Changes in cytoplasmic Ca2+
concentrations play crucial roles in many cell functions. Included among these functions are: electrical activity, secretion, muscle contraction, membrane permeability, enzyme activity, apoptosis, proliferation, protein synthesis, and gene expression.15
In addition to its intracellular roles, Ca2+
also participates in cell-to-cell communication. Contact-dependent intercellular communication takes several forms, such as communication via gap junctions and receptor pathways.16,17
As mentioned above, cell contact-dependent immunoregulatory events participate in maternal immunomodulation during pregnancy. However, the existence of Ca2+
signaling responses during intercellular contact between maternal and fetal cells have not been reported, although it is known that trophoblasts may exhibit Ca2+
signals in certain circumstances.18,19
In the present study, we have tested the hypothesis that leukocytes and trophoblast cell lines elicit Ca2+
signals in one another during their interaction. In order to study Ca2+
signals, it was necessary to use an in vitro
model, in this case trophoblast cell lines and peripheral blood leukocytes. We have found that both neutrophils and monocytes stimulate Ca2+
signals in adherent trophoblast cell lines. The converse is also true; trophoblast cell lines induce Ca2+
signals in bound neutrophils and monocytes. These signals were dependent upon Ca2+
and could not be elicited within HT1080 tumor cells. To the best of our knowledge, this is the first study to report Ca2+
signal transduction events during leukocyte-to-trophoblast cell line contact.
To provide the most reliable data possible, we employed several technically advantageous approaches. To detect Ca2+ changes, we used single cell microfluorometry incorporating super-quiet illumination and photon counting detection; this instrumentation provides very low noise excitation and fluorescence detection. The major source of noise in these experiments is the quantum (or shot) noise associated with photon emission by the dyes. Consequently, small changes in intensity are of a biological nature. In addition, we used ratiometric analysis of the Ca2+ dyes. Ratiometric methods have many advantages such as increased dynamic range, correction for variations in cell thickness, dye artifacts such as compartmentalization and leakage, and reduction of illumination noise. This approach was useful because the Ca2+ changes observed were not as large as those seen during other biological processes, such as antibody-dependent cellular cytotoxicity (data not shown). Furthermore, to insure that these biophysical analyses of signal transduction were biologically important, we tested our hypothesis using: two different fluorescent ratioing dyes, the Ca2+-buffering molecule BAPTA, two trophoblast-like cell lines, two types of leukocytes, and a non-trophoblast-like fibrosarcoma cell line. Our careful experimentation has shown clearly that trophoblast-like cells and leukocytes elicit Ca2+ signals in one another during physical contact.
Our work strongly supports the concept of bidirectional communication between cells of the mother and fetus. Bidirectional communication between the mother and fetus has been previously proposed by several laboratories.12,20-22
For example, this may take place in the form of cytokine cross-talk. In an important study, Jiang et al. demonstrated signaling in co-cultures of leukocytes and trophoblasts that could be transferred by incubation of JAR cells with leukocyte conditioned media suggesting that the effect is not contact-dependent.12
In the present study we have shown intercellular contact-dependent stimulation of Ca2+
signaling, as judged by microscopic observation and microfluorometry. It would seem likely that both contact-dependent and contact-independent mechanisms participate in immunoregulatory signaling at the maternal-fetal interface.
signals reported in this paper are reasonable from the standpoint of signal transduction. As mentioned above, Jiang et al. noted MAPK signaling in co-cultures of leukocytes and trophoblasts.12
This observation is consistent with our work inasmuch as both the MAPK cascade and Ca2+
changes can be affected by the same signaling stimulus. For example, Syk participates in tyrosine phosphorylation of proteins that lead to both MAPK and Ca2+
Therefore, stimulation of both pathways is not unreasonable.
signals noted above may contribute at several levels. For example, they may be directly related to the adherence events observed microscopically, which have also been observed by others.9
These findings may constitute a model for early events in syncytiotrophoblast damage.24,25
Alternatively, these Ca2+
signals may play an important role in the down-regulation of leukocyte function, which have been previously observed to protect trophoblasts.10,11
It may seem paradoxical that Ca2+
signals might play a role in both the prevention and promotion of trophoblast damage because it is widely known that Ca2+
signals broadly promote activation of immune cells. Included among the activation events are degranulation, production of reactive oxygen metabolites, cytokine release, migration and many others. However, in certain circumstances, Ca2+
signals may be inhibitory in nature (e.g., rod outer segments and pinealocytes).26,27
It seems possible that the inhibitory or stimulatory nature of the cell-cell interaction may depend upon its physiological context as well as the activation of additional signaling pathways. We are currently studying the physical and physiological attributes of these signals to further decode the nature of maternal-to-fetal cell signaling.