-CD45RO) is based on bibliography that supports these molecules are contained in cytoplasmic reservoirs [12], [13], [21], [22]. Let us remember NETs are constituted of granular components and their composition can vary depending on the stimulus [62].…”

“…On the other hand, unconventional molecules expression in PMN neutrophils, such as CD4 and CD45RO, has been reported, although there is very little bibliography on the matter [21], [22].…”

“…In T cells, CD45 molecule dephosphorylates Lyck tyrosine kinase as part of the TCR activation signaling cascade [10]. CD45RO molecule is an activation state marker in effector and memory T cells [10], [31], but has also been observed in some PMN neutrophils phenotypes from hemodialysis patients and in vitro activated PMN neutrophils with fMLP formylated peptides [22], [32]. This molecule is expressed in PMN from healthy patients but no natural ligand has been found [32].…”

“…This molecule is expressed in PMN from healthy patients but no natural ligand has been found [32]. CD45RO localizes to PMN-specific granules [22]. It is known that CD45RO molecule isoform is expressed when T cell is activated, and it will associate with TCR and coreceptor (CD4 or CD8), making T cell more sensitive to stimulation by low concentrations of peptide-antigen complexes.…”