Inter-alpha inhibitor protein administration improves survival from neonatal sepsis in mice

Abstract

Inter-alpha inhibitor proteins (IaIp) are serine proteases inhibitors that modulate endogenous protease activity and have been shown to improve survival in adult models of sepsis. We evaluated the effect of IaIp on survival and systemic responses to sepsis in neonatal mice. Sepsis was induced in 2-d-old mice with lipopolysaccharide (LPS), Escherichia coli, and group B Streptococci. Sepsis was associated with 75% mortality. IaIp, given by i.p. administration at doses between 15 and 45 mg/kg from 1 to 6 h after the onset of sepsis, improved survival to approximately 90% (p = 0.0159) in both LPS-induced sepsis and with live bacterial infections. The greatest effect was on reversal of hemorrhagic pneumonitis. The effects were dose and time dependent. Systemic cytokine profile and tissue histology were examined. Survival was compared in IL-10 knock out animals. Systemic cytokine levels including TNF-[alpha] and IL-10 were increased after induction of sepsis and modulated significantly after IaIp administration. Because the effect of IaIp was still demonstrable in IL-10 deficient mice, we conclude the beneficial effects of IaIp is because of suppression of proinflammatory cytokines such as TNF-[alpha] rather than augmentation of IL-10. IaIp may offer significant benefits as a therapeutic

Figure 1

The effect of IaIp on mortality in 2 day old mice. (A) Survival following 5 (blue, n=5), 10 (green, n=4) or 25 (red, n=4) micrograms of LPS per pup. (B). Dose dependent effects of 15 (red, n=13), 30 (blue, n=18) or 45 (green, n=17) mg/kg IaIp intraperitoneally one hour later following LPS as compared to HSA controls at the same concentrations:15 (red-dotted; n=12), 30 (blue-dotted, n=19) and 45 (green-dotted; n=9) mg/kg. Both 30 and 45 mg/kg were significantly different from the 15 mg/kg group, p<0.004. (C) Effect of delayed administration. Animals received 30 mg/kg IaIp at one (blue, n=23), three (green, n=18) or six (red, n=16) hours after LPS. IaIp resulted in significantly better survival than each respective control, p<0.001. HSA controls at the same time points: 1 hour (blue-dotted, n=19), 3 hours (green-dotted, n=19), and 6 hours (red-dotted, n=13)

Figure 2

The effect of IaIp administration on survival following subcutaneous administration of live bacteria in 2-day-old BALB/c mice. (A) Survival following subcutaneous injection of 10⁴ (blue), 10⁵ (green),10⁶ (red), 10⁷ (black) CFU E. coli per pup. (B). Dose dependent effect of IaIp on survival following E. coli administration. Control animals (red, n=28) received intraperitoneal HSA, and IaIp treated animals (blue, n=26) received 30 mg/kg IaIp intraperitoneally one hour later. IaIp treated animals had significantly better survival than either control group, p<0.05. (C) The effect of IaIp administration on survival following subcutaneous administration of group B streptococci. IaIp treated animals (blue, n=17) received 30 mg/kg IaIp intraperitoneally one hour later. IaIp treated animals had significantly better survival than the HSA control group (red, n=16).

Figure 3

The effect of IaIp on lung histology following LPS. Animals were given LPS followed by HSA (A) or 30 mg/kg IaIp (B). Hematoxylin and eosin stain. Magnification 40X.

Figure 4

Western blot (A) showing changes in endogenous, mouse IaIp (gray) and the Pre-alpha Inhibitor (dark gray) following administration of LPS. (B) The differences between each time point are all significant, p<0.05.

Figure 5

The effect of LPS on systemic cytokine levels. Animals received LPS, HSA and IaIp as described in Methods. Cytokines were measured by radioimmunoassay 3, 6, 12, 24, 48 and 72 hours later. Results were compared by Two-way ANOVA. A) TNF-alpha, p=0.0313; B) IL-10, p=0.0377; C) IL-12p70, p=0.0024; D) IL-6, p=0.649; E) IFN-gamma, p=0.141; F) MCP-1, p=0.4948.

Figure 6

Survival following LPS administration in IL-10^−/− mice. Animals were treated with LPS, either 0.25, 2.5 or 10 μg per animal, as described in Methods. Animals were treated with IaIp (IaIp + LPS 0.25 (blue, n=8); IaIp + LPS 2.5 (green, n=5); IaIp + LPS 10 (red, n=10)) or HSA (HSA + LPS 0.25 (blue-dotted, n=8); HSA + LPS 2.5 (green-dotted, n=5); HSA + LPS 10 (red-dotted, n=10)) as described in Methods 1 hour following LPS. Animals were examined every 4 hours for survival. All IaIp treated animals had significantly better survival than each of their respective control groups, p = 0.006.