INFECTION AND IMMUNITY, July 1996, p. 2850–2852 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 64, No. 7
Interleukin-10 Protects Neonatal Mice from Lethal Group B Streptococcal Infection VITALIANO CUSUMANO, FRANCESCO GENOVESE, GIUSEPPE MANCUSO, MARIA CARBONE, MARIA TERESA FERA, AND GIUSEPPE TETI* Istituto di Microbiologia, Facolta ` di Medicina e Chirurgia, Universita ` degli Studi di Messina, I-98122 Messina, Italy Received 15 December 1995/Returned for modification 21 February 1996/Accepted 19 April 1996
We investigated the role of interleukin-10 (IL-10) in a neonatal mouse model of lethal group B streptococci (GBS) sepsis. Plasma IL-10 levels significantly increased at 24 and 48 h after GBS inoculation. Neutralization of IL-10 with specific antibodies had no effect on lethality. Administration of recombinant IL-10 at 20 or 4 h before challenge, but not at later times, resulted in decreased tumor necrosis factor alpha levels and improved survival. IL-10 could be potentially useful for the treatment of GBS sepsis. tralization of cytotoxicity with TNF-a-specific rabbit serum (Genzyme, Cinisello Balsamo, Italy) confirmed that plasma TNF activity is entirely accounted for by TNF-a in this GBS infection model (12). Plasma IL-10 levels were determined with an enzyme-linked immunosorbent assay kit (Bender MedSystems, Vienna, Austria). The lower limits of detection were 10 IU/ml and 100 pg/ml for TNF-a and IL-10 assays, respectively, taking into account that sera were diluted 1:10 and 1:4, respectively, before testing. Data were expressed as means 6 standard deviations of three independent measurements, each performed on pooled plasma from four animals. Plasma IL-10 values were below the limit of detection of the assay in uninfected controls (not shown). In infected animals, IL-10 plasma levels significantly increased to 302 6 183 and 1,330 6 947 pg/ml at 24 and 48 h, respectively, after GBS challenge. In order to assess the role of endogenous IL-10 in GBS-induced lethality, pups were inoculated, at 6 h before challenge, with neutralizing rat anti-mouse IL-10 monoclonal antibodies (MAbs). These were purified from culture supernatants of hybridoma SXC1 (15) (kindly provided by L. Romani, University of Perugia, Perugia, Italy) and inoculated subcutaneously in 25-ml volumes. Control animals received an equal amount of PBS vehicle. Pretreatment with anti-IL-10 MAb (125 or 250 mg per pup) at 6 h before challenge totally prevented the increase in circulating IL-10 levels but did not significantly affect survival or blood colony counts in pups infected with 15 or 150 CFU (not shown). These doses produced 21 and 79% lethality, respectively, in controls (not shown). Since IL-10 protected mice from death by endotoxin (8, 11, 19) or staphylococcal enterotoxin B (7), we investigated whether administration of mouse rIL-10 (0.5 U/ng [Genzyme]) would be beneficial in our model. The rIL-10 was inoculated subcutaneously neat or diluted in PBS with 0.1% bovine serum albumin in 25- or 50-ml volumes at various times before challenge with 150 CFU. Pretreatment with 25 ng per pup at 20 h before challenge resulted in significant protection (P , 0.05), while no effect was observed with lower doses (Table 1). The time of rIL-10 pretreatment relative to challenge was critical (Table 2). No effects were noted when rIL-10 was given at the time of challenge or at later times. An increase in survival time, but not permanent protection, was observed when rIL-10 was given at 4 h before challenge (Table 2). Since IL-10 can modulate host defenses against infection (9, 10, 18), it was of interest to ascertain whether treatment with rIL-10 would affect the severity of infection. Colony counts performed on
Interleukin 10 (IL-10), initially described as cytokine synthesis inhibitory factor, is an important regulator of the functions of lymphoid and myeloid cells (14). This cytokine, mainly produced by the Th2 subset of helper T cells (6, 21), macrophages (4), and B cells (16, 17), exhibits anti-inflammatory and immunosuppressive functions (14). Recombinant IL-10 (rIL-10) protects adult mice from lipopolysaccharide (LPS)- or staphylococcal enterotoxin B-induced lethal shock, presumably through its ability to reduce the release of pathophysiologic mediators, including tumor necrosis factor alpha (TNF-a) and gamma interferon (2, 7, 8, 11, 19). Little is known about the role of IL-10 in neonatal septic shock, a major cause of morbidity and mortality. Therefore, we investigated the effects of IL-10 blockade and rIL-10 administration in mouse pups infected with group B streptococci (GBS), the most frequent cause of neonatal sepsis (1). Neonatal (#24 h old) BALB/c mice were used. Parental mice were obtained from Harlan-Nossan (Milan, Italy). Pups from each litter were randomly assigned to control or experimental groups, marked, and kept with the mother. GBS strain COH1, a highly virulent strain originally isolated from a septic neonate, was kindly provided by Craig Rubens, University of Washington, Seattle (13). Bacteria were grown to the late logarithmic phase in Todd-Hewitt broth (Difco, Diagnostic International Distribution, Milan, Italy) diluted in phosphatebuffered saline (PBS; 0.01 M phosphate, 0.15 M NaCl [pH 7.2]) and were inoculated subcutaneously (25 ml) in neonatal mice. Inocula were adjusted photometrically to give 15 or 150 CFU in 25 ml of PBS, which produced 20 to 35 and 70 to 90% mortality, respectively, over a 96-h period. Deaths rarely occurred after this time (12). To measure circulating levels of TNF-a and IL-10, animals were killed by decapitation under ether anesthesia at different times after challenge with 150 CFU. Mixed venous-arterial blood was collected in heparinized containers and centrifuged after saving 10 ml for colony counts. The latter were performed by standard pour plate methods. Pooled plasma from four animals was stored at 2708C until assayed for TNF-a and IL-10 levels. TNF activity was assessed by cytotoxicity in WEHI 164 clone 13 cells and expressed in units per milliliter, exactly as described previously (5). In selected samples, complete neu-
* Corresponding author. Mailing address: Istituto di Microbiologia, Piazza XX Settembre, 4, I-98122 Messina, Italy. Phone: 39-90-712110/ 672508. Fax: 39-90-719910. Electronic mail address:
[email protected]. 2850
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TABLE 1. Effects of rIL-10 on lethality induced by GBS in neonatal micea
a b
No. dead/total (%) at h:
IL-10 dose (ng/mouse)
24
48
1
0 (vehicle) 10 25
3/14 (21) 2/14 (14) 1/14 (7)
5/14 (35) 3/14 (21) 3/14 (21)
2
0 (vehicle) 5 25
2/14 (14) 3/14 (21) 1/14 (7)
4/14 (29) 11/14 (79) 11/14 (79) 4/14 (29) 10/14 (71) 10/14 (71) 2/14 (14) 4/14 (29)b 4/14 (29)b
Expt no.
72
96
9/14 (64) 10/14 (71) 6/14 (43) 7/14 (50) 3/14 (21)b 3/14 (21)b
Mouse pups were challenged with 150 CFU of strain COH1. P , 0.05 by Fisher exact test, compared with the respective vehicle controls.
blood samples obtained at 12, 24, and 48 h after challenge in animals treated with rIL-10 (25 ng) or anti-IL-10 (125 mg at 20 h before challenge) were not significantly different from those of control animals (not shown). Because IL-10 can modulate TNF-a production, an important pathophysiologic mediator in this model (20), the effects of rIL-10 or anti-IL-10 on circulating TNF-a levels in septic pups were investigated. Figure 1 shows that TNF-a elevations were measured at 12, 24, and 48 h after GBS challenge and in control animals, confirming our previous results (12). Pretreatment with IL-10 significantly (P , 0.05) decreased TNF-a levels at 24 h. Pretreatment with anti-IL-10 (125 mg per pup), however, did not affect plasma TNF-a (Fig. 1). In further experiments, increasing the dose of anti-IL-10 to 250 mg per pup also had no significant effect on TNF-a levels. The present data demonstrate for the first time induction of IL-10 by GBS, as evidenced by increased plasma levels in neonatal mice at 24 and 48 h after infection. Whether these increased endogenous IL-10 levels are essential in determining neonatal responsiveness to GBS remains to be clarified. AntiIL-10 treatment with neutralizing anti-IL-10 MAbs did not modulate survival or modify CFU. Thus, the IL-10 elevations in GBS-infected pups had apparently no major influence on septic outcome. High levels of IL-10 have been observed during the initial phase of fulminant meningococcal septic shock (3). However, the high levels of IL-10 were found both in survivors and in those who died. In addition, the IL-10 levels correlated weakly with the severity of the disease and the prognostic score. Our previous studies have demonstrated that the GBS neonatal model of infection is TNF-a dependent in that administration of anti-TNF-a is greatly protective in this model (20). Thus, we were particularly interested in determining the effects
FIG. 1. Effects of anti-IL-10 or rIL-10 administration on plasma TNF-a levels in neonatal mice at various times after GBS challenge. Plasma was collected at the indicated times after challenge from pups (four per group) pretreated with vehicle (■), 25 ng of rIL-10 (F), or 125 mg of anti-mouse IL-10 MAb (å) at 20 h before challenge. Points and bars represent means 6 standard deviations of three observations. Each of these was conducted on a different pooled plasma sample obtained from four animals. p, significantly (P , 0.05) different from vehicle controls by one-way analysis of variance and StudentNewman-Keuls test.
of IL-10 blockade or rIL-10 treatment on TNF-a levels. Indeed, previous studies have shown that IL-10 is a potent inhibitor of TNF-a production (4, 8). In this study, neutralizing anti-IL-10 antibodies did not modify TNF-a levels in GBS-infected pups. Conversely, pretreatment with rIL-10 was quite effective in decreasing plasma TNF. This suggests that (i) unlike IL-6 (12), endogenous IL-10 is not involved in regulation of TNF-a production in GBS infection and (ii) delayed appearance of endogenous IL-10 relative to TNF may at least partially account for this lack of regulatory effects. Unlike TNF-a levels, which peaked at 24 h and were declining by 48 h, the IL-10 levels were increased at 24 h and were continuing to rise at 48 h. Prophylactically, rIL-10 when given up to 20 or 4 h before challenge was protective against GBS infections, and there was a correlation between improved survival and TNF suppression. However, we cannot ascertain from the present data whether
TABLE 2. Effects of time of administration of rIL-10 on lethality induced by GBS in neonatal micea
a
No. dead/total (%) at h:
Time of administration (h)b
24
48
72
96
1
2 (vehicle) 24 224
2/14 (14) 0/14 (0) 2/14 (14)
4/14 (29) 2/14 (14) 2/14 (14)
10/14 (71) 3/14 (21)c 3/14 (21)c
12/14 (86) 8/14 (57) 3/14 (21)c
2
2 (vehicle) 0 124
3/14 (21) 2/14 (14) 2/14 (14)
5/14 (35) 3/14 (21) 5/14 (35)
9/14 (64) 7/14 (50) 10/14 (71)
11/14 (79) 9/14 (64) 12/14 (86)
Expt no.
Mouse pups were challenged with 150 CFU of strain COH1. Relative to time of challenge. c P , 0.05 by Fisher exact test, compared with the respective vehicle controls. b
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this was the only or even the most essential anti-inflammatory action of IL-10. The sustained protection for up to 20 h before challenge suggests a profound change in the host inflammatory response similar to that initiated by endotoxin tolerance (22). This requirement for a longer period of prophylaxis is quite different from endotoxin shock in which IL-10 could be protective when given simultaneously or immediately post-LPS (11). This difference may be accounted for by differences in the inflammatory response and kinetics of cytokine production after bolus injection of LPS versus infection with a small number of live virulent bacteria. Regardless of the mechanisms of action, the present data clearly demonstrate a protective role of IL-10 in a severe grampositive sepsis model. Since IL-10 has been shown at pharmacological doses to be highly effective in protection against staphylococcal enterotoxin B as well as endotoxemia, this suggests therapeutic application. It will be interesting to compare the efficacy of IL-10 with that of other potential candidates for treatment of sepsis. We thank James A. Cook, Medical University of South Carolina, for valuable critical review and suggestions in preparation of the manuscript. This study was supported in part by grants “40%” and “60%” of the Ministero della Sanita` e della Ricerca Scientifica and AIDS grant no. 931 of the Ministero della Sanita` of Italy. REFERENCES 1. Baker, C. 1986. Group B streptococcal infections in newborns. N. Engl. J. Med. 314:1702–1708. 2. Bean, R., A. Freiberg, S. Andrade, S. Menon, and A. Zlotnik. 1993. Interleukin-10 protects mice against staphylococcal enterotoxin B-induced lethal shock. Infect. Immun. 61:4937–4939. 3. Derkx, H. H. F., A. Marchant, M. Goldmann, R. P. G. M. Bijemer, and S. J. H. Van Deventer. 1995. High levels of interleukin-10 during the initial phase of fulminant meningococcal septic shock. J. Infect. Dis. 171:229–232. 4. de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, and J. E. de Vries. 1991. Interleukin-10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174: 1209–1229. 5. Espevik, T., and J. Nissen-Meyer. 1986. A highly sensitive cell line WEHI 164 clone 13 for measuring cytotoxic factor/tumor necrosis factor from human monocytes. J. Immunol. Methods 95:99. 6. Fiorentino, D. F., M. W. Bond, and T. R. Mosmann. 1989. Two types of mouse T helper cell: IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J. Exp. Med. 170:2081–2086. 7. Florquin, S., Z. Amraoui, D. Abramowicz, and M. Goldman. 1994. Systemic release and protective role of IL-10 in staphylococcal enterotoxin B-induced
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