Bi-directional calcium signaling between adjacent leukocytes and trophoblast-like cells

Abstract

Problem: Trophoblasts are believed to play an important role in mitigating immunological responses against the fetus. To better understand the nature of trophoblast-leukocyte interactions, we have studied signal transduction during intercellular interactions.

Method of study: Using a highly sensitive microfluorometric ratioing method and Ca²(+) -sensitive dyes, we measured Ca²(+) signals in trophoblast-like cell lines (JEG-3 and JAR) or in leukocytes (neutrophils and monocytes) during intercellular contact.

Results: Trophoblast cell lines exhibit Ca²(+) signals during leukocyte contact. In contrast, leukocytes cannot elicit Ca²(+) signals in non-opsonized tumour cells, suggesting that Ca²(+) signaling is not a general feature of cell-cell encounters. Similarly, leukocytes demonstrate Ca²(+) signals during contact with trophoblast cell lines. Ca²(+) signals were confirmed using three dyes and with the Ca²(+) buffer BAPTA.

Conclusion: We suggest that leukocyte-to-trophoblast interactions lead to mutual Ca²(+) signaling events in both cell types, which may contribute to immunoregulation at the materno-fetal interface.

Fig. 1

DIC micrograph of a neutrophil-to-JEG-3 trophoblast-like conjugate. Neutrophils were added to a sub-confluent culture of JEG-3 cells. After 15 min. at 37°C, the plates were gently washed to remove unbound neutrophils. The site of cell-cell interaction is indicated by an arrow. (Bar = 25 μm.)

Fig. 2

Super-quiet microfluorometry of Ca²⁺ signaling in JEG-3 trophoblast-like cells during contact with leukocytes. The Indo-1 ratio is plotted at the ordinate whereas time is given at the abscissa. All experiments were performed at 37°C using a highly stabilized microfluorometry system. Panel A shows a recording of Indo-1-labeled JEG-3 cells during neutrophil binding. An early spike followed by a gradual increase in the ratio was observed. Inclusion of the Ca²⁺ buffer BAPTA abrogated these changes, as indicated in the lower trace. Experiments with the JAR cell line are shown in panel B. Indo-1-labeled JAR cells also exhibited an increase in the fluorescence ratio during incubation with neutrophils, indicating that the observed changes were not restricted to JEG-3 cells. Signaling in JAR cells was similarly blocked by inclusion of BAPTA (lower trace). Although increases in the fluorescence ratio were noted for JAR cells, these changes were not as pronounced as those noted for JEG-3 cells. (The relative noise in the measured ratio increases at longer times due to mechanisms of dye loss, such as photobleaching, during the observation period.)

Fig. 3

Quantitative microfluorometry of Ca²⁺ signaling in JEG-3 cells during contact with leukocytes. The Fluo-4/Fura Red ratio is plotted at the ordinate whereas the time is given at the abscissa. All experiments were performed at 37°C. Panel A: The upper trace shows Fluo-4/Fura Red-labeled JEG-3 cells during incubation with neutrophils. The Fluo-4/Fura Red ratio increases, thus indicating a rise in intracellular Ca²⁺ concentration. Negative controls using the Ca²⁺ buffer BAPTA are shown. Panel B: Fluo-4/Fura Red-labeled JEG-3 cells were incubated with monocytes. Again, the Fluo-4/Fura Red ratio increases over time (upper trace), but is BAPTA-inhibitable (lower trace). Panel C: Although leukocytes elicit Ca²⁺ signals in JEG-3 cells, they do not promote Ca²⁺ signals in other cells. HT-1080 fibrosarcoma cells were labeled with Fluo-4 and Fura Red then incubated with unlabeled monocytes. No significant changes in the Fluo-4/Fura Red ratio were observed.

Fig. 4

Microfluorometry studies of Ca²⁺ signaling in neutrophils during contact with JEG-3 trophoblasts. The Fluo-4/Fura Red ratio is plotted at the ordinate whereas time is given at the abscissa. All experiments were performed at 37°C, as described above. Panel A: The upper trace shows Fluo-4/Fura Red-labeled neutrophils during incubation with JEG-3 cells. The increase in the Fluo-4/Fura Red ratio indicates a rise in intracellular Ca²⁺ concentration. Inclusion of the Ca²⁺ buffer BAPTA abrogated these changes, as shown in the lower trace. Panel B: Indo-1-labeled neutrophils were incubated with JEG-3 cells. As described for Fluo-4/Fura Red, the Indo-1 Red ratio increases in a BAPTA-inhibitable fashion. Panel C: The upper trace shows Fluo-4/Fura Red-labeled neutrophils during incubation with HT-1080 cells. No significant change in the Fluo-4/Fura Red ratio is observed. A control using the Ca²⁺ buffer BAPTA is shown in the lower trace.

Fig. 5

Microfluorometry studies of Ca²⁺ signaling in neutrophils during contact with JAR trophoblasts. The fluorescence ratio is plotted versus time in minutes. All experiments were performed at 37°C. Panel A: In these experiments, Fluo-4 and Fura Red were used as Ca²⁺ dyes to label neutrophils. The dye ratio increases during contact with JAR cells (upper trace), but not when cell were treated with BAPTA (lower trace). Panel B: The upper trace shows the Indo-1 ratio of neutrophils during contact with JAR trophoblasts. The ratio increases and oscillates over time, indicating increases in cytoplasmic Ca²⁺ concentrations. The matched control experiment using BAPTA is given in the lower trace. Panel C: The upper trace shows an Indo-1-labeled neutrophil adherent to an HT-1080 tumor cell. Only relatively small changes in the Indo-1 ratio are observed in comparison to the large changes noted for trophoblasts. The BAPTA control is given in the lower trace.