Abstract
Abstract. Lectin (PHA-P) activated nonadherent spleen cells from uninfected inbred strains of mice known to exhibit high parasitemias when infected with Trypanosoma cruzi (A/J and C3H/HeJ), released in vitro significantly less lymphotoxin (LT) than did a mouse strain (C57BL/6) known to exhibit low parasitemia when infected with T. cruzi. The capacity of mice to release LT in vitro changed upon infection with T. cruzi. Cells from infected C57BL/6 mice released LT levels well above that from uninfected C57BL/6 animals within 4 days after initial infection, and their capacity to release LT remained high even after the parasites were not detectable in the blood. Cells from infected C3H/HeJ mice were not able to respond as rapidly as those from C57BL/6 animals; however, they were able to release LT at the same levels as the infected C57BL/6 by day 18. The parasitemia induced by Trypanosoma cruzi in C3H/HeJ and C57BL/6 mice was compared with the in vitro ability of their spleen cells to spontaneously release LT. Spontaneous release of LT was significantly higher by spleen cells from infected C57BL/6 mice than by cells from infected C3H/HeJ mice. The kinetics of natural LT release differed from that of mitogen-(PHA-P)-stimulated LT release; LT activity peaked at 3–6 hours of incubation in the former and then declined whereas LT activity in the latter reached a plateau after 3 hours of incubation and did not decline for at least 18 hours in the presence of the inducer. Supernatants from infected C57BL/6 splenocytes, when concentrated 5× by ultrafiltration, significantly inhibited both bloodstream trypomastigote motility in vitro and their infectivity (by 98%) for WI38 human embryonic lung cell cultures. Similar preparations from C3H/HeJ splenocytes only slightly inhibited bloodstream trypomastigote motility and delayed, but did not prevent, infection of WI38 cells. No agglutination of immobilized parasites was noted. Splenocyte supernatants did not affect culture epimastigote motility or growth. This is the first report of direct action by lymphokine-containing supernatants against T. cruzi bloodstream trypomastigotes.
Highlights
Present address: Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles, California 90024
Nogueira et al.4 demonstrated a correlation between the ability of spleen lymphocytes from infected inbred strains of mice to generate, in vitro a lymphokine(s) capable of activating macrophages to a trypanocidal state with susceptibility of the mice to the Y and CL strains of T. cruzi
Twenty-four hours after adding the incubated trypomastigotes to the Wl38 cells, the medium containing free parasites was poured off, the host cells were washed one time with basal medium (BME), 5 ml BME plus 10% FCS was added, the cultures were gassed with 5% CO2 and the flasks were incubated at 37°C for another 24 hours
Summary
Supernatants from non-adherent spleen cell cultures were collected at various times (1-18 hours), cleared of cells by centrifugation (500 x g) for 10 min) and immediately tested for LT activity against L-929 cells. Duplicate tubes containing 1 x 18 Ficoll-Hypaque cleaned bloodstream trypomastigotes obtained from BALB/c mice infected [14,15] days previously with trypomastigotes were incubated overnight (18-20 hours at 37°C) in 0.5 ml of 5x concentrated supernatants or 5x concentrated media controls. Twenty-four hours after adding the incubated trypomastigotes to the Wl38 cells, the medium containing free parasites was poured off, the host cells were washed one time with BME, 5 ml BME plus 10% FCS was added, the cultures were gassed with 5% CO2 and the flasks were incubated at 37°C for another 24 hours. Observations were made only for [3,4] days because trypomastigote release from infected cells followed by the invasion of new cells resulted in increasing numbers of parasitized cells when antitrypomastigote activity was not present
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