BRIEF COMMUNICATION
Immunology 1992 77 473-476
Lipopolysaccharide synergizes with tumour necrosis factor-a in
cytotoxicity assays H. PFISTER, T. HENNET & T. W. JUNGI Institute of Veterinary Virology, University of Berne, Berne, Switzerland
Acceptedfor publication 14 June 1992
SUMMARY Lipopolysaccharide (LPS) from Escherichia coli was found to synergize with human recombinant tumour necrosis factor-at (TNF-a) in the lysis of L929 and WEHI 164 (clone 13) murine fibroblasts, two cell lines classically used in TNF-a bioassays. The effect was noted with TNF-a at low (sublytic or lightly lytic) concentrations and was significant for LPS concentrations in the ng range. The LPS effect could be inhibited by polymyxin B, and was not observed when the TNF-a assay was performed in the absence of actinomycin D. Enhancement of TNF-a lysis by LPS occurred in several assays for determining TNF-a, including MTT cleavage, crystal violet staining and lactate dehydrogenase release. Synergism was obtained only when LPS and TNF-a were added to cells simultaneously, but not when applied in sequence. The reported synergism may be relevant for TNF-a determinations by bioassay, and for the understanding of pathophysiology of Gram-negative sepsis.
Tumour necrosis factor-a (TNF-cx) and TNF-# (lymphotoxin) are two well-characterized molecular mediators of cytotoxicity and other host-defence-related functions. ' Their mechanisms of cytotoxicity are ill defined. Nevertheless, cytotoxicity assays represent the standard method for measuring TNF-a activity in
bioassays.2 Bioassays generally lack specificity. Even when the factor under investigation can be neutralized completely by antibodies or other means, additional factors may synergize. We demonstrate here that a factor synergizing with TNF-a is lipopolysaccharide (LPS) of Gram-negative bacteria. This could lead to an overestimation of TNF-a in LPS-containing specimens. A synergism between TNF-x and one of its inciting stimuli might be of pathophysiological significance. In the cytotoxicity assay, we used as target cells the two fibroblastoid cell lines, L929 and WEHI 164 (clone 13), both kindly provided by Dr R. Keller (Group for Immunobiology, University of Zurich, Switzerland). Cells were cultured in Dulbecco's minimum essential medium (MEM) supplemented with penicillin (100 U/ml), streptomycin (100 pg/ml), neomycin (5 pg/ml), glutamin (2-5 mM) and 7% foetal calf serum (FCS) in 80 cm2 tissue culture flasks and passaged three times weekly. For the TNF-a assay, cells subcultured for 24 hr at 3-5 x 104 cells/ well in Iscove's modified Dulbecco's medium containing penicillin (100 IU/ml), streptomycin (100 pg/ml), glutamine (2 mM), HEPES (10 mM), MEM vitamin solution (1%; Gibco BRL, Basel, Switzerland), MEM non-essential amino acids (1%; Gibco), and 0-5% bactopeptone (Difco, Detroit, MI). Medium Correspondence: Professor T. W. Jungi, Institute of Veterinary Virology, University of Berne, Lainggass-Str. 122, CH-3012 Berne, Switzerland.
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was then replaced by 50 y1 of the same medium also containing actinomycin D (Act. D; final conc. 1 5 pg/ml) and either TNF-a, LPS (Escherichia coli 011 :B4, 055:B5 or Salmonella enteriti-
dis; Sigma, St Louis, MO), polymyxin B (Px B; Sigma) or combinations of these agents. In some experiments, Act. D was omitted. After 24 hr of culture, cytotoxicity was determined by the colorimetric MTT assay.3 In some assays, WEHI 164 (clone 13) cells were used as targets,4 and cytotoxicity was also determined by the crystal violet method,2 and by determining the content of the cytoplasmatic enzyme, lactate dehydrogenase (LDH), in the supernatant.5 For LDH determinations, a commercial kit was used (Sigma). Cytotoxicity was expressed in % lysis, using the formula: % cytotoxicity
OD control cells - OD test cells
GOD test cells - OD water-lysed cells
Cytotoxicity was also converted to U/ml, using a calibration curve made with recombinant human recombinant TNF-a (National Institute for Biological Standards and Control, Potters Bar, U.K.). Results with L929 cell batches showing 50% cytolysis at TNF-a concentrations of either < 5 or > 10 U/ml were disregarded. Using the MTT assay, human recombinant TNF-ac dosedependently abrogated the mitochondrial dehydrogenase activity in L929 murine fibroblasts, as assessed by the conversion of MTT to a formazane derivative. Fifty per cent cytotoxicity was obtained with 0 5-0-8 U/well of TNF-ac (5-8 U/ml; Fig. I a). Cytotoxicity induced by low amounts of TNF-a could be enhanced in a dose-dependent manner by the addition of LPS during the TNF-a-target cell interaction (Fig. 1b). The effect was significant (P
Immunology 1992 77 473-476
Lipopolysaccharide synergizes with tumour necrosis factor-a in
cytotoxicity assays H. PFISTER, T. HENNET & T. W. JUNGI Institute of Veterinary Virology, University of Berne, Berne, Switzerland
Acceptedfor publication 14 June 1992
SUMMARY Lipopolysaccharide (LPS) from Escherichia coli was found to synergize with human recombinant tumour necrosis factor-at (TNF-a) in the lysis of L929 and WEHI 164 (clone 13) murine fibroblasts, two cell lines classically used in TNF-a bioassays. The effect was noted with TNF-a at low (sublytic or lightly lytic) concentrations and was significant for LPS concentrations in the ng range. The LPS effect could be inhibited by polymyxin B, and was not observed when the TNF-a assay was performed in the absence of actinomycin D. Enhancement of TNF-a lysis by LPS occurred in several assays for determining TNF-a, including MTT cleavage, crystal violet staining and lactate dehydrogenase release. Synergism was obtained only when LPS and TNF-a were added to cells simultaneously, but not when applied in sequence. The reported synergism may be relevant for TNF-a determinations by bioassay, and for the understanding of pathophysiology of Gram-negative sepsis.
Tumour necrosis factor-a (TNF-cx) and TNF-# (lymphotoxin) are two well-characterized molecular mediators of cytotoxicity and other host-defence-related functions. ' Their mechanisms of cytotoxicity are ill defined. Nevertheless, cytotoxicity assays represent the standard method for measuring TNF-a activity in
bioassays.2 Bioassays generally lack specificity. Even when the factor under investigation can be neutralized completely by antibodies or other means, additional factors may synergize. We demonstrate here that a factor synergizing with TNF-a is lipopolysaccharide (LPS) of Gram-negative bacteria. This could lead to an overestimation of TNF-a in LPS-containing specimens. A synergism between TNF-x and one of its inciting stimuli might be of pathophysiological significance. In the cytotoxicity assay, we used as target cells the two fibroblastoid cell lines, L929 and WEHI 164 (clone 13), both kindly provided by Dr R. Keller (Group for Immunobiology, University of Zurich, Switzerland). Cells were cultured in Dulbecco's minimum essential medium (MEM) supplemented with penicillin (100 U/ml), streptomycin (100 pg/ml), neomycin (5 pg/ml), glutamin (2-5 mM) and 7% foetal calf serum (FCS) in 80 cm2 tissue culture flasks and passaged three times weekly. For the TNF-a assay, cells subcultured for 24 hr at 3-5 x 104 cells/ well in Iscove's modified Dulbecco's medium containing penicillin (100 IU/ml), streptomycin (100 pg/ml), glutamine (2 mM), HEPES (10 mM), MEM vitamin solution (1%; Gibco BRL, Basel, Switzerland), MEM non-essential amino acids (1%; Gibco), and 0-5% bactopeptone (Difco, Detroit, MI). Medium Correspondence: Professor T. W. Jungi, Institute of Veterinary Virology, University of Berne, Lainggass-Str. 122, CH-3012 Berne, Switzerland.
473
was then replaced by 50 y1 of the same medium also containing actinomycin D (Act. D; final conc. 1 5 pg/ml) and either TNF-a, LPS (Escherichia coli 011 :B4, 055:B5 or Salmonella enteriti-
dis; Sigma, St Louis, MO), polymyxin B (Px B; Sigma) or combinations of these agents. In some experiments, Act. D was omitted. After 24 hr of culture, cytotoxicity was determined by the colorimetric MTT assay.3 In some assays, WEHI 164 (clone 13) cells were used as targets,4 and cytotoxicity was also determined by the crystal violet method,2 and by determining the content of the cytoplasmatic enzyme, lactate dehydrogenase (LDH), in the supernatant.5 For LDH determinations, a commercial kit was used (Sigma). Cytotoxicity was expressed in % lysis, using the formula: % cytotoxicity
OD control cells - OD test cells
GOD test cells - OD water-lysed cells
Cytotoxicity was also converted to U/ml, using a calibration curve made with recombinant human recombinant TNF-a (National Institute for Biological Standards and Control, Potters Bar, U.K.). Results with L929 cell batches showing 50% cytolysis at TNF-a concentrations of either < 5 or > 10 U/ml were disregarded. Using the MTT assay, human recombinant TNF-ac dosedependently abrogated the mitochondrial dehydrogenase activity in L929 murine fibroblasts, as assessed by the conversion of MTT to a formazane derivative. Fifty per cent cytotoxicity was obtained with 0 5-0-8 U/well of TNF-ac (5-8 U/ml; Fig. I a). Cytotoxicity induced by low amounts of TNF-a could be enhanced in a dose-dependent manner by the addition of LPS during the TNF-a-target cell interaction (Fig. 1b). The effect was significant (P
