Evaluation of the in vitro pyrogen test system based on proinflammatory cytokine release from human monocytes: comparison with a human whole blood culture test system and with the rabbit pyrogen test

Abstract

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I x C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I. C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.

FIG. 1.

Induction of release of proinflammatory cytokines from MM6-CA8 cells by various pyrogens. The cells were primed with 10 ng of calcitriol/ml. After incubation for 72 h at 37°C with calcitriol, the cells were plated in a 24-well plate at 10⁶ cells/0.9 ml/well, and 0.1 ml of pyrogen at various concentrations was added to each well. After incubation for 17 h at 37°C, the release of IL-6, TNF-α, or IL-1 in culture supernatants was evaluated by an ELISA. The amounts of cytokines released from MM6-CA8 cells cultured in a medium containing 0 μg of pyrogen/ml were as follows: IL-6, 2.1 ± 0.7 pg/ml; TNF-α, 0.6 ± 0.3 pg/ml; and IL-1, 2.5 ± 0.6 pg/ml. Data are expressed as the mean and SEM (n = 6 to 12).

FIG. 2.

Determination of the detection limit for endotoxin in the MM6-CA8 culture test system. The cells were incubated for 17 h at 37°C with endotoxin after being primed for 72 h with 10 ng/ml of calcitriol. The release of IL-6 in the culture supernatants was evaluated by an ELISA. The detection limit for endotoxin was defined as the minimum concentration inducing a significant amount of cytokine production (greater than 2 standard deviations above that obtained with 0 ng/ml). The arrow indicates the detection limit for IL-6. Data are expressed as the mean and SEM (n = 6).

FIG. 3.

Induction of the release of proinflammatory cytokines from human whole blood cells by various pyrogens. Freshly drawn heparinized human peripheral blood (225 μl) was incubated for 4 h at 37°C with 25 μl of saline containing various concentrations of pyrogens. After incubation, the blood samples were diluted with 750 μl of saline and centrifuged for 5 min at 15,000 × g. The release of IL-6, TNF-α, or IL-1 in the supernatants was evaluated by an ELISA. The amounts of cytokines released from human whole blood cells cultured in blood containing 0 μg of pyrogen/ml were as follows: IL-6, 1.0 ± 0.5 pg/ml; TNF-α, 1.8 ± 0.2 pg/ml; and IL-1, 2.1 ± 1.3 pg/ml. Data are expressed as the mean and SEM for three donors.

FIG. 4.

Correlation between the detection limit in the MM6-CA8 culture test system and that in the human whole blood culture test system. The detection limit for each pyrogen was defined as the minimum concentration of that pyrogen inducing a significant amount of cytokine production (greater than 2 standard deviations above that obtained with 0 ng of pyrogen/ml). The correlation is expressed for every cytokine; r, linear correlation coefficient.

FIG. 5.

Pyrogenicities of various pyrogens in rabbits. Rabbits were injected intravenously with the indicated doses of pyrogens. Rectal temperatures were measured for 5 h after injection. ΔT, change in temperature. Data are expressed as the mean and SEM (n = 6 to 12).

FIG. 5.

Pyrogenicities of various pyrogens in rabbits. Rabbits were injected intravenously with the indicated doses of pyrogens. Rectal temperatures were measured for 5 h after injection. ΔT, change in temperature. Data are expressed as the mean and SEM (n = 6 to 12).

FIG. 6.

Determination of the MPD of each pyrogen in the rabbit pyrogen test. ΔT max, maximum change in body temperature after pyrogen injection. Data are expressed as the mean and SEM (n = 6 to 12). Symbols: ○, endotoxin; •, PG; ▵, SAC; ▴, poly(I · C).

FIG. 7.

Relative potencies of pyrogens determined by the MM6-CA8 culture system, the human whole blood (HWB) culture system, and the rabbit pyrogen test. The potency of each pyrogen is expressed relative to that of endotoxin.