Caspase-1-mediated cytokine release from gestational tissues, placental, and cord blood

Abstract

Distinguishing between fetal and maternal inflammatory responses is necessary for understanding the immune interplay either side of the placenta. Fetal immunity reaches maturity during extrauterine life and while basic inflammatory responses afford a certain degree of protection, fetuses are vulnerable to infection. With the discovery of inflammasomes-intracellular scaffolds that facilitate the elaboration of reactions resulting in the release of mature interleukin-1β (IL-1β)-it is necessary to consider how inflammatory stimuli are processed. The purinergic P2X7 receptor located on haematopoietic cells is a key intermediary in signal transduction initiated at Toll-like receptors (TLR) terminating in release of the mature IL-1β product. We demonstrate herein that IL-1β release from fetal membranes and mononuclear cells isolated from cord, placental, and maternal blood, obtained at term, is P2X7- and caspase-1 dependent. The P2X7-dependent release of the cytokine, which was highest from choriodecidua, was attenuated by progesterone (P4), prolactin and an NFkB inhibitor. The NLRP3 inflammasome appears necessary for the processing of IL-1β in gestational tissues and leukocytes.

Keywords: IL-1β; P2X7 receptor; caspase-1; inflammasome; parturition; pregnancy.

Figure 1

Leukocyte populations in fetal and maternal blood. (A) Differences between cell marker expression in preparations of MBMCs, CBMCs, and PLBMCs using flow cytometry (n = 6–10 in each group). Leukocyte count in whole cord venous blood of L group and NL group was significantly different between the two the groups (P < 0.05). (B) Scatter plot showing the percentage of CBMCs, PLBMCs, and MBMCs expressing (B) CD45, (C) CD14, and (D) CD68, determined by flow cytometry.

Figure 2

Dot plot of leukocyte markers in isolated cells. Cells were prepared for flow cytometry then analyzed using fluorescence leukocyte markers CD45 (A), CD14 (B) and CD68 (C). Levels of CD14 (B) were lowest in PLBMCs, CBMCs, and MBMCs. (D) Dot plot for MBMCs.

Figure 3

Expression of TLR4 and P2X₇ proteins in gestational tissues. (A) CBMCs and PLBMCs express TLR4 (anti-TLR4 antibody at 1:200); (B) CBMCs and PLBMCs express the P2X₇ receptor (anti-P2X₇ antibody at 1:200). Immunofluorescence was detected using fluorescence-tagged secondary antibodies following fixation and incubation with a primary antibody. No background fluorescence was visible in cells that had been preincubated with control IgG instead of the primary antibody (C) Representative western blots obtained using lysates prepared from myometrium (M; n = 5), choriodecidua (D; n = 5), PLBMCs (n = 7), and CBMCs (n = 7) identifies an 85 kDa using anti-TLR4 antibody and (D) an anti-P2X₇ antibody that recognizes 74–78 kDa protein. (E) Using flow cytometry, a higher percentage of CBMCs (n = 7) expressed the P2X₇ receptor (80.2%) compared with TLR4 where 55.0% were positive (n = 7). (F) Concentration-dependence of IL-1β release in response to LPS from CBMCs.

Figure 4

Expression of NLRP3 in MBMCs, PLBMCs, and CBMCs. (A) Expression of P2X₇ was detected by immunofluorescence in MBMCs (LH panel), CBMCs (middle panel) and PLBMCs (right hand panel) with CBMCs requiring prior incubation with LPS + BzATP before NLRP3 was detectable. (B) Flow cytometry demonstrated that costimulation with LPS + BzATP resulted in 92.4% of CBMCs showing NLRP3 expression as seen in the single parameter histogram.

Figure 5

CBMCS and PLBMCs from laboring (L) and non-laboring (NL) women incubated with LPS and BzATP leads to increased IL-1β release. Costimulation with LPS and BzATP led to increased release of IL-1β than either LPS or BzATP alone from (A) CBMCs and (B) PLBMCs. Cells from laboring women produced significantly greater amounts of IL-1β. IL-1β release was inhibited following preincubation with the P2X₇ antagonists KN-62, oATP and the caspase -1 inhibitor YVAD-CHO following costimulation with LPS and BzATP as indicated on graphs by a “+” prefix before treatment. A combination of YVAD and oATP led to greater inhibition of IL-1β than either inhibitors alone, Both LPS and BzATP_evoked IL-1β release which was inhibited by P4 and prolactin in (A) CBMCs and (B) PLBMCs. (^*P < 0.05).

Figure 6

IL-1β release from maternal blood mononuclear cells (MBMCs). (A) Coincubation with LPS and BzATP elicited greater production of IL-1β from MBMCs. P4, prolactin, the NFkB inhibitor QNZ and the caspase-1 inhibitor YVAD-CHO all inhibited IL-1β release. (B) Western blotting showed positive expression for the P4 receptor in choriodecidua (CD; n = 6), some expression in PLBMCs (n = 6) but undetectable levels in CBMCs (n = 6). ^*P < 0.05 when compared using Dunnett's test.

Figure 7

Gestational membranes produce IL-1β. (A) Intact membranes assembled in transwells stimulated with LPS, BzATP, and LPS+ BzATP led to increased IL-1β release which was highest in the latter and from laboring samples. KN-62, YVAD-CHO, progesterone (P4), prolactin (PRL), and (QNZ) all caused a reduction in IL-1β release in cells stimulated with LPS + BzATP combined. (B) Using intact membranes and sampling media on both sides (upper) and (lower) of the transwell, amnion produced IL-1β only in the upper compartment (C) when choriodecidua was uppermost and upper and lower chambers tested for IL-1β production, higher levels of the cytokine were produced from the upper chamber and were higher than those produced when amnion was uppermost (n = 6; P < 0.05). IL-1β amounts are expressed per/cm². ^*P < 0.05 comparing each treatment between NL and L groups.