Intraperitoneal injection of lactoferrin ameliorates severe albumin extravasation and neutrophilia in LPS-induced inflammation in neonatal rats

Abstract

Lactoferrin (LF) plays various anti-inflammatory roles in inflammation experimentally induced by lipopolysaccharides (LPS). But the effects of LF on albumin extravasation and neutrophilia have not been elucidated. We aimed to study the effects of LF on albumin extravasation, neutrophilia and/or on other symptoms in inflammation caused by LPS in rats. Human lactoferrin (hLF) was injected (10 mg/100 mL in PBS) 18 h, or 15 min prior to, or 60 min after intraperitoneal injection of LPS in 13 days old Sprague Dawley rats. Prophylactic injection of hLF significantly ameliorated albumin extravasation in ascitic fluid at 5 h and neutrophilia in the blood at 24 h after LPS injection, but the after-injection of hLF did not. Interestingly, an injection of rat anti-TNFα IgG 15 min prior to LPS injection did not ameliorate albumin extravasation. Prophylactic injection of hLF significantly ameliorated other symptoms like mortality, and the decrease of phagocytotic activity of peritoneal polymorpho-nuclear leukocytes (PMNL), but did not ameliorate the decrease of platelets in the plasma. These findings suggest that hLF may be available as a medical treatment prior to surgery for prophylaxis of side effects like albumin extravasation or neutrophilia.Lactoferrin (LF) has diverse homeostatic activities in relation to anti-bacterial, anti-viral and anti-inflammatory immunological activities (12, 13). It is reported that prophylactic treatment by injection of human lactoferrin (hLF) reduced lipopolysaccharide (LPS)-induced TNFα production and the death rate in mice (15). In general, intraperitoneal injection of LPS (LPS-ip-injection), a cell wall component of gram-negative bacteria, induces severe septic like inflammation with production of TNFα and neutrophilia (6) in the plasma of experimental animals. In clinical patients suffering from spontaneous bacterial peritonitis, ascites are frequently found. More than 92% of these patients have monomicrobial infections with aerobic gram-negative bacilli that contain LPS in their cell walls (7). However, it is not known whether a prophylactic injection of hLF has homeostatic effects on albumin extravasation or neutrophilia after LPS-ip-injection. In the present study, we showed gross albumin accumulation in ascitic fluid 5 h after LPS injection in neonatal rats. Using this neonatal model, we examined whether LF had the ability to ameliorate gross albumin extravasations in the peritoneal cavity that followed LPS injection. We also examined changes in blood components like platelets, and what does relate to rat anti-TNFα IgG injected at the same site just prior to LPS injection. We also examined whether hLF had prophylactic effects on the decrease of the phagocytotic activity after LPS injection.

Lactoferrin-lipopolysaccharide (LPS) binding as key to antibacterial and antiendotoxic effects

“…However, the dual effect of Lf as a permeabilizing compound that enhances the antibiotic effect and as an LPS neutralizing agent may make it a candidate as an adjunct in combination with this antibiotic for the prevention or treatment of diarrhea [55] and post-surgery sepsis [72,86]. Increasing evidence supports the application of lactoferrin in combination with probiotics and antimicrobials in therapies focused on the prevention of neonatal sepsis, more than curative approaches as an adjunct to antibiotics, as the latter has been shown to lead to bacterial resistance [87].…”

“…Assays on animals show that Lf or derived peptides administered prophylactically confer protection against endotoxemia by attenuating the increase of serum TNF levels normally induced by LPS [56,61,72,75]. This protective effect of Lf administered prophylactically also has been described in the therapeutic treatment against toxic shock caused by LPS intravenously injected in mice [56].…”

“…Lf showed a neutralizing effect against the pro-inflammatory activity of LPS in one study, resulting in the attenuation of the LPS-induced increase in TNF levels, which in turn is linked to the attenuation of mortality [61,62,72] and an increase in resistance to hypothermia [62]. These in vivo studies are in agreement with in vitro assays, suggesting that the mechanism of this reduction in TNF levels [61,73] is related to the capacity of Lf and derived peptides to prevent LPS from binding with monocytes [62].…”

Immunoregulatory role of lactoferrin-lipopolysaccharide interactions

“…Indeed, several studies (Kruzel et al 2000;Lee et al 1998;Talukder and Harada 2007;Yajima et al 2005;Zagulski et al 1989;Zhang et al 1999) have demonstrated that hLf, bLf and Lfcin-derived peptides administration protects animals against sublethal doses of LPS. Interestingly, the optimal protection against induced septicaemia required a 12-24 h pre-injection of hLf and bLf, suggesting that this protein may act by other mechanisms than simple LPS scavenging (Zagulski et al 1989).…”

Ascites’ neutrophil function is significantly impaired in patients with decompensated cirrhosis but can be restored by autologous plasma incubation

“…Appropriate neutrophil activation is closely regulated by a number of stimulatory, but also inhibitory, factors such as immunoglobulins, complement factors, especially C3b, cytokines (IFN-γ, IL-8, GM-CSF and TNFα) and endotoxin levels2627. At the time of data acquisition and the ex vivo experiments, we were not able to directly measure these factors.…”