Macrophages are a source of extracellular adenosine deaminase-2 during inflammatory responses

Abstract

Serum activity of the adenosine deaminase (ADA) isozyme, ADA2, has been reported to be elevated during various disease states. Macrophages have been suggested as the cellular source of extracellular ADA activity because they are one of the only cell types in which intracellular ADA2 activity has been measured, but extracellular secretion has never been demonstrated. Rat primary peritoneal macrophages (PPMs) and peripheral blood monocytes (PBMs) were harvested and incubated for 18 h in RPMI supplemented with horse serum. PPM and PBM lysates were assayed for intracellular ADA activity (ammonia production). In vitro and in vivo extracellular ADA activities were measured in media and rat serum, respectively. Activity of ADA1 was confirmed by selective inhibition with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). ADA2 activity was inhibited by 2'-deoxycoformcin only, and was increased at a low pH (6.5). Activity of both ADA isozymes was found in PPMs and PBMs, and their media. In a separate group of rats, peritonitis was induced by ip insertion of 400 mg/kg caecal slurry. PPMs were harvested 24 h later and incubated for 18 h. In PPMs from rats with peritonitis both isozymes were elevated by a similar proportion. In contrast, media from these PPMs had a lower ADA1 and a higher ADA2 activity compared to PPMs from nonseptic rats. This resulted in a greater proportion of ADA2 in media. The isozyme proportions in serum from septic rats more closely resembled that of the PPM media. The response of PBM was small relative to that of PPM. These results suggest that macrophages are a significant source of extracellular ADA isozymes, the activity of which increases during an inflammatory response. Because extracellular isozymes profiles differ from cellular concentrations, the data also suggest differential release of each isozyme from PPMs.

Fig. 1

ADA isozyme sensitivity to EHNA: Duodenal homogenates (•) and macrophage cell lysates (□) were treated in vitro with EHNA concentrations ranging from 10⁻⁸ to 5 × 10⁻⁴m and adenosine deaminase activity was subsequently determined. Inhibition of ADA activity by EHNA relative to basal activity is shown. There was a dose dependent decrease in ADA activity between 10⁻⁷ and 10⁻⁴m EHNA. At concentrations higher than 10⁻⁴m EHNA ADA activity was below detection limits in duodenal homogenates. In contrast, the inhibition of macrophge cell lysates ADA activity reached a nadir at around 20–30%. The activity that remained represents ADA2 activity.

Fig. 2

Macrophage lysate ADA activity: Peritoneal macrophages were lysed after 24 h incubation in vitro. ADA activity was measured and reported as nmoles NH₃/ h·10⁴cells. (a) Both ADA1 () and ADA2 () were measurable in macrophage lysates of rats, with ADA1 representing the predominant proportion of total ADA activity. Activity of both isozymes were significantly higher in macrophages from rats with peritonitis. (b) Activity attributable to ADA2 represented < 50% of the total activity, regardless of rat condition. *Significantly different relative to nonseptic naive values. P < 0·05. Samples sizes (n) were 4 for nonseptic (each sample representing the pooled cells from 2 to 3 rats) and 6 for septic groups, each sample representing macrophages from a single septic rat.

Fig. 3

Macrophage media ADA activity: Primary peritoneal macrophages were incubated with RPMI media supplemented with 10% HS and 5% PSN for 24 h. Media was collected and subsequently assayed for ADA activity. (a) Both ADA1 () and ADA2 () were measurable in macrophage media. In nonseptic rats, ADA1 represented the predominant proportion of total ADA activity. ADA1 activity in the media of septic macrophages was lower than in the media of naive macrophages. Conversely, ADA2 activity was higher in the media collected from septic macrophages. (b) Activity attributable to ADA2 was significantly higher in media of macrophages collected from rats with peritonitis. *Significantly different relative to nonseptic naive values. P < 0·05. Samples sizes (n) were 4 for nonseptic (each sample representing the pooled cells from 2 to 3 rats) and 6 for septic groups, each sample representing macrophages from a single septic rat.

Fig. 4

Peripheral blood monocyte lysate ADA activity: Peripheral blood monocytes were lysed after 24 h incubation in vitro. ADA activity was measured and reported as nmoles NH₃/ h·10⁴cells. (a) Both ADA1 () and ADA2 () were measurable in monocyte lysates of rats, with ADA1 representing the predominant proportion of total ADA activity. However, cell concentrations were 50–200-fold lower than that of macrophages (note axis scale difference compared to Fig. 2). Activity of only ADA2 was significantly higher in monocytes from rats with peritonitis. (b) Activity attributable to ADA2 was significantly higher in monocytes collected from rats with peritonitis. *Significantly different relative to nonseptic naive values. P < 0·05. Samples sizes (n) were 4 for nonseptic (each sample representing the pooled cells from 2 to 3 rats) and 6 for septic groups, each sample representing macrophages from a single septic rat.

Fig. 5

Peripheral blood monocyte media ADA activity: Peripheral blood monocytes were incubated with RPMI media supplemented with 10% HS and 5% PSN for 24 h. Media was collected and subsequently assayed for ADA activity. (a) Both ADA1 () and ADA2 () were measurable in monocyte media. In nonseptic rats, ADA1 represented the predominant proportion of total ADA activity. Only ADA2 activity was higher in the media collected from septic monocytes. (b) Activity attributable to ADA2 was significantly higher in media of monocytes collected from rats with peritonitis. *Significantly different relative to nonseptic naive values. P < 0·05. Samples sizes (n) were 4 for nonseptic (each sample representing the pooled cells from 2 to 3 rats) and 6 for septic groups, each sample representing macrophages from a single septic rat.

Fig. 6

Serum ADA activity: ADA activity is reported as nmol ammonia/ h·ml serum. (a) Both ADA1 () and ADA2 () were measurable in serum of rats, with ADA1 representing the predominant proportion of total ADA activity in nonseptic rats. In rats with peritonitis, serum ADA1 and ADA2 activity levels were comparable. (b) The proportion of total ADA activity represented by ADA2 activity in the serum is represented. ADA2 became the predominant isozyme in serum collected from septic rats as compared to nonseptic rats. *Significantly different relative to nonseptic naive values. P < 0·05. n = 9 and 12 for nonseptic and septic groups, respectively.