INTRODUCTION

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Interleukin-10 (IL-10) is a pleiotropic cytokine produced by monocytes, macrophages, T helper-2 (Th2) cells, and B cells (26) and has been shown to inhibit cytokine secretion by Th1 cells and NK cells (13). Furthermore, IL-10 downregulates macrophage functions such as the secretion of proinflammatory cytokines like tumor necrosis factor alpha (TNF) and interleukin-12 (IL-12) (10) and the production of reactive oxygen intermediates and reactive nitrogen intermediates (3). However, IL-10 stimulates B cells (14), enhances T-cell proliferation (7, 23) and differentiation (6), increases FcR expression on monocytes (35), and enhances phagocytosis by monocytes (5).

As a consequence of the downregulation of macrophage function, endogenous IL-10 decreases the innate resistance against an infection with Mycobacterium avium (2, 11), Brucella abortus (12), Trypanozoma cruzi (32), and Salmonella choleraesuis (1). However, neutralization of endogenous IL-10 did not affect the course of a Salmonella enterica serovar Typhimurium infection (30).

Listeria monocytogenes is a facultative intracellular pathogen which induces a macrophage- and T-cell-dependent response by the host (16). During the first phase of a primary Listeria infection in mice, when granulocytes and macrophages limit the proliferation of bacteria in the liver and spleen (8, 15, 31, 33), resident and exudate macrophages secrete TNF and IL-12, which together induce the production of gamma interferon (IFN-) by NK cells (37, 38). During the second phase of a primary Listeria infection, IFN- stimulates T cells which in cooperation with macrophages and granulocytes ensure the eradication of bacteria from the liver and spleen (21).

When mice that have acquired resistance to L. monocytogenes are reinfected with the organism (i.e., subjected to a secondary Listeria infection), the elimination of bacteria is also dependent on granulocytes, resident and exudate macrophages, and the activation of macrophages by T cells (15, 21, 25, 31). During the course of a secondary infection, TNF, IL-12, and IFN- are secreted earlier than during a primary infection (27, 39). Recently, we reported that TNF is essential for the elimination of L. monocytogenes from the livers and spleens of Listeria-immune mice (34), while IFN- (27, 34) and IL-12 (39) play a less prominent role.

During a primary Listeria infection, neutralization of endogenous IL-10 has been shown to have a dual effect on the proliferation of Listeria in mice: the proliferation of bacteria in the liver and spleen decreased during the first 5 days of infection but increased from days 7 to 11 (41). The mechanisms by which IL-10 affects resistance to Listeria infection were not resolved.

The aim of this study was to investigate the effect of IL-10 on the course of a primary and a secondary L. monocytogenes infection in mice.

	MATERIALS AND METHODS

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Mice. Female, specific-pathogen-free CBA/J mice and BALB/c mice, aged 6 to 8 weeks, were purchased from IFFA Credo (Saint Germaine-sur-l'Abersle, France) and given dry food (Hope Farms, Woerden, The Netherlands) and tap water ad libitum. CBA/J mice were used for all experiments unless otherwise indicated.

Bacteria. L. monocytogenes strain EGD was maintained virulent by repeated passage through CBA/J mice and stored on blood agar plates at 4°C. The bacteria were cultured in tryptose phosphate broth for 18 h at 37°C, collected by centrifugation (10 min; 900 × g), washed in phosphate-buffered saline (pH 7.4), and resuspended in pyrogen-free saline.

Induction of a primary or a secondary L. monocytogenes infection in mice. A primary L. monocytogenes infection was induced in naive CBA/J mice by injecting 5 × 10³ bacteria (1 50% lethal dose [LD₅₀]) intravenously (i.v.) and in naive BALB/c mice by injecting 5 × 10⁴ bacteria i.v. CBA/J mice were rendered immune by injection of 0.1 LD₅₀ of L. monocytogenes i.v. After 3 weeks, these mice were reinfected with 10 LD₅₀ of L. monocytogenes i.v. (secondary infection).

Treatment with IL-10 during L. monocytogenes infection. Based on the results of another study (20), IL-10 was given once a day in a dose of 70 U/mouse i.v. during a primary infection, starting 1 day before infection and continuing until the end of the experiment. In a preliminary study, two different batches of IL-10 were compared and found to have the same effects (data not shown). In some experiments, each mouse was given 280 U of IL-10 to check whether a larger dose had a greater effect. Control mice received the same dilution of supernatant from mock-transfected cells (mock supernatant) during a primary Listeria infection. During a secondary infection, IL-10 was given once a day in a dose of 70 U/mouse i.v., starting 1 day before reinfection with 10 LD₅₀ of L. monocytogenes and continuing until the end of the experiment. Control mice received the same dilution of mock supernatant during a secondary Listeria infection.

Macrophages. Resident peritoneal macrophages of naive mice or mice with a primary Listeria infection were collected by peritoneal lavage with 2 ml of ice-cold saline containing 50 U of heparin/ml and cultured in a concentration of 10⁶ macrophages/ml in HEPES-buffered RPMI 1640 medium (Flow Laboratories, Irvine, United Kingdom) supplemented with 10% heat-inactivated fetal calf serum (GIBCO Laboratories, Grand Island, N.Y.), 2 mM L-glutamine (Flow Laboratories), streptomycin (50 µg/ml; Biochemie GmbH, Vienna, Austria), and sodium penicillin G (1,000 U/ml; Yamanouchi, Leiderdorp, The Netherlands), hereafter referred to as RPMI.

Inhibition of Toxoplasma gondii proliferation. The most appropriate measure of the effect of IL-10 treatment on macrophage activation would be the listericidal activity of macrophages. However, preliminary results indicated that peritoneal macrophages isolated from mice with a primary Listeria infection contain a variable number of viable bacteria and thus are unsuitable for such an assay. Therefore, we used inhibition of T. gondii proliferation, which is a reliable indicator of macrophage activation in vitro or in vivo (22). Peritoneal macrophages of mice treated with IL-10 or mock supernatant during a primary Listeria infection received no additional stimulus prior to infection with T. gondii. T. gondii proliferation is expressed as the fold increase in the number of T. gondii tachyzoites, i.e., the ratio of the number of T. gondii tachyzoites per 100 infected macrophages after 18 h of incubation to the number of T. gondii tachyzoites per 100 infected macrophages at the start of the assay (22).

Cytokines. Supernatant from COS cells transfected with murine IL-10 cDNA and mock supernatant were a generous gift from J. E. de Vries (DNAX, Palo Alto, Calif.). The IL-10 supernatant contained 7,000 U of IL-10/ml, where 1 U/ml gives 50% of the maximum response measured on MC/9 cells (36, 40). Recombinant rat IFN- was provided by P. H. van der Meide (Department of Immunobiology, Biomedical Primate Research Center, Rijswijk, The Netherlands). Recombinant mouse TNF was kindly provided by P. de Waele (Innogenetics NV, Gent, Belgium). The endotoxin concentration in the cytokine solutions and mock supernatant was below 20 pg/ml, as determined with a Limulus assay.

Antibodies. Rat anti-mouse TNF monoclonal antibodies (MAbs) (clone MP6-XT3) and biotin-conjugated rat anti-mouse TNF MAb (MP6-XT22) for enzyme-linked immunosorbent assay were obtained from Pharmingen, San Diego, Calif. Fluorescein isothiocyanate-conjugated anti-mouse CD3 MAb (clone 145-2C11), phycoerythrin (PE)-conjugated anti-mouse T-cell receptor (-TCR) MAb (clone H57-597), PE-conjugated anti-mouse -TCR MAb (clone GL3), and PE-conjugated anti-mouse CD8 MAb (clone 01045B) for fluorescence-activated cell sorting analysis were purchased from Pharmingen. PE-conjugated anti-mouse CD4 MAb (clone L3T4) was obtained from Becton Dickinson, San Jose, Calif. Anti-mouse IL-10 MAb (clone 2A5) for neutralization of endogenous IL-10 was purchased from Endogen, Cambridge, Mass.

Cytokine measurements in plasma. The concentration of IFN- in plasma was measured with a sandwich enzyme-linked immunosorbent assay as instructed by the manufacturer (Endogen). The standards had been calibrated to the reference standard of the National Institute for Biological Standards and Controls (NIBSC; South Mimms, United Kingdom), where 1 pg of IFN- equals 1 NIBSC unit (reference lot Gg-02-901-533).

Neutralization of endogenous IL-10 during a primary L. monocytogenes infection. Once every 2 days, the mice received 25 µg of anti-IL-10 MAb i.v. beginning 18 h prior to infection with 1 LD₅₀ of L. monocytogenes i.v. until the end of the experiment. Control mice received saline.

Flow cytometric analysis of splenocytes. On various days of a primary infection, a segment of the spleen was taken and gently pressed through a nylon filter to obtain a single-cell suspension. The cells were passed over a cotton-wool column at 4°C to remove cell clumps, washed twice with ice-cold RPMI, counted, and resuspended in ice-cold Boseral (Organon Teknika, Boxtel, The Netherlands) to a final concentration of 10⁷ cells/ml. Aliquots of 10⁶ cells were incubated on ice for 30 min with optimal concentrations of fluorescein isothiocyanate-conjugated anti-mouse CD3 MAb in combination with PE-conjugated anti-mouse -TCR MAb, PE-conjugated anti-mouse -TCR MAb, PE-conjugated anti-mouse CD4 MAb, or PE-conjugated anti-mouse CD8 MAb. After incubation, the cells were washed and resuspended in Boseral, and fluorescence was measured using a FACScan (Becton Dickinson); 30,000 events were analyzed per sample. Cells not incubated with MAb, but otherwise treated similarly, were used to determine background fluorescence. Dead cells were identified with propidium iodide and excluded from analysis.

Statistical analysis. Results were expressed as the mean ± standard deviation (SD) of at least six mice per time point unless otherwise indicated. Differences between various groups at one time point were assessed using a one-way analysis of variance with a Fisher least-significant-difference comparison report. Differences between various groups during the time course were assessed by a two-way analysis of variance. For all analyses, the level of significance was set at 0.05.

	RESULTS

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Effect of IL-10 on the course of a primary Listeria infection. First, the effect of treatment with 70 U of IL-10 on the proliferation of bacteria during a primary infection was assessed. On the first day of infection, the number of bacteria recovered from the spleens of IL-10-treated CBA/J mice was approximately 1 log₁₀ lower than that of control mice which received mock supernatant, a difference which remained over a period of 9 days (Fig. 1A and inset). On days 1 and 3 of infection, the ratio of spleen weight to body weight was significantly higher for the IL-10-treated mice than for the control mice (day 1, [2.89 ± 0.34] × 10³ for control mice and [3.63 ± 0.62] × 10³ for IL-10-treated mice; day 3, [4.81 ± 0.51] × 10³ for control mice and [6.19 ± 0.30] × 10³ for IL-10-treated mice). In the livers of IL-10-treated mice, the number of bacteria was not significantly different from that in the livers of control mice (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Numbers of L. monocytogenes organisms in spleens and livers of IL-10-treated CBA/J mice with a primary or secondary infection. A primary infection was induced by injection of 1 LD50 of L. monocytogenes i.v. (A and B). A secondary infection was induced by injecting first 0.1 LD50 and 3 weeks later 10 LD50 of L. monocytogenes i.v. (C and D). Starting 1 day before infection, the mice were treated once a day with 70 U of IL-10 () or mock supernatant () i.v. until the end of the experiment. On various days of infection, the numbers of bacteria in the spleen (A and C) and liver (B and D) were assessed. In another experiment (inset), the time of observation during a primary infection was prolonged to 9 days. Values are log10 means (±SD) of viable bacteria in the organs of six mice.

Effect of IL-10 on the course of a secondary Listeria infection. During an infection in immune mice, the number of listeriae recovered from the spleens of IL-10-treated CBA/J mice was significantly lower on days 1 and 2 than the number recovered from control immune mice (Fig. 1C). However, on days 3 and 4, the numbers of bacteria in the spleens of IL-10-treated and control immune mice did not differ significantly (Fig. 1C). The number of L. monocytogenes cells recovered from the livers of immune mice treated with IL-10 during a secondary infection was significantly higher than in control immune mice during days 2, 3, and 4 of infection (Fig. 1D).

Histology. The spleens of control mice with a primary Listeria infection showed numerous necrotic cells with pyknotic nuclei in the inner periarteriolar lymphocyte sheaths (PALS) on day 2 (Fig. 2a). The distribution of cells in the inner PALS of control mice was less dense than in the outer PALS (Fig. 2a). In the spleens of IL-10-treated mice, such lesions could not be found (Fig. 2b). The livers of IL-10-treated mice showed small inflammatory foci on day 2 of a primary infection that were similar to the foci in the liver of control mice (data not shown). The livers and spleens of mice during a secondary Listeria infection revealed no clear differences between IL-10-treated and control immune mice (data not shown).

View larger version (163K): [in this window] [in a new window]   FIG. 2.   Morphology of the spleen of an IL-10-treated mouse during a primary L. monocytogenes infection. A primary infection was induced by injection of 1 LD50 of L. monocytogenes i.v. Starting 1 day before infection, the mouse was treated once a day with mock supernatant (a) or 70 U of IL-10 (b) i.v. until the end of the experiment. On day 2 of infection, a segment of the spleen was removed, fixed in 10% formaldehyde, and embedded in paraffin. The segments were sectioned and stained with hematoxylin and eosin. The inset shows the area surrounding the central arteriole in the inner PALS (iP) in greater detail. MZ, marginal zone. Magnifications: A, ×50; B, ×80; insets, ×200.

Toxoplasmastatic activity as a measure of macrophage activation. On days 2 and 4 of a primary Listeria infection, peritoneal macrophages of IL-10-treated mice did not inhibit T. gondii proliferation as efficiently as macrophages of control mice treated with mock supernatant (Fig. 3). These results indicate that treatment of mice with IL-10 reduces activation of macrophages in vivo.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Inhibition of T. gondii proliferation by peritoneal macrophages of IL-10-treated mice with a primary L. monocytogenes infection. A primary infection was induced by injection of 1 LD50 of L. monocytogenes i.v. Starting 1 day before infection, the mice were treated once a day with 70 U of IL-10 or mock supernatant i.v. until the end of the experiment. On days 2 and 4 of infection, peritoneal macrophages were collected for assessment of toxoplasmastatic activity after 18 h of culture with T. gondii. Fold increase of the number of T. gondii parasites was calculated as the ratio of the number of T. gondii parasites per 100 infected macrophages after 18 h of incubation to the number of T. gondii parasites per 100 infected macrophages at the start of the assay. Values are means ± SD (n = 6).

Effect of IL-10 on the plasma concentration of IFN- during primary and secondary L. monocytogenes infections in mice. During a primary infection in control mice, the mean plasma concentration of IFN- increased on day 2 of infection and decreased thereafter (Fig. 4A). On day 2 of infection in IL-10-treated mice, the mean concentration of IFN- was approximately eight times lower than in control mice and remained at that level on later days (Fig. 4A).

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Concentration of IFN- in plasma of mice treated with IL-10 during a primary or secondary L. monocytogenes infection. A primary infection was induced in mice by injection of 1 LD50 of L. monocytogenes i.v. (A). A secondary infection was induced by injecting first 0.1 LD50 and 3 weeks later 10 LD50 of L. monocytogenes i.v. (B). Starting 1 day before infection, the mice were treated once a day with 70 U of IL-10 () or mock supernatant () i.v. until the end of the experiment. On various days of infection the concentration of IFN- in plasma were assessed. Values are means for three mice from one representative experiment. An asterisk indicates a significant difference (P < 0.05).

Analysis of splenocytes of mice treated with IL-10 during a primary L. monocytogenes infection. Since IL-10 can enhance T-cell proliferation and since T cells can be distinguished from other spleen cells by the expression of CD3, we investigated the expression of this marker on spleen cells. At all time points, three populations could be distinguished: a CD3-negative (CD3^neg) population of cells that varied in size, a population of CD3 mildly positive (CD3^lo) cells which were larger in size than the CD3^neg cells, and a population of strongly positive CD3 (CD3^hi) cells which were of intermediate size (Fig. 5).

View larger version (70K): [in this window] [in a new window]   FIG. 5.   Flow cytometric analysis of CD3neg, CD3lo, and CD3hi cells in spleens of IL-10-treated mice with a primary L. monocytogenes infection. A primary infection was induced by injection of 1 LD50 of L. monocytogenes i.v. Starting 1 day before infection, the mice were treated once a day with 70 U of IL-10 or mock supernatant i.v. until the end of the experiment. On various days of infection, the splenocytes were isolated and analyzed by flow cytometric analysis. At all time points, three distinct populations could be distinguished on the basis of forward scatter (FSC) and CD3 staining. Data of a representative experiment are shown.

Effect of treatment with anti-IL-10 MAb on the percentage of CD3^lo spleen cells during a primary L. monocytogenes infection. Since the percentage of CD3^lo spleen cells in control mice increased from day 2 until day 6 of infection and since the percentage of CD3^lo spleen cells was already increased on day 2 of infection in IL-10-treated mice (Table 1), we investigated whether neutralization of endogenous IL-10 affected the percentage of CD3^lo spleen cells. On day 6 of infection, the percentage of CD3^lo cells in the spleens of anti-IL-10-treated mice (18.38% ± 1.67%) was significantly lower (P < 0.05, n = 5) than in phosphate-buffered saline-treated mice (23.33% ± 1.52%). These results suggest that endogenous IL-10 stimulates an increase in the percentage of CD3^lo cells in the spleens of mice with a primary Listeria infection.

	DISCUSSION

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The results of this study show that treatment of mice with IL-10 has divergent effects on the proliferation of Listeria in the spleen and liver. During a primary and a secondary infection, treatment with IL-10 decreases the number of bacteria in the spleen. However, IL-10 did not affect the number of Listeria in the liver during a primary infection and increased the number of bacteria during a secondary infection. Biological activity of IL-10 has also been demonstrated by a decrease of the toxoplasmastatic activity of macrophages obtained from IL-10-treated, Listeria-infected mice, which had decreased IFN- levels in the circulation compared to mock-treated animals.

Our finding that during a primary infection treatment with IL-10 enhances the elimination of Listeria from the spleen and does not affect the number of bacteria in the liver is supported by the reports that neutralization of endogenous IL-10 during a primary Listeria infection increases the number of bacteria in the liver and spleen during later days of infection (28, 41). On the other hand, our finding is contradictory to reports that resistance to Listeria infection is enhanced in anti-IL-10-treated mice or IL-10-deficient mice during the initial phase of a primary infection (9, 41).

The increased anti-Listeria activity in the spleen is supported by the absence of necrotic lesions in the spleens of IL-10-treated mice with a primary infection, whereas numerous necrotic lesions were observed in control mice. The ratio of splenic to body weight in IL-10-treated mice was significantly increased compared to control mice, indicating an increased number of cells in the spleen. Several types of cell could account for this increase in cell number. First, influx of granulocytes and monocytes into the spleen, which is essential for the elimination of Listeria during an infection (33), may be increased by IL-10 (20, 42). However, in our study histological examination of the spleens of IL-10-treated mice showed no accumulation of granulocytes or monocytes. Second, the increase in the number of spleen cells may be due to an increase in T cells induced by IL-10. This is supported by our finding that IL-10 treatment induced an increase in the percentage of CD3^lo spleen cells as early as day 2 of infection, whereas in control mice an increase in the percentage of CD3^lo cells occurred on day 6 of infection. Since T cells are mediators of resistance to Listeria (17, 19), and IL-10 stimulates the cytolytic activity of CD8⁺ T cells in vitro (6), it is likely that CD3^lo CD8⁺ T cells are involved in the increased listericidal activity. It is not clear how IL-10 increases the percentage of T cells in the spleen. Possibly, IL-10 stimulates the differentiation of CD3^neg into CD3^lo T cells (7, 23, 26). Not only exogenous IL-10 affects the percentage of T cells in the spleen; endogenous IL-10 stimulates the early development of CD3^{lo lo} T cells during a Listeria infection. This has been demonstrated by our finding that the increase in the percentage of CD3^{lo lo} T cells on day 6 of a primary infection could be inhibited by neutralization of endogenously formed IL-10. These findings strongly agree with the report that neutralization of IL-10 increases the number of bacteria in the liver and spleen at later days of infection (41) and may explain why treatment of T-cell-deficient SCID mice with IL-10 leads to a decreased resistance only against a primary Listeria infection (20).

Treatment with IL-10 decreased the concentration of IFN- in plasma of mice with a primary Listeria infection, which is supported by the finding that addition of IL-10 to a culture of macrophages and T cells stimulated with heat-killed Listeria leads to inhibition of IL-12 release from macrophages which results in inhibition of IFN- secretion by T cells (18, 29). Since CBA/J mice have a cytokine response which is skewed to Th1-type cytokines like IFN-, it could be argued that these mice would be more sensitive to treatment with IL-10. BALB/c mice preferentially respond with a Th2-type cytokine response. Therefore, we investigated whether treatment of BALB/c mice with IL-10 during a primary Listeria infection had comparable effects on Listeria proliferation as in CBA/J mice. The proliferation of Listeria in the livers and spleens of BALB/c mice was comparable to that in the livers and spleens of CBA/J mice, indicating that the effect of IL-10 is independent of the genetic background of the mouse strain.

During a secondary Listeria infection, treatment with IL-10 led to an increase in the number of bacteria in the liver. This effect of IL-10 could be caused in part by the initial decrease in IFN- in the circulation although IFN- is not essential for resistance against a secondary Listeria infection (34). However, other mechanisms must play a role as well, such as the inhibitory effect of IL-10 on macrophage activation that may lead to a decrease in the anti-Listeria activity in the liver.

Previously, it was shown that IL-10 decreases IFN- secretion by splenocytes from SCID mice stimulated with heat-killed Listeria (37). However, 10 to 45% of the IFN- production by splenocytes from Listeria-immune mice stimulated with heat-killed Listeria is independent of IL-12 secretion (39). These findings could explain why in our study of IL-10-treated mice with a secondary Listeria infection the concentration of IFN- was reduced only on day 1 of infection and not affected at later times postinfection.

Taken together, the available data indicate that the effects of IL-10 on the proliferation of Listeria in the spleen and liver during a primary and a secondary infection are probably the result of a combination of stimulation of anti-Listeria resistance, possibly by an increase in the number of CD3^{lo lo} T cells, and inhibition of anti-Listeria resistance by downregulation of both Th1 cytokine production and macrophage activation.