Comparative study of the effects of acepromazine and structurally related compounds on the TNF-α release by monocytes stimulated by Chlamydia pneumoniae

Acepromazine (ACP), a member of the phenothiazine family, has antioxidant properties and interacts with reactive oxygen species produced by stimulated neutrophils ( Serteyn et al. 1999 ). We found that ACP reduced the differentiation of monocytes induced by an overnight incubation with a crude Chlamydia pneumoniae extract ( Serteyn et al. 2001 ). The same model was used to test the effects of phenothiazines on the TNF‐α release by activated monocytes. A crude Chlamydia pneumoniae extract was obtained by mechanical disruption and centrifugation (1 minute, 1500 r.p.m.) of 78 hours infected McCoy cells. Monocytes (THP1 cell line; 2 × 10⁶ cells by assay) were incubated overnight with 30 µL of Chlamydia pneumoniae crude extract (equivalent to an endotoxin charge of 3.5 pg) in the presence or absence of phenothiazines (from 10^?6 to 10^?4 M) ( Mouithys‐Mickalad et al. 2001 ). For estimation of TNF‐α release, the supernatants were collected, centrifuged (to eliminate the undifferentiated monocytes) and used for TNF‐α measurements (n = 6) (Quantikine HS human TNF‐α, R&D Systems, UK). Acepromazine was compared to other phenothiazines (chlorpromazine, trifluoperazine) or to structural analogues of phenothiazines (phenoxazine, thioxanthen‐9‐one and methylene blue). For each assay, cytotoxicity was evaluated by microscopic examination and blue trypan exclusion method. Mean values of TNF‐α were compared by a Student t‐test (p < 0.05). TNF‐α release by Chlamydia‐treated THP1 was significantly decreased by ACP in a dose‐dependent manner, 378 ± 30, 209 ± 38 and 189 ± 35 ng mL^?1 for 10^?6, 10^?5 and 10^?4 M compared to the control values 385 ± 9 ng mL^?1. Similar inhibitions of TNF‐α release were obtained with trifluoperazine (313 ± 25 and 265 ± 14 ng mL^?1 at 10^?6 and 10^?5 M) and chlorpromazine (323 ± 29 and 227 ± 13 ng mL^?1 at 10^?6 and 10^?5 M), but at 10^?4 M, these two drugs were cytotoxic. The other structurally parent compounds increased significantly the TNF‐α production: 630 ± 46 and 468 ± 60 ng mL^?1 for thioxanthen‐9‐one and 547 ± 17 and 331 ± 111 ng mL^?1 for methylene blue at 10^?5 and 10^?6 (M). At 10^?4 M, the two compounds were cytotoxic. Phenoxazine increased the TNF‐α production, slightly at 10^?6 and 10^?5 M (444 ± 39 and 424 ± 16 ng mL^?1, respectively) and significantly at 10^?4 M (959 ± 30 ng mL^?1). Further studies are needed to verify if the inhibition of TNF‐α release by some phenothiazines could be linked to a reduction of the signal transduction, especially the NF‐κB pathway. These results could be interesting for the anaesthesia or treatment of animals suffering from a systemic inflammatory reaction.