ABSTRACT
The objective of these studies was to assess the intracellular calcium (Ca2+) signal involved in the activation of ectromelia virus (EV)-specific cytotoxic T lymphocytes (CTL) upon stimulation with EV-sensitized (A20) target cells or concanavalin A (Con A). The CTL originated from mice previously infected with EV. The level of cytosolic Ca2+ in EV-specific CTL was measured using Fluo-3 AM. In both cases transient [Ca2+]i rise and a sustained plateau (1723 nM) were observed in buffer with 1 mM extracellular calcium. The [Ca2+]i response of EV-specific CTL to EV-sensitized target cells or Con A in extracellular calcium free buffer consisted of only one peak at 852 nm. The cytotoxic activity EV-specific CTL assessed in normal medium was 53.0%, but significantly reduced to 15% when verapamil was added to the buffer. No enhancement of [Ca2+]i response in EV-specific CTL was observed upon costimulation with PMA. Association of the level of intracellular [Ca2+]i in EV-specific CTL and the percentage of their cytotoxicity was found. These results show partial dependence of EV-specific CTL killing on extracellular calcium.
Subject(s)
Calcium/immunology , Ectromelia virus/immunology , Ectromelia, Infectious/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Cells, Cultured , Cytosol/immunology , Cytotoxicity Tests, Immunologic , Mice , Mice, Inbred BALB C , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/virology , Tumor Cells, CulturedABSTRACT
PMNL leukocytosis is a feature common to many types of infectious and inflammatory diseases. How PMNL are recruited to tissues is not yet clear although it is a question that has considerable clinical importance. We investigated the function of PMNL which migrated through an artificial barrier (Chinese hamster ovary (CHO) cells, collagen and nylon cloth membrane) subjected to CT or choleragenoid treatment toward plain medium (the same RPMI in the upper and lower chamber) or medium containing chemotactic factor (fMLP or LPS or ZAS). CT treatment significantly (P < 0.01) reduced the Fc gamma R expression on the surface of PMNL. The PMNL functions, namely, migration, phagocytic activity and intracellular killing of staphylococci, also have been reduced significantly (P < 0.01). Fc gamma R expression and some functions of PMNL that migrate to chemoattractants were reduced, irrespective of the presence or absence of CT; however, the inhibitory effect of CT on PMNL function was observed only when PMNL migrate to the lower chamber without chemotactic factor. On the other hand choleragenoid treatment of CHO cells did not have any significant influence on PMNL function and Fc gamma R expression. In conclusion, our experiments demonstrate that CT reduces EAFc rosetting and the Fc gamma R-dependent phagocytic and bactericidal activity of bovine blood PMNL.
