TABLE 2.

Differential effect of IL-10 or holotoxin pretreatment on LT-IIbB-induced cellular activation^a

Stimulus	Pretreatment	Cellular activation assay with:
		NF-κB p65 (OD450)	TNF-α (pg/ml)	IL-1β (pg/ml)
Medium	None	0.059 ± 0.032	9 ± 6	7 ± 4
LT-IIbB	None	1.132 ± 0.145	779 ± 113	312 ± 61
	IL-10	0.337 ± 0.054*	103 ± 24*	71 ± 33*
	LT-IIa	0.848 ± 0.088*	144 ± 42*	603 ± 87*
	Boiled LT-IIa	1.278 ± 0.132	723 ± 133	299 ± 81
	LT-IIb	0.778 ± 0.074*	101 ± 65*	584 ± 103*
	Boiled LT-IIb	1.084 ± 0.077	696 ± 157	287 ± 88
	CT	0.812 ± 0.123*	156 ± 72*	650 ± 99*
	Boiled CT	1.098 ± 0.101	687 ± 183	323 ± 45
Boiled LT-IIbB	None	0.102 ± 0.047	21 ± 10	18 ± 9
FimA (positive control)	None	1.798 ± 0.286	2,474 ± 465	343 ± 78
	IL-10	0.457 ± 0.098*	482 ± 76*	92 ± 27*
	LT-IIa	1.352 ± 0.167*	536 ± 97*	1,352 ± 282*
	Boiled LT-IIa	1.702 ± 0.208	2,547 ± 512	387 ± 78
	LT-IIb	1.211 ± 0.102*	687 ± 128*	1,408 ± 335*
	Boiled LT-IIb	1.694 ± 0.187	2,163 ± 334	362 ± 90
	CT	1.287 ± 0.129*	612 ± 110*	1,208 ± 198*
	Boiled CT	1.762 ± 0.225	2,348 ± 292	404 ± 98

^aTHP-1 cells were preincubated for 1 h with IL-10 (10 ng/ml) or holotoxins (either LT-IIa, LT-IIb, or CT; all at 2 μg/ml) prior to stimulation with LT-IIbB (2 μg/ml) or FimA (1 μg/ml), which was used as a positive control for NF-κB activation. Boiled LT-IIbB served as a negative control for stimulus, whereas boiled LT-IIb served as a negative control for pretreatment. After 90 min of stimulation, cellular extracts were analyzed for NF-κB p65 activation by using an ELISA-based kit (Active Motif). After 16 h, culture supernatants were analyzed by ELISA for TNF-α and IL-1β release. Data shown are means ± standard deviations, n = 3. *, Statistically significant (P < 0.05) differences between non-pretreated controls and groups pretreated with IL-10 or holotoxin. OD₄₅₀, optical density at 450 mm.