Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine

Abstract

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.

Keywords: ADP; AMP; ATP; Adenosine; Adenosine Receptor; Immunology; Infectious Diseases; Innate Immunity; Purine; Purinergic Agonists.

FIGURE 1.

Cell-associated enzymes dominate newborn and adult blood ADPase activity, whereas soluble enzymes dominate blood AMPase and ADA activities and are differentially expressed in newborns and adults. Whole blood, hemocytes, or plasma (MFP) were incubated with 50 μm [¹⁴C]ADP for 5 or 15 min (A). MFP was incubated with 50 μm [¹⁴C]AMP and 20 μm EHNA (B) or 50 μm [¹⁴C]adenosine (C) for 15 min prior to measurement of conversion to other purine metabolites by TLC. Shown is one representative of five (A) or six (B and C) independent experiments.

FIGURE 2.

Neonatal cord blood plasma demonstrates high AMPase and lower relative ADA activities. A, soluble plasma (MFP) AMPase activity was determined by adding 200 μm [¹⁴C]AMP for 5, 10, and 15 min in the presence of EHNA before reaction termination, and subsequent TLC was quantified by densitometry with the rate calculated based on the change in product between 5 and 10 min and between 10 and 15 min (which were equivalent and were averaged; n = 16 each population; Student's t test; *, p < 0.05). B, soluble plasma (MFP) ADA activity was determined by adding 200 μm [¹⁴C]adenosine for 30 and 60 min prior to reaction termination and TLC separation (n = 12 each population; Student's t test; *, p < 0.05, rate calculated based on the change in product between 30 and 60 min). Error bars, S.E.

FIGURE 3.

Newborn neutrophils express relatively high AMPase activity. Newborn washed hemocytes (A and C) or isolated peripheral blood neutrophils (4 × 10⁶/ml) (B and D) were incubated with or without inhibitors of TNAP (MLS0038949; 100 μm) and 5′-NT (APCP; 100 μm) before the addition of 50 μm [¹⁴C]AMP for 15 min in the presence of EHNA before reaction termination, and subsequent TLC separation was quantified by densitometry. A, n = 6 for both newborn and adult; B, n = 5 for newborn and n = 6 for adult. C, one representative of six independent experiments; D, one representative of five independent experiments. A, newborn versus adult control condition; Student's t test; ***, p = <0.001; all other conditions compared with appropriate population (newborn or adult) control by analysis of variance with Bonferroni's multiple comparison test (♢) with p < 0.01. B, newborn versus adult Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001, all other conditions compared with population control by analysis of variance with Bonferroni's multiple comparison test, with p < 0.01 unless indicated as not significant (ns). Error bars, S.E.

FIGURE 4.

TNAP and 5′-NT are key contributors to soluble plasma AMPase activity. The contributions of TNAP and 5′-NT were determined by the addition of a 100 μm concentration of the selective inhibitor MLS0038949 or APCP, respectively. 50 μm [¹⁴C]AMP was added in the presence of EHNA, and after 5 min, the reaction was terminated prior to TLC separation and quantified by densitometry (A; n = 11). TNAP did not influence total AMPase activity in plasma at low concentrations of substrate (B; 5 μm [¹⁴C]AMP, 1 min, n = 8) but did significantly contribute at high concentrations (C; 100 μm [¹⁴C]AMP; 5 min; n = 8; Student's t tests; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars, S.E.

FIGURE 5.

Soluble plasma ADA levels increase significantly during the first year of life. A, soluble ADA activity was determined by the addition of 50 μm [¹⁴C]adenosine for 5 min to microparticle-free plasma collected at 0, 1, and 2 years of age from 12 subjects (paired Student's t tests; 0 versus 1, p < 0.001; 0 versus 2, p < 0.0001; 1 versus 2, p < 0.01). B, a cohort of platelet-rich plasma samples from a previous study, including samples from cord blood and 1, 2, 3, 4, 6, 9, and 12 months of age (1 sample/subject), were processed to MFP and incubated with 50 μm [¹⁴C]adenosine for 15 min (analysis of variance with Bonferroni's multiple comparison test; 0 versus 3 months, p < 0.01; 0 versus 4 months, p < 0.05). Total ADA (C), ADA1 (D), and ADA2 (E) activity were evaluated by a chromogenic assay as described under “Materials and Methods.” Total ADA (n = 11 newborn, n = 14 adult; Student's t test; **, p < 0.01) (C) and ADA1 activity (n = 11 newborn; n = 14 adult; Student's t test; *, p < 0.05) (D) were significantly lower in newborn samples. Error bars, S.E.

FIGURE 6.

Extracellular AMP metabolism leads to diminished LPS-induced TNF-α production in newborn and adult blood (A). Newborn cord or adult peripheral blood was diluted 1:1 (final) with RPMI and treated with LPS (100 ng/ml) or mock-treated with RPMI and treated with 100 μm AMP or Ado once (at start) or twice (second addition at 2 h), as indicated, and all wells were mixed gently at the 2 h time point. After a total of 4 h of incubation (37 °C, 5% CO₂, 96-well round bottom plates), supernatants were evaluated by ELISA for TNF-α (n = 13 newborn, n = 15 adult; Student's t test; *, p < 0.05). Whole blood samples with higher plasma AMPase activity produce less TNF-α in response to LPS (B). Plasma AMP dephosphorylation to adenosine was determined as in Fig. 3; n = 11 for each population; statistical analysis by linear regression, significantly non-zero slope for adult and combined populations; *, p < 0.05; newborn population not significant. Error bars, S.E.

FIGURE 7.

Distinct purine metabolism at birth results in higher ambient adenosine concentrations. This figure presents a model that synthesizes both published and novel information from this current study highlighting age-specific differences in sequential cell- and plasma-based generation of Ado from ATP and of deamination of Ado to inosine (Ino). A, newborn blood contains a high density of CD39/ENTPD1-expressing leukocytes, contributing to high ATPase and ADPase activity (i); high plasma concentration of 5′-NT and AP that drive conversion of AMP to Ado (ii); and relative deficiency of ADA, resulting in a net increase in ambient adenosine concentrations and capacity to generate adenosine following release of purine substrates (iii). B, in marked contrast, adult blood features lower density of CD39/ENTPD1-expressing leukocytes (i), lower plasma 5′-NT and AP activity (ii), and relatively greater ADA activity, resulting in lower basal adenosine concentrations (iii).