Antibody‐dependent cell‐mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates

Abstract

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

Fasciola gigantica cathepsin L proteinase‐based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

“…Most importantly, these antibodies were capable of mediated killing of NEJ in conjunction with mouse peritoneal lavage adherent cells, or sheep complement in ADCC and antibody-dependent complement mediated cytotoxicity assays, respectively. Our findings are in agreement with Piedrafita et al 22 who have shown that up to 80% of F. hepatica NEJ are killed when incubated for 48-72 hr in the presence of sheep infection serum and rat peritoneal adherent cells. More importantly, FasAc14p-mediated generation of significant NEJ killing may be considered an in vitro correlate of protection, since cytotoxicity against NEJ was recently implicated as a major mechanism of Indonesian thin-tail sheep resistance against F. gigantica infection.…”

“…About 1 ml blood were obtained from each sheep 7 days after the last injection, to evaluate serum antibody responses of individual sheep to newly excysted juvenile worms (NEJ) by indirect membrane immunofluorescence (IF), antibody-dependent cell-mediated (ADCC) and antibody-dependent complement-mediated cytotoxicity assays using standard procedures, as described. [21][22][23] Briefly, metacercariae were used 5-10 days after release from laboratory-infected snails and encystment, and the outer cyst removed by shaking for 90 min at 378C in 2% (w/v) pepsin in 0.2% HCl. Following centrifugation, the metacercariae were incubated, with shaking, for 120 min at 378C, in the presence of 0.45% taurocholic acid (ICN Biomedicals, Costa Mesa, CA) to achieve final release of outer cyst, and in the presence of high CO 2, and low O 2 tension, which was achieved by addition of 0.3% L-cysteine (anti-oxidant).…”

Early and transient cytotoxic response of peritoneal cells from Fasciola hepatica-infected rats

“…In our case, however, SOD in the culture medium did not affect the action of peritoneal cells against F. hepatica juveniles (data not shown). This result confirms what was observed by Piedrafita et al [25].…”

“…A previous study established that PC of four-week infected rats does not affect the survival of juvenile flukes cultured for 24 h and subsequently transferred to the peritoneal cavity of normal rats [8]. Conversely, Piedrafita et al showed in a recent publication that naive rat PC are able to kill NEJ in vitro when cultured in the presence of immune serum of either rat or sheep origin, and that the cytotoxic mechanism largely involves NO production [25]. The discrepancies between these results and ours could be the consequence of different experimental procedures: the effector/Target (E/T) ratio in Piedrafita's study was 1 NEJ/2 × 10 5 PC, while the E/T ratio in the present study was 6-20 times lower (3 to 10 NEJ/10 5 PC).…”

“…Moreover, juvenile F. hepatica has been described as being resistant to in vitro killing by free radicals as compared with Schistosoma mansoni [24]. However, more recent data suggested that peritoneal cells (PC) could destroy juvenile flukes by an antibodydependent, nitrogen intermediate-dependent mechanism [25]. But these studies focused on the interactions between juvenile flukes and peritoneal cells from naive rats.…”