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. 2005 Apr;140(1):11–21. doi: 10.1111/j.1365-2249.2005.02735.x

A defective Th1 response of the spleen in the initial phase may explain why splenectomy helps prevent a Listeria infection

N Kuranaga *, M Kinoshita †, T Kawabata *, N Shinomiya *, S Seki *
PMCID: PMC1809347  PMID: 15762870

Abstract

Listeria monocytogenes (Listeria) are known to grow and proliferate in the liver while a splenectomy induces host resistance against a Listeria infection despite the fact that a splenectomy inhibits the Th1 response. Therefore, the mechanism by which a splenectomy helps to prevent the growth of Listeria still remains to be elucidated. After an i.v. challenge of Listeria (1 × 106 CFU) in C57BL/6 mice, Listeria rapidly increased in the spleen but not in the liver until 48 h. However, after this initial phase, Listeria remarkably grew in the liver. In contrast, when the mice received a splenectomy beforehand, no remarkable growth of Listeria in the liver was observed after Listeria challenge despite the fact that serum IFN-γ and IL-12 levels at 24 h after Listeria challenge were significantly lower than those in the sham mice. However, the liver leucocytes from mice by 6 h after infection produced a substantial amount of IFN-γ while spleen MNC did not, whereas spleen leucocytes at 24 h after Listeria challenge did. Consistently, the IFN-γ and IL-12 levels in the tissue homogenates of the spleen were significantly lower than in those of the liver until 6 h after infection. This defective spleen Th1 response in the early phase of Listeria infection was corrected by an IL-18 i.p. injection just after the Listeria challenge. Our findings suggest that Listeria exploit the defective Th1 environment of the spleen in the initial phase and afterwards overcome the host defense mechanism of the liver.

Keywords: rodent, bacterial, cytokines, spleen, liver, Listeria

Introduction

The spleen plays an important role in the host resistance of many infections. Asplenic states are therefore usually characterized by defects in the host defense against bacterial invasions, particularly encapsulated organisms such as Pneumococcus, Meningococcus and Hemophilus influenza[1–5]. In contrast, however, hosts after a splenectomy tend to resist infections with the intracellular microbe, Listeria monocytogenes (Listeria) [6–9]. Listeria is associated with severe infections in newborns, the elderly, and immunocompromized individuals [10–15].

The course of Listeria infection has been well studied in the mouse. This pathogen can survive inside macrophages by escaping into the cytoplasm from the phagosomes [16–19]. Either CD4+ or CD8+ T cells help prevent listeriosis [20–24], and γδ T cells also play an important role in survival from listeriosis [25–30]. IFN-γ produced by T cells and NK cells is crucial for the primary response to Listeria infection [31–34], and IL-12 and IL-18 from activated macrophages/dendritic cells are essential inducers of IFN-γ[35–38]. Granulocytes may also produce IL-12 in mice infected with Listeria[39]. These Th1 cytokines and lymphocytes are thus indispensable for the elimination of Listeria. However, splenectomized mice still show a remarkable resistance to infection with Listeria despite the fact that they exhibit a failure to increase systemic Th1 cytokines [40]. As splenectomy indeed increases the phagocytic activity of the liver after a Listeria infection [8], and inflammatory response and the Listeria clearance in the liver have been shown to be more rapid and enhanced in splenectomized mice than in control mice [41]. Therefore, a reduced Th1 response and increased resistance toward Listeria infection in splenectomized mice have long been contradictory phenomena.

The aim of the present study is to elucidate the mechanism of these phenomena in splenectomized mice with Listeria infection and also to show how IL-18, as a Th1 response inducer, is effective against Listeria infection.

Materials and methods

This study was conducted according to the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College, Japan.

Mice and procedure of splenectomy

Male C57BL/6 mice (8 week-old, weight-matched, Charles River Inc, Yokohama, Japan) were purchased and maintained under specific pathogen free conditions. A splenectomy was aseptically performed using an electric scalpel under deep ether anaesthesia through a left-sided lateral 1·0 cm subcostral incision. A sham operation was also performed through the same incision, but the spleen was returned to the abdominal cavity without any ligation. At 2 weeks after the splenectomy, the mice were used for the experiments (10–11 week-old).

Reagents

Anti-NK 1·1 Ab (PK136) was prepared from hybridomas grown in our laboratory. Mouse recombinant IL-18 (MBL, Nagoya, Japan) was utilized for the experiments.

Listeria or E. coli challenge and collection of blood sample or tissue homogenate

Listeria monocytogenes (strain EGD) was grown in brain heart infusion broth (Difco Laboratories, Detroit, MI, USA). Inocula were prepared by growing cultures for 20 h at 37°C, followed by centrifugation at 3000 × g for 20 min and then resuspension in phosphate buffered saline (PBS) with glycerol. Samples were adjusted to a concentration of 1 × 109−1 × 1010 colony forming unit (CFU)/ml and frozen in 1 ml aliquots at −80°C until use. Listeria were suspended in an appropriate number in PBS at a concentration of 1 × 106/0·2 ml, and each 0·2 ml suspension was injected intravenously.

Avirulent strain of E. coli (B strain)(ATCC 11303, Sigma Co., St. Louis, MO, USA) was cultured in a similar way using brain heart infusion broth, and then they were adjusted to a concentration of 1 × 1012 CFU/ml, and stored at −80°C. Appropriate dilutions of the thawed preparations were made in PBS. The mice were infected intravenously with 1 × 109 of E. coli in a volume of 0·2 ml.

The mice were i.v. injected with 1 × 106 CFU of Listeria or 1 × 109 CFU of E. coli at 2 weeks after either a splenectomy or a sham operation. Blood samples were obtained from the retro-orbital plexus and then the sera were stored at −80°C until the assays were performed. Mice were bled from the subclavian artery and vein, and the liver and spleen were removed from the mice under ether anaesthesia to produce 1 ml of homogenized PBS suspension. The homogenates were centrifuged at 400× g for 15 min and then the supernatant was stored at −80°C until the assays were performed.

Viable bacterial counts

Under the deep ether anaesthesia, mice were bled from the subclavian artery and vein, and the liver and spleen were aseptically removed from mice to produce a homogenized PBS suspension. The serially 10-fold diluted homogenates by PBS of organs were placed on brain heart infusion agar plates and then were incubated at 37°C for 24 h. The number of bacterial colonies was then counted and expressed as either CFU/organ or CFU/mg of organs.

IL-18 treatment

IL-18 treatment was performed by an i.p. injection of IL-18 (0·2 µg/0·5 ml/body) immediately following the Listeria challenge. Sham treatment was also performed by an i.p. injection of PBS (0·5 ml) in the same way as the IL-18 treatment.

Isolation of the liver and spleen leucocytes

The liver and spleen leucocytes were obtained as previously described [42–44]. Briefly, the liver and spleen were removed from the mice under lethal ether anaesthesia. The liver was then minced and suspended in a RPMI 1640 medium containing 0·05% collagenese (Wako, Osaka, Japan) to shake for 20 min in a 37°C water bath. The liver specimen was passed through a 200-gauge stainless steel mesh. After washing, the cells were resuspended in 33% Percoll solution and centrifuged at 500× g for 20 min at room temperature. The pellet was resuspended in a RBC lysis solution and then was washed twice in 10% FBS RPMI 1640. Splenocytes were also passed through the stainless steel mesh and then were treated with the RBC lysis solution, and finally were washed twice in 10% FBS RPMI 1640.

Cell cultures

After counting the cells, 5 × 105 of the liver or spleen leucocytes in 200 µl of 10% FBS RPMI 1640 medium were cultured in 96 well flat-bottomed plates in 5% CO2 at 37°C for 24 h and then the culture supernatants were stored at −80°C.

Measurements of cytokine levels using sera, tissue homogenate and culture supernatants

The IFN-γ and IL-12 levels of the sera or tissue homogenates and the IFN-γ levels of the culture supernatants were measured by cytokine-specific ELISA kits (Endogen, Woburn, MA, USA) according to the manufacturer's instructions without any modifications. The sera were usually 10-fold diluted by the assay buffer included in the respective ELISA kit and then were used to perform the measurements.

In vivo NK and NK1·1 Ag+T (NKT)* cell depletion

Anti-NK1·1Ab (PK136) (200 µg/mouse) was i.v. injected into the mice 3 days before the Listeria challenge. Anti-NK1·1 Ab depletes both NK and NKT cells for approximately one week, as we previously reported [45,46].

Statistical analysis

The data are presented as the mean values ± SE. The statistical analyses were performed using an iMac computer (Apple, Cupertino, CA, USA) and the Stat View 4·02 J software package (Abacus Concepts, Berkeley, CA, USA). The survival rates were compared using the Wilcoxon rank test, while other statistical evaluations were compared using the standard one-way analysis of variance followed by the Student's t-test. P < 0·05 was considered to indicate a significant difference.

Results

A splenectomy increases mouse survival in spite of a down-regulation of the serum Th1 cytokine levels after Listeria challenge while a splenectomy does not have any effect on the mice after an E. coli challenge

The mice were i.v. injected with either 1 × 106 CFU of Listeria or 1 × 109 CFU of E. coli at 2 weeks after either a splenectomy or a sham operation. No splenectomized mice died after the Listeria challenge while all of the sham mice died within 5 days (Fig. 1a). Interestingly, splenectomized mice showed significantly lower peaks of both serum IFN-γ and IL-12 at 24 h after Listeria challenge than those of the sham mice (Fig. 1b,c). Regarding E. coli infection, 40% of the E. coli challenged sham mice died presumably due to either infection per se or endotoxin shock. In contrast with Listeria challenge, a splenectomy did not affect the survival or the change in the serum Th1 cytokine levels in the mice after E. coli challenge (Fig. 1d–f).

Fig. 1.

Fig. 1

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The effects of a splenectomy on mouse survival (a, d), the serum levels of IFN-γ (b, e) and IL-12 (c, f). The mice were i.v. challenged with Listeria (1 × 106 CFU) or E.coli (1 × 109 CFU) at 2 weeks after either a splenectomy (○) or a sham (▪) operation and then the survival was monitored. The sera were obtained from the mice immediately before the bacterial injections and at 1, 3, 6, 12, 24 and 48 h after bacterial injections to measure the IFN-γ and IL-12 levels. The data are the means ± SE from 10 mice in each group.

The spleen produces a small amount of either IFN-γ or IL-12 in comparison to the liver in the very early phase after the Listeria challenge and Listeria rapidly proliferate in the spleen by 48 h

The mice were i.v. injected with 1 × 106 CFU of Listeria at 2 weeks after either a splenectomy or a sham operation. We focused on the spleen and liver in the very early phase. The livers and spleens were obtained from the mice at 3, 6 and 12 h after Listeria challenge to count the number of bacteria and were expressed as per mg of the organs (CFU/mg). If the CFU number was expressed per organ, the CFU number in the livers of the sham mice was always larger than that in the spleen (data not shown). The number of Listeria in the spleen started to increase in number as early as 6 h after infection, whereas the Listeria number did not increase at all in the livers of both the sham operated mice and the splenectomized mice at this time point (Fig. 2a). Furthermore, although the tissue homogenates of the livers in both mouse groups contained substantial amounts of IFN-γ and IL-12 at this time point, the spleen of the sham-operated mice contained very low levels of these cytokines (Fig. 2b,c). However, although the spleen started to produce large amounts of IFN-γ as well as IL-12 by 12 h after infection (Fig. 2b,c), the Listeria number continued to increase in the spleen at 24 h after infection while the Listeria number also started to increase in the liver of sham-operated mice (Fig. 2d). The number of Listeria in the liver of sham mice thereafter extensively increase at 48 h and 72 h after Listeria challenge and Listeria number of the liver of sham mice was larger than that of spleen at 72 h while the Listeria number in the liver of splenectomized mice did not substantially increase (Fig. 2e,f). The tissue cytokine levels peaked at 24 h after the Listeria challenge while spleen contained a larger amount of IFN-γ and IL-12 than the liver (Fig. 2g,h). We also used nontreated control mice in these experiments and the results were essentially similar to those of the sham-operated mice (data not shown). Consistently, spleen leucocytes from mice at 3 h and 6 h after Listeria challenge produced little, if any, IFN-γ in comparison to that in the liver leucocytes in vitro while the spleen leucocytes from the mice at 24 h after the infection produced a substantial amount IFN-γ (Fig. 3a–c). We did not perfuse the liver because the blood contained only a very small number of Listeria[47] and the number of Listeria contained in the small amount of the blood within the liver is thought to be negligible (mice were already bled from the subclavian artery and vein). Although the liver has been reported to contain endogenous biotin which prevents to determine the cytokine levels in the liver homogenates by ELISA [48], the basal IL-12 level in the liver homogenates before the Listeria challenge was low and no significant difference was observed in comparison to the basal IL-12 level in the spleen (Fig. 2C). Even if the nonspecific binding of the endogenous biotin cannot be ruled out completely, however, the change in the cytokine levels is thought to have been accurately evaluated.

Fig. 2.

Fig. 2

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The Listeria number (a) and the cytokine levels in the spleen (▴) and liver (▪) of the sham-operated mice and the liver (○) of splenectomized mice (b, c) in very early phase after Listeria challenge. A comparison of the number of Listeria in the livers and spleens of the splenectomized mice or sham-operated mice at 24 h (d), 48 h (e) and 72 h (f) after the Listeria challenge. The IFN-γ and IL-12 levels in the tissue homogenates of the livers and spleens at 24 and 48 h (g, h) after Listeria challenge. The mice were i.v. challenged with Listeria (1 × 106 CFU) at 2 weeks after either a splenectomy or a sham operation. The livers and spleens were obtained from the mice at 3, 6, 12, 24, 48 and 72 h to count the number of bacteria. Similarly, the livers and spleens were also obtained from the other mice to measure the Th1 cytokine levels of the tissue homogenates. The data are the means ± SE from 5 mice in each group.

Fig. 3.

Fig. 3

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IFN-γ production from liver and spleen leucocytes after Listeria challenge. The mice were i.v. injected with 1 × 106 CFU of Listeria. The liver and spleen leucocytes were obtained at 3 (a), 6 (b) and 24 (c) h after Listeria challenge to measure the IFN-γ production in vitro. The data are the mean ± SE from 5 mice in each group.

IL-18 treatment increases mouse survival and bacterial clearance in both the liver and spleen after the Listeria challenge

The nontreated mice were i.p. injected with IL-18 or PBS immediately after the i.v. injections of Listeria (1 × 106 CFU). The IL-18-treated mice showed a significantly increased survival in comparison to the PBS-treated mice (Fig. 4a). The livers and spleens were obtained from the mice at 12 and 24 h to count the number of Listeria. The IL-18 treatment decreased the number of Listeria in the spleen at both 12 and 24 h significantly more than in the PBS-treated mice (Fig. 4b,c). The IL-18 treatment also decreased the number of Listeria in the liver at 12 h significantly more than in the PBS-treated mice (Fig. 4d,e).

Fig. 4.

Fig. 4

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The effects of IL-18 treatment on mouse survival (a) and the bacterial count in the spleen (b, c) and liver (d, e) after Listeria challenge. The mice were i.p. injected with IL-18 (0·2 µg) or PBS immediately after Listeria i.v. challenge (1 × 106 CFU) and then their survival was monitored. The liver and spleen were obtained from the mice at 12 and 24 h after Listeria challenge to count the number of bacteria. The data are the means ± SE from 10 mice in each group.

The IL-18 treatment increased the Th1 cytokine levels in both the sera and tissue homogenates of the liver and spleen after the Listeria challenge

The nontreated mice were i.p. injected with either IL-18 or PBS immediately following i.v. injections of Listeria (1 × 106 CFU). The sera were obtained from the mice at 3, 6 and 12 h after Listeria challenge. The IL-18 treatment significantly increased the serum IFN-γ level until 12 h than the PBS-treated mice (Fig. 5a). The tissue homogenates of the liver and spleen were also obtained from the mice at 3, 6 and 12 h. Consistently, IL-18 treatment increased the IFN-γ levels in both the liver and spleen homogenates until 12 h after the Listeria challenge significantly more than in the PBS-treated mice (Fig. 5b,c).

Fig. 5.

Fig. 5

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The effects of IL-18 treatment on the IFN-γ levels of the sera (a) and the tissue homogenates of the liver (b) and spleen (c) after Listeria challenge. The mice were i.p. injected with IL-18 (0·2 µg) (▪) or PBS (○) immediately after Listeria challenge (1 × 106 CFU). The sera were obtained from the mice immediately before the Listeria injections and at 3, 6 and 12 h to measure the IFN-γ levels. Similarly, the livers and spleens were also obtained from the mice to measure the IFN-γ levels of tissue homogenates. The data are the means ± SE from 7 mice in each group.

Depletion of NK and NKT cells decreases the Listeria clearance in the liver but not in the spleen accompanied with the decreases of IFN-γ levels in the sera and liver homogenates

Mice were i.v. injected with anti-NK1·1 Ab or PBS at 3 days before the Listeria (1 × 106 CFU) challenge. Although the depletion of NK and NKT cells by anti-NK1·1 Ab did not affect the number of Listeria in the spleen at both 12 and 24 h after the Listeria challenge (Fig. 6a,b), it significantly increased the number of Listeria in the liver at 24 h as compared to PBS-treated mice despite no any difference in the bacterial numbers at 12 h between the two groups (Fig. 6c,d). The sera were obtained from the mice at 3, 6, 12, 24 and 48 h after the Listeria challenge. The depletion of NK and NKT cells significantly decreased the serum IFN-γ level but not IL-12 level at 24 h as compared to PBS-treated mice (Fig. 7a,b). Supernatants of the tissue homogenates of the liver and spleen were also obtained from the other mice at 24 h. The results revealed that the depletion of NK and NKT cells decreased the IFN-γ level in the liver but not in the spleen after Listeria challenge (Fig. 7c,e) while it did not affect the IL-12 levels in both the liver and the spleen (Fig. 7d,f).

Fig. 6.

Fig. 6

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The effects of depletion of NK cells and NKT cells on the bacterial count in the spleen (a, b) and the liver (c, d) after the Listeria challenge. The mice were i.v. injected with anti-NK1·1 Ab (200 µg) or PBS at 3 days before the Listeria challenge (1 × 106 CFU). The liver and spleen were obtained from the mice at 12 and 24 h after the Listeria challenge to count the number of bacteria. The data are the means ± SE from 5 mice in each group.

Fig. 7.

Fig. 7

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The effects of depletion of NK and NKT cells on the IFN-γ and IL-12 levels of the sera (a, b) and the tissue homogenates of the spleen (c, d) and liver (e, f) after Listeria challenge. The mice were i.v. injected with anti-NK1·1 Ab (200 µg) or PBS at 3 days before the Listeria challenge (1 × 106 CFU). The sera were obtained from the mice immediately before the Listeria injections and at 3, 6, 12, 24 and 48 h to measure the IFN-γ and IL-12 levels. Similarly, the livers and spleens were also obtained from the mice to measure the IFN-γ and IL-12 levels of tissue homogenates. The data are the means ± SE from 5 mice in each group.

Discussion

In the present study, we showed that the Listeria rapidly proliferated in the very early phase in the spleen after a Listeria challenge. Consistently, the spleen produced a very small amount of IFN-γ and IL-12 until 6 h after the infection. However, the spleen started to extensively produce IL-12 and IFN-γ after the initial phase when the spread of Listeria to the spleen has already occurred. In contrast, the liver of either splenectomized or sham mice produced a substantial amount of Th1 cytokines in the early phase of infection. However, although the Listeria in the liver of sham mice started to vigorously increase beyond 24 h after Listeria injection, the Listeria did not increase in the liver of the splenectomized mice. IL-18 treatment immediately after Listeria challenge increased the cytokine production from the spleen and liver in the early phase and decreased the Listeria number of both organs and thereby increasing the mouse survival.

Although a splenectomy reduces the protective function of the hosts against various bacterial infections, it also renders the hosts extremely resistant to infection with Listeria. Actually, splenectomized mice showed a remarkably decreased amount of viable bacteria in the liver than the sham mice at 72 h after Listeria challenge and thereby may significantly improve their survival. However, the splenectomized mice failed to increase the serum Th1 cytokines after Listeria challenge, although the Th1 response is indispensable for eliminating Listeria from the host [37,49–52]. Wood et al. [40] also reported the decrease of Listeria-induced Th1 cytokines in the splenectomized mice. A down-regulation of the Th1 response in splenectmized mice seems to conflict with the increased resistance to a Listeria infection. In marked contrast, no difference in the mouse survival or the serum changes of Th1 cytokines were observed after an E. coli challenge between the mice with and without a splenectomy. A splenectomy thus might not seriously affect an E. coli infection. As reported previously, we also confirmed that splenectomized mice are extremely susceptible to Streptococcus pneumoniae infection (data not shown). Infections with Listeria, a facultative intracellular bacterial parasite, are thus very unique and quite different from E. coli infections and encapsulated bacteria, probably because Listeria can grow in the macrophages in the spleen as well as in the liver after i.v. injection.

It is noteworthy that Listeria rapidly grew in the spleen of the sham mice until 24 h, and furthermore, the infected spleen markedly increased the Th1 cytokine levels at 24 h when the serum Th1 cytokine levels peaked, regardless of the fact that no difference was observed in the number of Listeria or Th1 cytokine levels in the liver between the mice with and without a splenectomy. Nomura et al. reported that Listeriolysin O, a cytolytic virulence factor of Listeria monocytogenes, could induce the splenocytes to produce Th1 cytokines [35]. Listeria explosively grows in the spleen in the early phase after i.v. injection, and thereafter the infected spleen produces large amount of Th1 cytokines. We therefore suggest that the increase in the serum IL-12 and IFN-γ levels at 24 h after Listeria challenge might be the result of an overflow growth from the infected spleen, and thereby splenectomized mice could not increase their serum Th1 cytokine levels. However, cytokine production from the spleen is too late to protect mice from Listeria.

Although the number of Listeria in the liver was not different until 24 h between the mice with and without splenectomy, it markedly increased in the sham mice than in the splenectomized mice following 48 h. Skamene & Chayasirisobhon [9] and Lannigan et al. [41] also mentioned that the same number of Listeria lodge in the liver on the first day in the mice with and without splenectomy, however, the splenectomized mice subsequently significantly decrease the number of Listeria in the liver from the first day onwards. A remarkable growth of Listeria in the liver thus follows an initial vigorous growth in the spleen that occurs until 24 h after injection, thus suggesting that overwhelming Listeria in the spleen might flow into the liver through the splenic vein and then grow into the liver, because the splenic vein is not only a major drain of the spleen but also a major feeder of the liver. Consistent with this speculation, we observed the existence of bacteria in the splenic vein but not in the inferior vena cava at 24 h after Listeria challenge (our unpublished observation). We therefore consider that Listeria could not markedly grow in the liver of the splenectomized mice because the asplenic mice have no initial growth sites for Listeria.

The natural course of an experimental Listeria infection in mice is such that the liver captures over 90% of an i.v. injected inoculum of bacteria and the remaining 10% can be found in the spleen [53]. However, the initial explosive growth of Listeria occurs in the spleen but not in the liver. It is noteworthy that the Th1 cytokine levels of tissue homogenate were significantly lower in the spleen than in the liver within the initial 6 h. Furthermore, spleen leucocytes did not produce IFN-γ in response to the Listeria infection in this initial period, although the liver leucocytes promptly produced IFN-γ, thus suggesting that Listeria might exploit the insufficient Th1 environment of the spleen and then explosively grow there. The liver contains abundant NK cells and NKT cells that promptly produce a large amount of IFN-γ after Listeria challenge [54]. In contrast, NK cells and NKT cells are only minor populations in the spleen. The liver leucocytes, in particular NK and NKT cells could therefore markedly increase the IFN-γ production at 3 and 6 h after the Listeria challenge than the spleen leucocytes. We also found that the deletion of NK cells and NKT cells increased the number of Listeria in the liver of the mice but decreased the serum IFN-γ levels of the mice at 24 h after the infection. The liver NK and NKT cells might thus contribute to the up-regulation of Th1 environment in the liver and thereby decrease Listeria growth during the initial period in the liver. On the other hand, the spleen does not have such a quickly up-regulated Th1 response because of an insufficient amount of NK and NKT cells. The Th1 cytokine-mediated immune reactions are no doubt essential for the host defense against Listeria infections, however, the increase in the levels of systemic Th1 cytokines does not always reflect the local immunological events.

IL-18 induces production of a large amount of IFN-γ[49,50,55–57]. IL-18 treatment may therefore promptly increase the tissue IFN-γ levels of the spleen as well as of the liver during this initial period and thereby suppress Listeria growth in the spleen. Taken together, our findings demonstrated an important mechanism by which Listeria exploit the spleen, which acts as an initial incubator.

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