INFCTION AND IMMUNITY, Jan. 1977, p. 337-339 Copyright 1977 American Society for Microbiology

Vol. 15, No. 1 Printed in U.S.A.

Protection Against Endotoxin-Induced Mortality in Mice Treated with Transition Metal Salts STEPHEN L. SNYDER,* RICHARD I. WALKER, AND JOSEPH V. MONIOT Experimental Pathology Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20014

Received for publication 21 July 1976

Administration of manganese or chromium chloride 1 h before challenge with single bolus of Salmonella typhosa endotoxin completely obviated death in endotoxin-challenged mice. a

The release of lysosomal mediators from specialized cells involved in endotoxin clearance may be important in inflammation and shock (1, 2, 6, 9). Increases in serum acid hydrolase activities (9, 10) as well as increased fragility of hepatic lysosomes (9, 20) have been reported during endotoxemia. Furthermore, endotoxin can stimulate the release of lysosomal hydrolases from polymorphonuclear leukocytes (PMNs) in vitro (3, 8). The uncontrolled release of lysosomal mediators (particularly proteases) can initiate coagulation and fibrinolytic disorders (11, 13), complement and Hageman factor activation (19), kinin production (12, 19), and tissue necrosis (1, 15). Therefore, any agent that would suppress release of lysosomal contents from target cells and/or block the action of lysosomal substances already released should be beneficial to an animal suffering endotoxemia. The protection afforded by treatment with zinc (16) or cortisone (7), both of which have membrane-stabilizing properties, as well as the reduction in mortality achieved with protease inhibitors (2, 16) is consistent with this notion. We hypothesized that transition metals other than zinc may also suppress the response of phagocytic cells to inflammatory stimuli (5, 17, 18). Therefore, selected di- and trivalent cations were tested for their usefulness as protective agents against bacterial endotoxin. Female B6CBF1 mice (Cumberland View Farms, Clinton, Tenn.) weighing 20 to 25 g (8 to 10 weeks old) were acclimated to laboratory conditions for 2 weeks and determined to be free from murine pneumonia complex and oropharyngeal Pseudomonas spp. They were injected intraperitoneally with metal salts dissolved in distilled water and 1 h later challenged intraperitoneally with Salmonella typhosa lipopolysaccharide (Difco, Detroit, Mich.). We found that chromium (5.88 gmol) and

manganese chloride (8.82 umol) completely obviated the lethal effect of endotoxin as determined by mortality at 48 h (Table 1). At this time, 0.85 mg of endotoxin caused 90% mortality in untreated animals. Some protection (50% survival) was also afforded by NiCl2 (0.74 to 1.47 gmol). The effectiveness of Cr3+ and Mn2+ as protecTABLE 1. Mortality ofB6CBF, mice, at 48 h, treated with various metal salts and challenged with Salmonella typhosa endotoxina % Mortality at dose

(jkmol) specified'

0.74

2.94

4.41

5.88

8.82

50 70 70

10 100 80 100 100 100 70 100 80

or 40

0"

80

90

Metal salts NaCl (control)

CrCl3 MnCl2 NiCl2 SnCl2

CoC12

CuS04 FeCl3 FeSO4

AuCl3

40

1.47 90 100 100 40r 100 100 100 80 100 100

75"

100" 100 100 100 80

80

Each mouse received 0.85 mg of endotoxin intraperitoneally. b Ten mice were employed at each concentration, except where noted. e Two separate experiments employed 10 mice per experiment. a

tive agents against endotoxin-induced lethality was studied further (Fig. 1). When mortality was plotted as a function of the amount of endotoxin administered to treated or untreated mice, the mean lethal dose at 48 h for untreated animals was 0.55 mg of endotoxin. The mean lethal dose at 48 h for Cr3+- and Mn2+-treated mice was approximately 1.75 mg. Thus, resistance to endotoxin-induced mortality was increased greater than threefold by prior treatment with these metals. This compared to a protective factor of 2.5 found for zinc-treated mice (16). 337

338

INFECT. IMMUN.

NOTES

We reported that zinc chloride reduced the increases in serum 8-glucuronidase seen in untreated mice undergoing endotoxin shock and that this reduction may reflect suppression of the release of harmful lysosomal hydrolases (16). We were unable to obtain a similar correlation using chromium or manganese. Manganese enhanced the levels of serum 83-glucuronidase seen 30 min after challenge with endotoxin, whereas Cr3+ and Fe3+ reduced the increase of f-glucuronidase at this time (Fig. 2). The metals had little effect on the activity of this enzyme 5 h postchallenge with endotoxin. eCONTROLS YMnCI2 -TREATED *CrCI3 -TREATED

100

/

80

,, a

4

060

b/

z

A /

V 40

2. 20

1.0

0.5

2.0

1.5

Sklmonelln typhoso endotoxin (mg) FIG. 1. Percent mortality at 48 h of control and

and chromium chloride-treated mice endotoxin dose. Mice received 5.88 pmol of chromium and 8.82 ,umol of manganese chloride intraperitoneally 1 h before challenge with endotoxin. Ten mice were used for each point. manganese-

versus

Serum, -glucuronidase increases observed in this study may reflect action of the various metals and/or endotoxin at multiple sites. Therefore, to determine whether transition metal ions could inhibit enzyme release, in vitro experiments were performed with isolated cells thought to be important in the inflammatory response. In these studies we determined the effect of transition metal salts on the zymosan-stimulated release of 8-glucuronidase from human PMNs purified by Ficoll-Hypaque density centrifugation (4). As reported by others (21), incubation of 1.6 X 106 to 2.0 x 106 PMNs with zymosan for 1 h at 370C significantly increased (40 to 75%) /3-glucuronidase release above that seen in unstimulated cells. Addition of transition metal chlorides to the mixture inhibited release of the enzyme (Table 2). For those metal salts found to protect against endotoxin-induced death, the concentration of metals needed to inhibit zymosan-induced release of 3-glucuronidase by 65% or greater was: Ni2+, 0.6 x 10-3 M; Zn2+, 1.1 x 10-3 M; Cr3+, 1.1 x 10-3 M; and Mn+2, 3.3 x 10-3 M. It is noteworthy that the relative doses of these metals required to achieve maximum protection in the survival experiments follow approximately this same order (Table 1) (16). Conjugation of endotoxin with Fe3+ and Cr3+ reduces toxicity of the molecule (14). Since protection could not be achieved with Fe3+ when used in our survival experiments, attenuated iron-endotoxin complexes are probably not formed in vivo (11). Direct mixing with Mn2+ in the peritoneal cavity is unlikely, as we were able to obtain increased survival by administering Mn2+ subcutaneously. However, we have not been able to achieve protection by the ad-

0a_ Uj 0

o9

I

C.

UNTREATED

(0

Z- E

4

IL

Cr

Cr

Fe Mn

Fe Mn

Cr

Fe Mn

0

ML FIG.

2. Serum

with endotoxin. endotoxin.

CONTROLS

f3giu'curonidase Samples

were

30

minot

activities in untreated and Cr3

collected

by retroorbital bleeding

post-ET Mn2 30

min

or

Fe3t -treated mice

and 5 h

challenged after challenge with

VoL. 15, 1977

NOTES

TABLE 2. Percent inhibition of zymosan-stimulated release of f3glucuronidase from human PMNs by transition metal saltsa Concn (M x Added Added salt %

~10-3)

Inhibition

85 ± 12 1.1 67 ± 12 3.3 1.1 30 10 89 ± 15 0.6 NiCi2 40 ± 7 1.1 FeCl, 71 ± 3 1.1 CrC13 77 ± 16 1.1 ZnCl2 54 ± 7 0.1 AuC13 a Incubation mixture contained 1.0 ml of 1.6 x 106 to 2.0 x 106 PMNs suspended in Hanks balanced salts, pH 7.4, 0.1 ml of zymosan particles (5 mg/ml), 0.1 ml of serum, and 0.1 ml of the appropriate salt. Temperature of incubation was 370C. b Average ofthree determinations. Percent inhibition = 100 x (Z - C) - (Zm - C)/Z - C, where C, Z, and Zm symbolize the micromoles of phenolphthalein produced by incubating 0.2 ml of supernatant from resting (C) and zymosan-stimulated PMNs with (Zm) and without (Z) added heavymetal ions for 18 h with 1.0 ml of 1.5 mM phenolphthalein 3-D-glucosiduronate in acetate buffer (pH 4.5) at 370C.

3.

4.

CoCl, MnCl2

ministration of chromium by any alternate route. Further investigations are required to clarify the mechanisms by which some metal ions protect mice against endotoxin-induced mortality. We have shown that metal ions can moderate the release of lysosomal hydrolases from isolated PMNs in vitro. It is possible that endotoxin triggers the disruption of homeostasis, ultimately resulting in death, by causing phagocytic cells to degranulate at an accelerated rate. If this is the case, the protection afforded by some metal ions could be related to the attenuation of the release of lysosomal mediators via interaction with phagocytic cells or with humoral agents necessary for their activation. Until more information is available regarding the site of action of these metals as well as the nature of their inherent toxicities, any interpretation as to the mechanism of protection remains speculative. This work was supported by grant no. M4 305.05-3100 from the Naval Research and Development Command.

5. 6. 7.

8.

9.

10.

11.

12. 13.

14.

15.

16. 17. 18. 19.

20.

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