ADP and other metabolites released from Acanthamoeba castellanii lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines

Abstract

Monocytes/macrophages are thought to be involved in Acanthamoeba infections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released by Acanthamoeba castellanii trophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium by A. castellanii, interacting with specific P2y(2) purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca(2+)](i), morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-alpha). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-alpha release induced by amoebic ADP were blocked by the P2y(2) inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1beta. These findings suggest that pathogenic free-living A. castellanii by release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.

FIG. 1.

Time courses of the [Ca²⁺]_i evoked in THP-1 cells, loaded with Fura 2-AM (3 μM) by 80 μl of heat-treated filtered cell-free PBS (cPBS) conditioned for 2 h by the addition of 6 × 10⁶ A. castellanii trophozoites/ml (A), by the same volume of PBS containing 20 μM ADP₀ (B), by cPBS (C) or 20 μM ADP₀ (D) after chelation of extracellular Ca²⁺ with 5 mM EGTA, by cPBS (E) or 20 μM ADP₀ (F) after exposition to 10 μM thapsigargin, or by cPBS (G) or 20 μM ADP₀ (H) after 20 min of loaded THP-1 cells exposure to 10 μM ryanodine.

FIG. 2.

Phase-contrast microscopy of THP-1 cells incubated for 7 h at 37°C in 5% CO₂ atmosphere with RPMI-20 mM HEPES alone (A) and with fractions of conditioned medium containing amoebic metabolites (>30 kDa; pRPMI) (B), amoebic metabolites (30 to 10 kDa; rRPMI) (C), amoebic ADP (sRPMI) (D), heat-inactivated amoebic metabolites (>10 kDa; mRPMI) (E), heat-treated amoebic ADP (cRPMI) (F), or containing 20 μM ADP₀ (G).

FIG. 3.

Activation of intracellular caspase-3 on THP-1 cells exposed, both in the presence or in the absence of 20 μM suramin, for 14 h at 37°C in 5% CO₂ atmosphere to RPMI medium (Control), entire conditioned medium (iRPMI), or fractions of conditioned medium containing, respectively, amoebic metabolites of >30 kDa (pRPMI), amoebic metabolites of from 30 to 10 kDa (rRPMI), amoebic ADP (sRPMI) or heat-treated amoebic ADP (cRPMI), amoebic metabolites of >10 kDa heat inactivated (mRPMI), or to RPMI containing 20 μM ADP₀. Values are means ± the standard error of at least four experiments. ✽, P < 0.05 versus the control; ✽✽, P < 0.01 versus control.

FIG. 4.

Release of TNF-α (A), IL-1β (B), and IL-6 (C) from THP-1 cells exposed, both in presence or in absence of 20 μM suramin, for 14 h at 37°C in 5% CO₂ atmosphere to RPMI medium (Control), entire conditioned medium (iRPMI), or fractions of conditioned medium containing amoebic metabolites of >30 kDa (pRPMI), amoebic metabolites included of from 30 to 10 kDa (rRPMI), amoebic ADP (sRPMI) or heat-treated amoebic ADP (cRPMI), amoebic metabolites of >10 kDa heat inactivated (mRPMI), or to RPMI containing 20 μM ADP₀. Values are means ± the standard error of at least four experiments. ✽, P < 0.01 versus the control.