Leukocyte pyruvate kinase expression is reduced in normal human pregnancy but not in pre-eclampsia

Abstract

Problem: Emerging evidence suggests that metabolism influences immune cell signaling and immunoregulation. To examine the immunoregulatory role of glycolysis in pregnancy, we evaluated the properties of pyruvate kinase in leukocytes from non-pregnant women and those with normal pregnancy and pre-eclampsia.

Method of study: We evaluated pyruvate kinase expression in lymphocytes and neutrophils from non-pregnant, pregnant, and pre-eclampsia patients using fluorescence microscopy and flow cytometry. Leukocyte pyruvate kinase activity and pyruvate concentrations were also evaluated. To study pyruvate's effect on signaling, we labeled Jurkat T cells with Ca(2+) dyes and measured cell responses in the presence of agents influencing intracellular pyruvate.

Results: The expression of pyruvate kinase is reduced in lymphocytes and neutrophils from normal pregnant women in comparison with those of non-pregnant women and pre-eclampsia patients. Similarly, the activity of pyruvate kinase and the intracellular pyruvate concentration are reduced in leukocytes of normal pregnant women in comparison with non-pregnant women and women with pre-eclampsia. Using Jurkat cells as a model of leukocyte signaling, we have shown that perturbations of intracellular pyruvate influence Ca(2+) signals.

Conclusion: Normal pregnancy is characterized by reduced pyruvate kinase expression within lymphocytes and neutrophils. We speculate that reduced pyruvate kinase expression modifies immune cell responses due to reduced pyruvate concentrations.

Fig. 1

Pyruvate kinase activity assay. Pyruvate kinase activity was measured using phosphoenolpyruvate as substrate and coupling pyruvate production to the disappearance of NADH, which can be quantified using fluorescence detection.

Fig. 2

Fluorescence microscopy of pyruvate kinase. CD3⁺ lymphocytes were prepared from peripheral blood samples obtained from non-pregnant women (A), pregnant women (B), and pre-eclampsia patients (C). Cells were labeled with an anti-pyruvate kinase antibody. Images were collected under identical conditions, as described in the Materials and Methods. (Bar = 10.6 mm)

Fig. 3

Flow cytometry histograms of pyruvate kinase abundance in human CD3⁺ lymphocytes are shown. Panel A: Representative histograms showing pyruvate kinase or isotype control labeling are shown. Pyruvate kinase expression is reduced in maternal lymphocytes (black dashed line) relative to lymphocytes from non-pregnant adults (black solid line). Fluorescence histograms of cells stained with an isotype control antibody are shown in gray (dashed line = maternal; solid = normal adult). Only CD3⁺ cells are shown in the histograms (see inset showing CD3 PE vs. side scatter dot plot). Panel B: Representative histogram overlay of cells stained with an anti-lactate dehydrogenase or isotype control antibodies. Samples from pregnant women (black dashed line) and non-pregnant women (black solid line) are shown. Isotype controls are shown in gray on the left hand side levels between maternal blood and normal adult peripheral blood CD3⁺ lymphocytes (dashed line = maternal; solid = normal adult). Lymphocyte pyruvate kinase expression, but not LDH expression, is substantially reduced during pregnancy.

Fig. 4

Summary of flow cytometry studies of CD3⁺ lymphocytes (A) and neutrophils (B) from non-pregnant (open bars), pregnant (gray bars) and pre-eclamptic (black bars) women. Panel A: Pyruvate kinase expression is reduced in lymphocytes from pregnant women in comparison to non-pregnant women (P=0.00001). Pyruvate kinase expression in cells from pre-eclampsia patients was significantly elevated in comparison to normal pregnant patients (P=0.00046), but not non-pregnant women. Ten individuals in each category were studied. No significant differences are detected in lactate dehydrogenase levels. Panel B: Maternal peripheral blood neutrophils have significantly less pyruvate kinase than neutrophils from both non-pregnant women (0.000037) as well as women with preeclampsia (0.0036). Eight non-pregnant women were studied. Nine normal pregnant women and nine pre-eclampsia patients were studied. As noted above for lymphocytes, no significant effect was noted for lactate dehydrogenase. Error bars represent the SD.

Fig. 5

Flow cytometry histograms of pyruvate kinase labeling of human CD3⁺ lymphocytes are shown. Cells were untreated (control) or treated with LPS (100 ng/ml, 30 min.). Isotype controls are shown on the left hand side. No differences in pyruvate kinase levels could be noted in the presence of LPS.

Fig. 6

Evaluation of intracellular pyruvate concentrations. Intracellular pyruvate concentrations were determined by measuring pyruvate levels in cell extracts then correcting for cell volume. The pyruvate concentration in PBMCs from non-pregnant women (1.58±0.22 mM; mean±SEM; n=8) was significantly higher than that of pregnant women (0.95±0.11 mM; n=11) (P=0.01). The pyruvate concentration in cells from pre-eclamptic patients (1.45±0.19; n=10) was significantly higher than that of normal pregnant women (P<0.05). As indicated by the standard error of the means, these values are a good measure of the underlying population means, thus supporting the proposed mechanistic contribution to signaling.

Fig. 7

Effect of pyruvate and phenylalanine, a pyruvate kinase inhibitor, on the Fluo-4/Fura Red ratio (a measure of intracellular Ca²⁺) in unstimulated Jurkat cells. The Fluo-4/Fura Red ratio is listed at the ordinate (mean±SD). Experiments were performed as described in the Materials and Methods. Phenylalanine, a pyruvate kinase inhibitor, is known to perturb the concentration of metabolites in Jurkat cells. At 6 mM, phenylalanine significantly reduced the Fluo-4/Fura Red ratio. On the other hand, addition of pyruvate to cells promoted an increase in the Fluo-4/Fura Red ratio. Although significant, these changes were not dramatic and required multiple repetitions to reach these high levels of significance. (n=10)

Fig. 8

Effect of pyruvate and phenylalanine on the Fluo-4/Fura Red ratio (mean±SD) in stimulated Jurkat cells. In the first three columns, cells were activated using PHA. Addition of phenylalanine promoted a decrease in the Fluo-4/Fura Red ratio whereas the addition of pyruvate promoted an increase in the Fluo-4/Fura Red ratio. Untreated cells are shown on the right hand side for comparison. (n=10)

Fig. 9

Proposed mechanism of pyruvate-mediated influence on cell growth and immunologic modifications. Pyruvate is produced by pyruvate kinase, the final enzyme of glycolysis. When pyruvate kinase levels are depressed at this rate-limiting step of glycolysis, upstream glycolytic phosphometabolite intermediates accumulate to higher levels. Higher levels of glycolytic intermediates support the synthesis of carbohydrates, nucleic acids, amino acids, and lipids, as indicated in the diagram. Pyruvate promotes Ca²⁺ signaling by inhibiting the negative Ca²⁺ feedback at the CRAC channel. Ca²⁺ directly influences NFAT. However, when pyruvate levels are reduced, signaling is reduced.