Involvement of tumour necrosis factor-alpha in Clostridium perfringens beta-toxin-induced plasma extravasation in mice

Abstract

Background and purpose: Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK(1) receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation.

Experimental approach: Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA.

Key results: Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-alpha and interleukin (IL)-1beta levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-alpha and IL-1beta. The plasma extravasation and the release of TNF-alpha induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-alpha. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-alpha, but not anti-IL-1beta. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody.

Conclusions and implications: These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-alpha via the mechanism involving tachykinin NK(1) receptors, but not via TLR4.

Figure 1

β-Toxin-induced plasma extravasation and release of cytokines in the dorsal skin of mice. ¹²⁵I-labelled BSA (0.1 ml of 2.5% solution) was injected into the tail vein. After 5 min, β-toxin (25–100 ng) was injected i.d. (0.05 ml per site). Plasma extravasation was measured 60 min after the injection of β-toxin (a). TNF-α (b) and IL-1β (c) levels were determined in skin samples collected 1 h after the injection of β-toxin. Values are mean±s.e.mean, n=6. BSA, bovine serum albumin; i.d., intradermally; IL-1β, interleukin-1β; TNF-α, tumour necrosis factor-α.

Figure 2

Effect of anti-β-toxin antibody on β-toxin-induced plasma extravasation and release of cytokines in the dorsal skin of mice. Anti-β-toxin antibody (1 μg per site) and control mouse IgG (1 μg per site) were administered i.d. 5 min before the experiment. β-Toxin (50 ng per site) or saline (0.05 ml per site) was injected i.d. Plasma extravasation and release of cytokines were measured 60 min after the injection of β-toxin or saline. Values are mean±s.e.mean, n=6. ^*P<0.01, compared with β-toxin with control mouse IgG. I.d., intradermally.

Figure 3

Effect of SR140333 on β-toxin-induced release of cytokines in the dorsal skin of mice. SR140333 (1 or 5 nmoles per site) or vehicle was given i.d. 5 min before the intradermal injection of β-toxin (50 ng per site) or saline (0.05 ml per site). TNF-α (a) and IL-1β (b) levels were determined in skin samples collected 1 h after the injection of β-toxin or saline. Values are mean±s.e.mean, n=6. ^*P<0.05 and ^**P<0.01, compared with β-toxin with vehicle. I.d., intradermally; IL-1β, interleukin-1β; TNF-α, tumour necrosis factor-α.

Figure 4

Effect of capsaicin on β-toxin-induced release of cytokines in the dorsal skin of mice. After the dorsal skin was shaved, capsaicin solution or the diluent alone was painted for 4 days as described in Methods. TNF-α (a) and IL-1β (b) levels were determined in skin samples collected 1 h after the injection of β-toxin (50 ng per site) or saline (0.05 ml per site). Values are mean±s.e.mean, n=6. ^*P<0.01 and ^**P<0.05, compared with β-toxin with vehicle. IL-1β, interleukin-1β; TNF-α, tumour necrosis factor-α.

Figure 5

Effect of anti-TNF-α, anti-IL-1α and anti-IL-1β on β-toxin-induced plasma extravasation in the dorsal skin of mice. Anti-TNF-α antibody (1 μg per site), anti-IL-1α antibody (1 μg per site), anti-IL-1β antibody (1 μg per site) antisera and control goat IgG (1 μg per site) were administered i.d. 12 h before the experiment. Mice received β-toxin (50 ng per site) or saline (0.05 ml per site), and plasma extravasation was measured 60 min after the injection of β-toxin. Values are mean±s.e.mean, n=6. ^*P<0.01, compared with β-toxin with control goat IgG. I.d., intradermally; IL-1α, interleukin-1α; IL-1β, interleukin-1β; TNF-α, tumour necrosis factor-α.

Figure 6

β-Toxin-induced plasma extravasation in the dorsal skin of C3H/HeN and C3H/HeJ mice. ¹²⁵I-labelled BSA (0.1 ml of 2.5% solution) was injected intravenously into the tail. After 5 min, β-toxin (25–100 ng) was injected i.d. (0.05 ml per site). Plasma extravasation was measured 60 min after the injection of β-toxin. Values are mean±s.e.mean, n=6. BSA, bovine serum albumin; i.d., intradermally.

Figure 7

Effect of anti-TLR4/MD-2 antibody on β-toxin-induced plasma extravasation in the dorsal skin of C3H/HeN mice. Anti-TLR4/MD-2 antibody (1 μg per site) and control rat IgG (1 μg per site) were administered i.d. 12 h before the experiment. Mice received β-toxin (50 ng per site) or saline (0.05 ml per site), and plasma extravasation was measured 60 min after the injection. Values are mean±s.e.mean, n=6. I.d., intradermally; TLR4, toll-like receptor 4.