Haloperidol Suppresses NF-kappaB to Inhibit Lipopolysaccharide-Induced Pro-Inflammatory Response in RAW 264 Cells

Abstract

BACKGROUND Haloperidol, a tranquilizing agent, is administered both to treat symptoms of psychotic disorders and to sedate agitated and delirious patients. Notably, haloperidol has been suggested to inhibit the immune response through unknown mechanisms. We hypothesized that the sedative modulates the immune response via NF-κB. MATERIAL AND METHODS Using flow cytometry, we analyzed the effects of haloperidol on expression CD80 and CD86 in RAW 264 cells and in primary macrophages derived from bone marrow. Secretion of interleukin (IL)-1β, IL-6, and IL-12 p40 was measured by enzyme-linked immunosorbent assay. In addition, NF-κB activation was evaluated using a reporter assay based on secretory embryonic alkaline phosphatase. Finally, synthetic antagonists were used to identify the dopamine receptor that mediates the effects of haloperidol. RESULTS Haloperidol inhibited NF-κB activation, and thereby suppressed expression of CD80, as well as secretion of IL-1β, IL-6, and IL-12 p40. CD80 and IL-6 levels were similarly attenuated by a D2-like receptor antagonist, but not by a D1-like receptor antagonist. CONCLUSIONS The data strongly suggest that haloperidol inhibits the immune response by suppressing NF-kB signaling via the dopamine D2 receptor.

Figure 1

(A) Effect of haloperidol on expression of the co-stimulatory molecules CD80 and CD86 in LPS-stimulated RAW 264 cells. Cells were treated with or without haloperidol on day 2 of culture, stimulated with 300 ng ml⁻¹ LPS on day 3, and analyzed by flow cytometry on day 4. CD80 and CD86 expression was measured in CD11b-gated cells. Data are mean ±SD (n=6). ** P<0.01. (B) Haloperidol suppresses CD80 expression in a dose-dependent fashion. * P<0.05; ** P<0.01. MFI, mean fluorescence intensity.

Figure 2

Haloperidol suppresses LPS-induced expression of CD80 and CD86 in primary macrophages derived from bone marrow. Cells were treated with or without haloperidol on day 5, and with 100 ng mL⁻¹ LPS on day 6. Expression of CD80 and CD86 was measured by flow cytometry in CD11b-gated cells 24 h after LPS treatment. Data are mean ±SD (n=6). ** P<0.01. MFI, mean fluorescence intensity.

Figure 3

Secretion of IL-1β, IL-6, and IL-12 p40 in RAW 264 cells treated with or without haloperidol on day 2 and exposed to 300 ng mL⁻¹ LPS on day 3. The culture supernatant was collected 24 h after LPS induction, and levels of IL-1β, IL-6, and IL-12 p40 were measured by enzyme-linked immunosorbent assay. Data are mean ±SD (n=6). ** P<0.01.

Figure 4

NF-κB activation in RAW-Blue cells cultured with or without haloperidol at 1×10⁵ cells well⁻¹, and stimulated for 24 h with 300 ng ml⁻¹ LPS. Expression of the reporter enzyme secretory embryonic alkaline phosphatase was measured by colorimetry at 655 nm. Data are mean ±SD (n=6). * P<0.05; ** P<0.01.

Figure 5

Effect of dopamine D1-like and D2-like receptor antagonists on (A) CD80 expression and (B) IL-6 secretion in RAW 264 cells exposed to LPS. Cells were treated on day 2 with haloperidol; SCH23390, a D1-like receptor antagonist; and L750.667, a D2-like receptor antagonist. On day 3, RAW 267 cells were stimulated with 300 ng mL⁻¹ LPS for another 24 h. Flow cytometry was used to measure CD80 expression in CD11b-gated cells, while enzyme-linked immunosorbent assay was used to measure IL-6 secreted into the culture supernatant. Cells that were treated with LPS, but not with drugs, were used as control. Data are mean ±SD (n=6). ** P<0.01.