Adrenaline suppression of the macrophage nitric oxide response to lipopolysaccharide is associated with differential regulation of tumour necrosis factor-alpha and interleukin-10

Abstract

Adrenaline is a catecholamine hormone secreted by the adrenal medulla in response to acute stress. Previous studies have shown that adrenaline suppresses the nitric oxide (NO) response of murine macrophages (M phi s) stimulated in vitro with lipopolysaccharide (LPS). We have now extended these studies to examine the effects of adrenaline on the production of tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10). Our results showed that NO, TNF-alpha and IL-10 were concurrently produced following in vitro LPS (10 micrograms/ml) stimulation of murine peritoneal M phi s. Adrenaline suppressed both NO and TNF-alpha with concomitant up-regulation of the IL-10 response above that seen with LPS alone. In this in vitro model of LPS stimulation we demonstrated that TNF-alpha was required for NO production, as the TNF-alpha neutralizing monoclonal antibody, TN3.19.12, abolished the response; in contrast, IL-10 suppressed NO. In order to determine any functional consequence of adrenaline-mediated IL-10 augmentation on NO production, M phi s were stimulated with LPS and specific neutralizing anti-IL-10 antibodies were added to the cultures. The LPS NO response was suppressed to 43% of the control value by adrenaline (10(-8) M) and an irrelevant control antibody had no effect on the adrenaline-mediated inhibition of NO, but anti-IL-10 treatment restored the NO response to levels similar to those observed with LPS alone. Furthermore, we demonstrated that exogenous TNF-alpha, at a dose range of 1.9-50 ng per ml, also restored the nitrite response to LPS in the presence of adrenaline. Together, the observations that neutralization of IL-10 and addition of TNF-alpha abrogate adrenaline's inhibition of NO, suggest that this hormone suppresses NO partly through up-regulation of IL-10 which, in turn, may suppress TNF-alpha that is required for NO production. Finally, we also observed that the M phi-activating cytokine, interferon-gamma (IFN-gamma), attenuated the inhibitory effect of adrenaline on the LPS NO response.

Figure 1

Effects of adrenaline on macrophage (Mφ) nitrite, tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) responses to lipopolysaccharide (LPS) stimulation. Murine peritoneal Mφs were cultured in medium alone, with LPS (10 µg/ml), or with LPS in the presence or absence of adrenaline. Additional control wells were cultured with adrenaline alone. After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction, and TNF-α and IL-10 by specific enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean±SEM and are representative of three similar experiments.

Figure 2

Effects of propranolol on adrenaline-mediated modulation of macrophage (Mφ) nitrite, tumour necrosis factor-α (TNF-α) and interleukin-10 (IL-10) responses to lipopolysaccharide (LPS). Murine peritoneal Mφs were cultured in medium alone, with LPS (10 µg/ml), or with LPS plus adrenaline (10⁻⁸ m) in the presence or absence of the β-receptor antagonist propranolol. Additional control wells were cultured with either adrenaline or propranolol alone. After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction, and TNF-α and IL-10 by specific enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean±SEM and are representative of three similar experiments.

Figure 3

Effect of tumour necrosis factor-α (TNF-α) neutralization on the macrophage (Mφ) nitrite response to lipopolysaccharide (LPS). Murine peritoneal Mφs were cultured in medium alone, or with LPS (10 µg/ml) in the presence or absence of the neutralizing anti-TNF-α antibody TN3.19.12. After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction. Results are expressed as mean±SEM and are representative of two similar experiments. *P < 0·01, TN3.19.12 treatment compared to the LPS-alone control.

Figure 4

Effects of interleukin-10 (IL-10) on the macrophage (Mφ) nitrite response to lipopolysaccharide (LPS). Murine peritoneal Mφs were cultured in medium alone, with LPS (10 µg/ml), or with LPS in the presence of either IL-10 (a), or the anti-IL-10 neutralizing antibody SXC-1 and its irrelevant isotype-matched control antibody, J51D (b). After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction. Results are expressed as mean±SEM and are representative of two similar experiments. *P < 0·01 treatment groups compared to the LPS-alone control.

Figure 5

Interleukin-10 (IL-10) neutralization restores the nitrite response in the presence of adrenaline. Murine peritoneal macrophages (Mφs) (10⁵) were cultured in medium alone, with lipopolysaccharide (LPS) alone (10 µg/ml), or with LPS plus adrenaline (10⁻⁸ m) in the presence or absence of either the anti-IL-10 neutralizing antibody, SXC-1, or its irrelevant isotype-matched control antibody, J51D. After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction. Results are expressed as mean±SEM and are representative of two similar experiments. *P < 0·01, SXC-1 treatment compared to LPS plus adrenaline.

Figure 6

Exogenous tumour necrosis factor-α (TNF-α) restores the nitrite response in the presence of adrenaline. Murine peritoneal macrophages (Mφs) (10⁵) were cultured in medium alone, with lipopoysaccharide (LPS) alone (10 µg/ml), or with LPS plus adrenaline (10⁻⁸ m) in the presence or absence of recombinant murine TNF-α. After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction. Results are expressed as mean+SEM and are representative of three similar experiments. *P < 0·01, TNF-α treatment compared to LPS plus adrenaline.

Figure 7

Interferon-γ (IFN-γ) abolishes the suppressive effect of adrenaline on nitrite. Murine peritoneal macrophages (Mφs) (10⁵) were cultured in medium alone, with lipopolysaccharide (LPS) alone (10 µg/ml), with LPS plus IFN-γ (at 10 U/ml or 100 U/ml), or with LPS plus adrenaline (10⁻⁷ m) in the presence or absence of recombinant murine IFN-γ. After 48 hr, culture supernatants were harvested and nitrite levels determined by the Griess reaction. Results are expressed as mean+SEM and are representative of three similar experiments. *P < 0·01, IFN-γ treatment compared to LPS plus adrenaline.