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Published on: June 16, 2011
Role of function-associated molecules in target cell lysis: analysis of rat adherent lymphokine-activated killer
L Jaso-Friedmann1, J H Leary, D L Evans
1Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602.
Abstract:
Monoclonal antibodies (MAbs) against fish nonspecific cytotoxic cells (NCC) inhibit the lysis of target cells by NCC and by human and rat natural killer (NK) cells. We now show that the anti-function-associated molecule (anti-FAM) MAb 5C6 also inhibits the cytolytic activity of rat (F344) nonadherent lymphokine-activated killer (LAK) cells and adherent LAK (ALAK) cells. Rat nylon-wool-nonadherent (NWNA) cells were cultured for 48 h in recombinant human interleukin-2 (IL-2). Nonadherent cells were then removed and the plastic-adherent (ALAK) cells were recultured for 9 days. Cells were daily analyzed by cytometry (using MAb 5C6 and the rat NK cell MAb 3.2.3) and cytotoxicity. Approximately 40% of the initially cultured NWNA cells, 50-60% of the LAK cells and 87-98% of the ALAK cells were positive for MAb 5C6. Tissue culture supernatants containing MAb 5C6 caused a dose-dependent inhibition of cytotoxicity against P815 and YAC-1 targets. Approximately 50% inhibition was obtained at MAb concentrations of 1.25-2.5 micrograms/ml. Maximum inhibition of ALAK cytotoxicity was 86% on day 4 of culture. Two-dimensional SDS-PAGE and Western blot analysis of acetone-precipitated cell membrane preparations identified a 55-kD molecule (pI 6.1-6.3) on NWNA and ALAK cells and an additional slightly more acidic 45-kD protein on ALAK cells. These data indicate that rat ALAK cells may initiate lysis of target cells via receptor binding.
Insights
Monoclonal antibodies targeting function-associated molecules inhibit rat adherent lymphokine-activated killer (ALAK) cell cytotoxicity. These antibodies bind to specific molecules on ALAK cells, suggesting a receptor-mediated cytotoxic mechanism.
Area of Science:
- Immunology
- Cell Biology
Background:
- Monoclonal antibodies (MAbs) against fish nonspecific cytotoxic cells (NCC) inhibit target cell lysis.
- Function-associated molecule (FAM) MAbs, like 5C6, are known to affect natural killer (NK) cell activity.
Purpose of the Study:
- To investigate the effect of anti-FAM MAb 5C6 on rat lymphokine-activated killer (LAK) and adherent LAK (ALAK) cell cytotoxicity.
- To identify the molecular targets of MAb 5C6 on rat LAK and ALAK cells.
Main Methods:
- Rat nonadherent lymphokine-activated killer (NWNA) cells were cultured with interleukin-2 (IL-2) to generate LAK and ALAK cells.
- Flow cytometry was used to analyze MAb 5C6 and MAb 3.2.3 (rat NK cell MAb) binding.
- Cytotoxicity assays were performed using P815 and YAC-1 target cells.
- Two-dimensional SDS-PAGE and Western blot analysis identified target molecules.
Main Results:
- MAb 5C6 inhibited the cytolytic activity of rat LAK and ALAK cells in a dose-dependent manner.
- MAb 5C6 positive cells increased during culture, with ALAK cells showing high positivity (87-98%).
- A 55-kD molecule was identified on NWNA and ALAK cells, and an additional 45-kD protein on ALAK cells.
Conclusions:
- Rat ALAK cells express molecules recognized by anti-FAM MAb 5C6.
- These molecules are involved in the cytotoxic activity of ALAK cells.
- Data suggest that rat ALAK cells may initiate target cell lysis through receptor binding.
