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Activation of the immunoinflammatory system during infections with gram-negative bacteria is a common cause of septic shock, a life-threatening condition characterized by functional derangements in many organs (4, 10). Gram-negative bacteria provoke septic shock by releasing their cell wall component lipopolysaccharide (LPS) into the circulation (4). Here, LPS directly activates monocytes and neutrophils and indirectly activates T-lymphocytes, causing release of both type 1 cytokines, such as interleukin-1 (IL-1), IL-12, tumor necrosis factor alpha (TNF-), and gamma interferon (IFN-), and type 2 cytokines, such as IL-6 and IL-10 (5).

It is generally accepted that type 1 cytokines play a pivotal role in the pathogenesis of endotoxic shock conditions through their proinflammatory and vasoactive properties (5). However, the production and the action of type 1 cytokines may be antagonized by type 2 anti-inflammatory cytokines, and the balance between these two cytokine subsets may therefore influence the host response to endotoxemia (22). Thus, LPS-induced lethality in mice is prevented by blockade of endogenous IL-1, IL-12, TNF- or IFN- with specific antagonists or by administration of type 2 cytokines, such as IL-4, IL-10, or IL-13 (1-3, 11, 12, 14, 16-19). Pharmacological compounds capable of inhibiting the production or action of type 1 cytokines while at the same time up-regulating the production of type 2 cytokines may therefore be suitable candidates for the prevention or treatment of endotoxemia.

Multifunctional protein 14 (MFP-14) is a ubiquitous 14-kDa protein consisting of 137 amino acids, present in all animal species and maximally expressed in the liver. This protein has pleiotropic effects; it can act as a tumor antigen, a selective protein synthesis inhibitor, or a specific calpain activator (6, 13, 15, 20, 23). In addition, we have recently demonstrated that MFP-14 modulates concanavalin A-induced ex vivo cytokine production in BALB/c mice towards a type 2 response, as it suppresses secretion of IFN-, TNF-, and IL-2 while increasing that of IL-4 (21). These properties of MFP-14 may be related to its capacity to ameliorate the course of type 1 cytokine-dependent immunoinflammatory diseases such as adjuvant-induced arthritis in rats and type 1 diabetes mellitus in NOD mice (21).

This immunopharmacological profile of MFP-14 prompted us to study its effects in murine endotoxemia. The data show that MFP-14 successfully counteracted LPS-induced lethality in mice regardless of whether it was given prior to or 1 h after endotoxin challenge. MFP-14 also counteracted the effect of LPS on the blood levels of TNF- and IFN- in the blood.

Recombinant MFP-14 (7) was kindly provided by Sicor SpA (Rho, Italy). MFP-14 was checked for endotoxin contamination by the Limulus amebocyte lysate assay, given that 1 U/ml is equal to 0.1 ng of U.S. Pharmacopeia standard Escherichia coli/ml. E. coli endotoxin (8) MFP-14 had an endotoxin concentration of <0.005 IU/mg of protein. LPS from E. coli (serotype O127:B8) and phosphate-buffered saline (PBS), pH 7.2, were purchased from Sigma Chemicals (St. Louis, Mo.).

Soluble TNF receptor type I (sTNF-RI) was previously described. It consists of the two extracellular domains of the p55 TNF-receptor covalently linked to polyethylene glycol. sTNF-RI blocks both soluble and cell-associated TNF-mediated events and ameliorates the course of murine immunoinflammatory arthritis and pneumonitis (25). The anti-mouse IFN- monoclonal antibody (MAb) AN-18 was produced as described elsewhere (26). MAb AN-18 has been shown to block the activity of endogenous IFN- in mice (26).

Six- to 8-week-old female BALB/c mice were purchased from Charles River (Calco, Italy). They were kept under standard laboratory conditions with free access to food and water and were allowed to adapt to their environment for at least 1 week before the experiments began. All animal procedures were carried out in accordance with the institutional guidelines, which are in compliance with national laws for the care and use of laboratory animals. To induce lethal endotoxemia, the mice were injected intraperitoneally (i.p.) with 750 µg of LPS diluted in 1 ml of PBS. This dose of LPS was selected on the basis of previous experiments showing its capacity to induce lethality within 3 days in 75 to 100% of the mice.

The effects of MFP-14 on the development of LPS-induced lethality were evaluated both under a prophylactic and under a therapeutic regimen. For prophylaxis, the mice received i.p. injections with either 2.5 or 25 µg of MFP-14, diluted in 0.5 ml of PBS, 24 and 1 h prior to LPS challenge. Control mice were treated either with PBS alone or with heat-inactivated MFP-14 (hi-MFP14; boiled for 45 min) under similar conditions or left untreated. The therapeutic capacity was tested by treating the mice with a single i.p. injection of 25 µg of MFP-14 given 1 h after LPS. Lethality was assessed at 1-day intervals for 3 consecutive days.

Heparinized blood was obtained by cardiac puncture under ether anesthesia. Blood samples were obtained prior to, and 2 and 8 h after, i.p. injections of 100 µg of LPS. This dose of LPS was chosen on the basis of previous studies showing its ability to induce abundant release of cytokines in the blood without killing the mice (9). Plasma TNF-, IFN-, and IL-10 levels were measured by specific solid-phase enzyme-linked immunosorbent assays purchased from Endogen (Cambridge, Mass.). Samples were run in duplicate and according to the manufacturer's instructions. The lower limits of sensitivity of the assays were 10 pg/ml for TNF- and IFN- and 5 pg/ml for IL-10. The intra- and interassay coefficients of variations were within 10 and 20%, respectively.

Lethalities were compared using the log rank (Mantel Cox) test. The cytokine levels were not normally distributed, and these are therefore shown as medians and quartiles. Comparisons were evaluated by two-tailed Wilcoxon's signed rank test. P values of <0.05 were considered significant.

As expected, most of the control mice died within 3 days of LPS injection (Fig. 1). The group lethalities were comparable regardless of whether they were left untreated, treated with PBS, or treated with or hi-MFP-14. The cumulative incidences of lethality were 80% in untreated animals, 72% in PBS-treated animals, and 73% in hi-MFP-14-treated animals. In contrast, prophylactic treatment with MFP-14 greatly improved the survival of the mice, with only 20% dying during the observation period (Fig. 1). MFP-14 did not merely delay the lethal action of LPS, as none of the remaining mice from the controls or from the MFP-14-treated group died during a follow-up period of 1 week. The effect of MFP-14 was seen only with the high dose of the protein, as mice pretreated with 2.5 µg two times prior to LPS challenge showed a rate and kinetics of lethality indistinguishable from that of controls with a cumulative lethality of 89% (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Effect of prophylactic MFP-14 treatment on LPS-induced lethality in mice. Mice were either left untreated () (n = 25) or treated with PBS () (n = 25), hi-MFP-14 () (n = 15), or MFP-14 given 24 and 1 h prior to LPS challenge at either 2.5 µg per dose () (n = 18) or 25 µg per dose () (n = 20). The protective effect of MFP-14 at 25 µg per dose was significant compared with all control groups: P < 0.0001 for each comparison (Mantel-Cox log rank test). Data from three independent experiments were merged, since there was less than 10% variation between experiments.

To evaluate whether MFP-14 also had a therapeutic capacity, experiments were carried out where the protein was first administered to the mice 1 h after they had been injected with LPS. As shown in Fig. 2, the protection afforded by therapeutic MFP-14 also diminished LPS-induced lethality. The cumulative incidence of mortality was 84% in PBS-treated controls and 40% in the MFP-14-treated mice. Again, none of the mice died during the 1-week follow-up period. However, delaying MFP-14 administration until 3 h after LPS injection was no longer effective, the rate and kinetics of mortality of the so-treated mice being comparable to those of the controls (data not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Effect of "early therapeutic" MFP-14 treatment on LPS-induced lethality in mice. Mice were treated with PBS (open circles) (n = 25) or 25 µg of MFP-14 given 1 h after LPS challenge (solid circles) (n = 20). Data from three independent experiments were merged, since there was less than 10% variation between experiments. The protective effect of MFP-14 was significant: P < 0.05 (Mantel-Cox log rank test).

The effects of MFP-14 on the cytokine release pattern induced by LPS were studied using two groups of mice treated with either 25 µg of MFP-14 or PBS 24 h and 1 h prior to LPS challenge. Animals in both groups were killed before LPS injection (time zero) and 2 and 8 h thereafter. As shown in Fig. 3, the levels of TNF-, IFN-, and IL-10 in plasma, all below the limit of sensitivity of the assays at time zero, increased considerably 2 and/or 8 h after LPS in mice given PBS alone. Mice treated with MFP-14 had significantly lower levels of TNF- and IFN- 2 and 8 h after LPS, respectively. In contrast, MFP-14 had no effect on the plasma levels of IL-10.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Effects of MFP-14 prophylaxis on levels of LPS-induced TNF-, IFN-, and IL-10 in blood. Blood samples were obtained from mice treated with PBS (open circles) or MFP-14 (solid circles) prior to (time zero), and 2 and 8 h after, i.p. injections of 100 µg of LPS. Ten mice were studied at each time point. Data from two independent experiments were merged, since there was less than 10% variation between experiments. *, P < 0.002 (Wilcoxon's two-tailed signed rank test).

To ascertain whether the therapeutic efficacy of MFP-14 could be related to its inhibitory effects on TNF- and IFN- synthesis, parallel experiments were carried out out where BALB/c mice were pretreated with sTNF-RI (0.5 mg/kg of body weight) and MAb AN-18 (0.5 mg/mouse), either alone or in combination, 1 h after LPS challenge. Control mice received irrelevant rat immunoglobulin and heat-inactivated (by boiling for 45 min) sTNF-RI. Similar doses of sTNF-RI and MAb AN-18 were prevously found to be effective in other murine models of immunoinflammatory diseases (25, 26). The cumulative rate of mortality at the end of the study (72 h after LPS) was comparable between the different groups, 12 of 15 (80%) in the control mice, 11 of 15 (73.3%) in the mice pretreated with sTN-RI, 12 of 15 (80%) in those pretreated with MAb AN-18, and 13 of 15 (86.7%) in those treated with sTNF-RI plus MAb AN-18. The kinetics of mortality were also indistinguishable among the different groups (data not shown).

Our data show for the first time that MFP-14 when given both as a prophylactic and as an early therapeutic regimen successfully counteracts the lethal effect of LPS in BALB/c mice. This effect was associated with a significant modification of the cytokine response in that mice receiving MFP-14 had lower levels of TNF- and IFN- in plasma at 2 and 8 hours, respectively, after LPS administration. Because TNF- and IFN- are essential mediators of lethality in murine endotoxemia (2, 11), it is possible that the beneficial effects of MFP-14 were causally related to its suppressive effects on the synthesis or release of these two type 1 proinflammatory cytokines. However, while MFP-14 also efficiently prevented LPS-induced lethality when administered 1 h after LPS challenge, blockade of endogenous TNF- and/or IFN- with a soluble receptor or MAb failed to do so. Because, in agreement with literature data (2, 11), both sTNF-RI and MAb AN-18, either alone or in combination, reduced the rate of LPS-induced lethality when injected into mice prophylactically from 24 to 48 h prior to LPS injection (data not shown), the above may indicate that the therapeutic efficacy of MFP-14 depends on immunopharmacological mechanisms other than the inhibition of TNF- and IFN- synthesis. For example, MFP-14 could have down-regulated other type 1 cytokines, such as IL-1 and IL-12 (1, 14, 19), and/or up-regulated the production of type 2 anti-inflammatory cytokines, apart from IL-10, which was not affected in our study (3, 16, 17, 21). Studies to test these hypotheses are in progress.

In conclusion, MFP-14 prevented LPS-induced lethality in mice when given either before or, less effectively, early after LPS administration. Because experimental endotoxemia may not entirely mirror the pathogenic pathways operating during infections with live bacteria, caution should be exercised in the application of these findings to the clinical setting. Nonetheless, these data provide further in vivo evidence for the powerful biological effects of MFP-14 in a well known immunoinflammatory model and strengthen its emerging immunopharmacological profile. Studies may therefore be warranted aimed at considering the possible testing of MFP-14 in the prevention and treatment of different forms of shock and multiple organ failure not only in patients with endotoxemia because of infections with gram-negative bacteria, but also in high-risk patients, for example, those subjected to major surgery and other forms of physical trauma, including burns.