Roles for Tumor Necrosis Factor and Gamma Interferon in Resistance to Enteric Listeriosis

Abstract

Listeria monocytogenes normally infects the host by translocating from the intestinal lumen. Experiments were carried out to determine if, when, and where tumor necrosis factor (TNF) and gamma interferon (IFN-γ) function in antibacterial resistance during enteric listeriosis. Groups of normal mice and severe combined immunodeficient (SCID) mice were injected with neutralizing monoclonal antibodies (MAb) specific for each cytokine and then inoculated intragastrically with L. monocytogenes. The course of infection was monitored by enumerating listeriae in gut-associated lymphoid tissues, livers, and spleens. By the third day of infection, bacterial numbers in infected tissues and organs were greatly exacerbated in all mice treated with anti-TNF MAb, whereas bacterial numbers in the organs of mice treated with anti-IFN-γ MAb did not differ from those present in the respective organs of control mice. However, by the fifth day of infection, bacterial numbers in the organs of anti-IFN-γ MAb-treated normal mice and SCID mice were much greater than in the corresponding organs of control mice. Experiments with Listeria-immune mice revealed that TNF and IFN-γ are involved in the expression of anti-Listeria memory immunity; however, it was also found that the anti-IFN-γ MAb was relatively ineffective in inhibiting the expression of anti-Listeria immunity, whereas a polyclonal anti-IFN-γ was quite effective.

Mouse listeriosis is a widely used model for the study of host resistance mechanisms that are expressed against intracellular bacteria. The importance of gamma interferon (IFN-γ) and tumor necrosis factor (TNF) in host anti-Listeria resistance in mice has been extensively studied. Buchmeier and Schreiber (4) showed that listeriosis was exacerbated in mice treated with anti-IFN-γ monoclonal antibody (MAb). Work carried out in this laboratory (15, 16) and in others (28) showed that anti-TNF antibody treatment of mice converted a normally immunizing infection initiated by the intravenous injection of a sublethal Listeria monocytogenes inoculum into a lethal infection. Likewise, anti-TNF antibody treatment exacerbated listeriosis in immunoincompetent nude (nu/nu) mice (14, 15). Gene knockout mice lacking functional membrane receptors for either IFN-γ or TNF were found to be considerably more susceptible to listeriosis than the wild-type mice (20, 32, 35).

Most studies investigating host cells and cytokines in anti-Listeria resistance have involved mice infected by parenteral routes of inoculation; however, L. monocytogenes normally infects the host by translocating from the intestinal lumen and then spreading to internal organs. MacDonald and Carter (24) reported that listeriae present in the lumens of the gastrointestinal tracts of mice were capable of infecting Peyer’s patches (PP) in a manner similar to that of Salmonella typhimurium (5). PP-associated listeriae were shown to be capable of entering mesenteric lymph nodes (MLN), from which listeriae are capable of infecting other internal organs, including the liver and spleen (24). Moreover, invading listeriae caused intestinal intraepithelial lymphocytes to secrete IFN-γ (42). In light of these observations and of the knowledge that TNF acts as a cofactor in the induction of IFN-γ, experiments were carried out to determine whether IFN-γ and TNF play roles in innate resistance and specifically acquired antibacterial resistance in the gut-associated lymphoid tissues and other infected organs following the translocation of listeriae from the intestinal lumens of mice.

Mice.

Male BALB/c mice 8 to 12 weeks of age were purchased from either Charles River Laboratories (Wilmington, Mass.) or Taconic Farms (Germantown, N.Y.). C.B-17 severe combined immunodeficient (SCID) mice were purchased from Jackson Laboratories (Bar Harbor, Maine). BALB/c mice were maintained under pathogen-free husbandry conditions, while immunoincompetent SCID mice were maintained in autoclaved microisolator cages provided with sterile food and water.

L. monocytogenes.

L. monocytogenes (strain EGD, serotype 1/2a) was grown overnight at 37°C in Trypticase soy broth (BBL Microbiology Systems, Becton Dickinson, Cockeysville, Md.). The culture broth was centrifuged at 800 × g for 20 min, and the pelleted bacteria were resuspended in Dulbecco’s phosphate-buffered saline (DPBS), pH 7.4. The stock culture, having a titer of 6.6 × 10⁹ CFU/ml, was aliquoted in tubes and stored at −70°C. Immediately before use, stock preparations were quick-thawed and diluted in DPBS (pH 7.4). The intravenous 50% lethal dose for L. monocytogenes in BALB/c Crl mice was determined to be 4 × 10³ CFU. The standard intragastric (i.g.) inoculum was 2 × 10⁸ CFU in 0.2 ml of DPBS. Mice were gavaged i.g. with an 18-gauge feeding needle (Popper, Long Island City, N.Y.).

Enumeration of organ-associated bacteria.

Organ homogenates of livers, spleens, MLN, and PP were prepared by grinding organs suspended in iced sterile saline (0.85%) with a motorized Teflon pestle. Enumeration of bacterial CFU in the organ homogenates was determined by plating serial 10-fold dilutions of liver, spleen, or mesentery or mesenteric lymph node homogenates on Trypticase soy agar (BBL Microbiology Systems, Becton Dickinson). Bacterial CFU in homogenates of the Peyer’s patches were plated on Listeria-selective phenylethanol (PEA) agar consisting of 1.5% Noble agar, 1.5% Trypticase peptone, 0.5% phytone peptone, 0.5% NaCl, 1.0% glycine, 0.05% LiCl, and 0.25% PEA (24). When illuminated with oblique light, L. monocytogenes colonies on PEA agar were identified by their characteristic light-blue color. Tests for esculin, catalase, and/or motility were performed to ensure that questionable colonies on PEA agar were indeed L. monocytogenes.

Anticytokine antibodies.

The R4-6A2 hybridoma (ATCC HB170), which secretes a rat anti-murine IFN-γ MAb (immunoglobulin G1 [IgG1]), and the XT3.11 hybridoma (DNAX Research Institute, Palo Alto, Calif.), which secretes rat anti-murine TNF-alpha (α) MAb (IgG1) were grown as ascites in the peritoneal cavities of pristane-primed CB6F₁ mice according to our published procedures (1, 17). The R4-6A2 anti-IFN-γ MAb and the XT3.11 anti-TNF MAb were purified from ascitic fluids according to previously published procedures (17). A rabbit anti-IFN-γ polyclonal IgG antibody was generated by immunizing a New Zealand White female rabbit with pure recombinant mouse IFN-γ having a specific activity of 10⁷ antiviral U/ml, the kind gift of Genentech, Inc. (South San Francisco, Calif.). The rabbit anti-IFN-γ IgG or rabbit control IgG was purified from serum according to published procedures (18). The various purified antibody preparations were assayed for endotoxin concentrations by means of a quantitative chromogenic Limulus amebocyte lysate assay (Whittaker Bioproducts, Walkersville, Md.). The quantification of the anti-IFN-γ- and anti-TNF MAb-neutralizing activities was performed as previously reported (18, 38).

Anticytokine antibody treatment of mice.

Mice were injected intraperitoneally with a given antibody preparation 4 h prior to the i.g. inoculation of bacteria. Mice were injected with 10⁵ neutralizing units (NU) of the R4-6A2 rat anti-IFN-γ MAb (specific activity, 1.8 × 10⁵ NU/mg) in PBS (pH 7.4). Mice injected with the XT3.11 rat anti-TNF MAb received 2 × 10⁴ NU (specific activity, 6 × 10³ NU/mg) in PBS (pH 7.4). The mice that were injected with the rabbit anti-IFN-γ IgG were given 2 × 10⁴ NU (specific activity, 2 × 10³ NU/mg), while the corresponding control mice were injected with an equivalent amount (in milligrams) of control rabbit IgG. At the time of sacrifice, antibody-treated mice were anesthetized and bled by cardiac puncture, and sera were collected and assayed to ensure that excess amounts of the anticytokine were present in the blood throughout the course of the experiments. In all cases, antibody titers exceeded 10³ NU/ml of blood.

Statistical analysis.

The experimental results were compared with Student’s t test, which requires that the populations be normally distributed and have equal variances. A significant difference between experimental groups was defined by a P value of <0.05. Experiments involving statistical comparisons were performed with 3 to 5 mice per group.

The importance of TNF and IFN-γ in resistance to an immunizing Listeria enteric infection.

The inhibition of cytokine-mediated effects in vivo by the administration specific antibodies has proven effective for establishing the importance of a cytokine in host resistance to infectious agents (4, 8, 15, 16). Experiments were carried out with specific anti-TNF- or anti-IFN-γ MAb-treated mice to determine if TNF and IFN-γ are involved in resistance to enteric listeriosis. Groups of BALB/c mice were injected intraperitoneally with anti-TNF MAb or anti-IFN-γ MAb and inoculated i.g. 4 h later with 2 × 10⁸ CFU of L. monocytogenes. The course of enteric listeriosis was then monitored at progressive times by enumeration of the listerial CFU in the PP, MLN, livers, and spleens of the treated mice and control mice. By the end of the first day of infection, no significant differences existed in numbers of listeriae present in the corresponding organs (MLN, livers, and spleens) of mice in the different experimental groups of mice (data not shown). Moreover, based on the limits of detection of the assay, listeriae were absent from the PP of control mice groups (results not presented). However, by day 3 of infection (Fig. 1), the numbers of listeriae in the organs of the anti-TNF MAb-treated mice were greatly exacerbated, whereas numbers of listeriae were elevated only in the MLN of the anti-IFN-γ-treated mice. Also, at this time anti-TNF MAb-treated mice were lethargic and hypothermic, and most died by day 5 of infection. Enumeration of listeriae in the organs of the one remaining anti-TNF MAb-treated mouse on day 5 of infection revealed overwhelming numbers of listeriae in the organs (Fig. 1). On day 5 of infection, the infected organs of the anti-IFN-γ MAb-treated mice had greater numbers of listeriae than did the corresponding organs of the control mice. These results establish that the effect of TNF is important in antilisterial resistance during the first 3 days of enteric listeriosis, whereas IFN-γ mediates an important effect in resistance after this time.

FIG. 1.

The effect of anti-TNF MAb or anti-IFN-γ MAb treatment on the course of enteric listeriosis. BALB/c mice were injected intraperitoneally with 10⁵ NU of anti-IFN-γ MAb, 3 × 10⁴ NU of anti-TNF, or PBS and all mice were inoculated i.g. 4 h later with 2 × 10⁸ CFU of L. monocytogenes. Organ L. monocytogenes CFU were determined on days 3 and 5 following the i.g. inoculation of bacteria. Data are presented as the means (bars) ± standard deviations of organ CFU for an experimental group. Means lacking standard deviations indicate that either bacterial CFU were below detection limits in one or more organs from an experimental group or that insufficient numbers of mice survived treatment, as for the anti-TNF MAb-treated group (one survivor) on day 5. Dashed horizontal lines represent the detection limits of the assay. Single and double asterisks indicate significant differences from the value for the control mice at P values of <0.05 and <0.01, respectively.

The importance of TNF and IFN-γ in innate immunity to enteric listeriosis.

Results from the foregoing experiment established that an IFN-γ-mediated effect is expressed in Listeria-infected organs of mice after the time (day 3) when the host normally begins to generate a T-cell-mediated anti-Listeria immune response that is capable of resolving infection (25–27). In an attempt to dissociate IFN-γ- or TNF-mediated effects in innate antibacterial immunity from possible effects which could be important in the generation and/or expression of specifically acquired anti-Listeria immunity, immunoincompetent SCID mice were used to determine the importance of these cytokines in innate antibacterial immunity to enteric listeriosis. Groups of C.B-17 SCID mice were inoculated intraperitoneally with anti-TNF MAb or anti-IFN-γ MAb and inoculated i.g. with 2 × 10⁸ CFU of L. monocytogenes 4 h later. Since SCID mice lack discernible PP and MLN, listeriae were enumerated in the mesenteries, livers, and spleens of the MAb-treated SCID mice and control SCID mice on days 3 and 5 of listeriosis. As shown in Fig. 2, anti-TNF MAb treatment, but not anti-IFN-γ MAb treatment of SCID mice greatly enhanced numbers of listeriae in the mesentery, liver, and spleen on day 3 of infection. By day 5 of infection, all anti-TNF MAb-treated SCID mice had succumbed to overwhelming infection, whereas infected SCID mice treated with anti-IFN-γ MAb were only beginning to show signs of morbidity associated with overwhelming bacterial infection. Bacterial numbers present in the mesenteries, livers, and spleens of the anti-IFN-γ MAb-treated SCID mice and control SCID mice on day 5 of listeriosis are also presented in Fig. 2, in which the numbers of listeriae in the organs of the anti-IFN-γ-treated mice are shown to be much greater than in the respective organs of control mice. Thus, the results presented in Fig. 1 and 2 collectively establish that the magnitude and temporal manifestation of TNF- and IFN-γ-mediated antibacterial effects are, respectively, similar in the organs of immunocompetent and immunoincompetent mice during enteric listeriosis.

FIG. 2.

The effect of anti-TNF MAb and anti-IFN-γ MAb treatment on the course of enteric listeriosis in immunoincompetent SCID mice. C.B-17 SCID mice were injected intraperitoneally with 10⁵ NU of anti-IFN-γ MAb, 3 × 10⁴ NU of anti-TNF, or PBS, and 4 h later all mice were inoculated i.g. with 2 × 10⁸ CFU of L. monocytogenes. Organ L. monocytogenes CFU were determined on days 3 and 5 following the i.g. inoculation of bacteria. All anti-TNF MAb-treated mice were dead by day 5 of infection. Data are presented as the means (bars) ± standard deviations of organ CFU. Means lacking standard deviations indicate that bacterial CFU were below detection limits in one or more organs from an experimental group of mice. Dashed horizontal lines represent the detection limits of the assay. Single and double asterisks indicate significant differences from the values for the control mice at P values of <0.05 and <0.01, respectively.

The importance of TNF and IFN-γ in the expression of anti-Listeria memory immunity in the intestine.

The results of the preceding experiments do not provide evidence as to whether TNF and IFN-γ function in the expression of anti-Listeria immunity, because similar results were obtained with normal mice and SCID mice undergoing a primary Listeria enteric infection. To determine whether TNF or IFN-γ plays a role in anti-Listeria immunity in the intestine, Listeria-immune mice immunized by an i.g. inoculation of 2 × 10⁸ CFU of L. monocytogenes 28 days earlier were injected intraperitoneally with either anti-TNF MAb or anti-IFN-γ MAb and challenged with an i.g. dose of 6 × 10⁹ CFU of L. monocytogenes 4 h later. Presented in Fig. 3 are the results of this experiment: the CFU in the PP, MLN, livers, and spleens of the MAb-treated Listeria-immune mice on days 3 and 5 of a secondary challenge. On day 3 of infection, listerial numbers were elevated in the organs of the anti-TNF MAb-treated immune hosts relative to those in the corresponding organs of immune control mice (P < 0.01). At this time, listerial numbers in the respective organs of nonimmune control mice, Listeria-immune control mice, and anti-IFN-γ MAb-treated Listeria-immune mice were similar. However, by day 5 of infection, listeriae were not detected in the organs of the control Listeria-immune mice, whereas substantial numbers of listeriae were present in the corresponding organs of the nonimmune control mice, indicating that memory anti-Listeria immunity is not expressed until after the first 3 days of infection. All anti-TNF MAb-treated Listeria-immune mice were dead by day 5 of infection. On day 5, the bacterial numbers in the organs of the anti-IFN-γ MAb-treated and Listeria-immune mice were only marginally higher than those in the Listeria-immune control mice and were not as high as the bacterial numbers in the organs of the nonimmune control mice. Moreover, the anti-IFN-γ MAb treatment of immune mice did not completely prevent the expression of immunity, since these mice survived the secondary challenge. This result was not due to a lack of serum anti-IFN-γ MAb-neutralizing activity during the course of the experiment, for it was found that substantial quantities of anti-IFN-γ MAb were present in the peripheral circulation of treated mice on day 5 of infection (data not shown).

FIG. 3.

The effect of anti-TNF MAb and anti-IFN-γ MAb treatment on the expression of memory anti-Listeria immunity. BALB/c mice rendered Listeria immune by i.g. inoculation of 2 × 10⁸ L. monocytogenes 28 days earlier were injected intraperitoneally with 10⁵ NU of anti-IFN-γ MAb, 3 × 10⁴ NU of anti-TNF, or PBS. Four hours later, all treated Listeria-immune mice and a group of nonimmune mice were challenged with an i.g. inoculum of 6 × 10⁹ CFU of L. monocytogenes. On days 3 and 5 following rechallenge, organ CFU were enumerated. Data are presented as means (bars) and ± standard deviations. Means lacking standard deviations indicate that bacterial CFU were below detection limits in one or more organs from an experimental group of mice or that insufficient numbers of mice survived treatment, as for the anti-TNF MAb-treated group (one survivor) on day 5. Dashed horizontal lines represent the detection limits of the assay. Single and double asterisks indicate significant differences from the value for the control mice at P values of <0.05 and <0.01, respectively.

Comparative analysis of the abilities of different anti-IFN-γ antibody preparations to block the expression of anti-Listeria memory immunity.

The failure of the anti-IFN-γ MAb treatment of Listeria-immune mice to completely subvert the expression of immunity (Fig. 3) conflicts with previous reports indicating that anti-IFN-γ MAb treatment of Listeria-immune mice converted what would be normally a sublethal infection initiated by an extravascular challenge into a lethal one (41). One possible explanation for the apparent discrepancy between the results reported here and those reported elsewhere is that anti-IFN-γ antibody preparations may have specificities for different molecular domains mediating distinct IFN-γ activities (6, 37). To test this possibility, the abilities of the anti-IFN-γ MAb used in the preceding experiments and a rabbit anti-IFN-γ polyclonal antibody (PAb) to inhibit the expression of memory immunity in Listeria-immune mice were compared. The results presented in Fig. 4 show Listeria CFU in the PP, MLN, livers, and spleens of immune mice treated with anti-IFN-γ MAb, rabbit anti-IFN-γ PAb, or control rabbit IgG on day 5 following the i.g. inoculation of listeriae. The number of Listeria CFU in the organs from the immune host treated with the anti-IFN-γ MAb did not differ from that in the immune control mice, whereas the organs of immunized mice treated with the anti-IFN-γ PAb preparation possessed greatly augmented numbers of bacteria compared to listerial numbers in the corresponding organs of the immune control mice and the anti-IFN-γ MAb-treated immune mice. This result indicates that the anti-IFN-γ PAb is more effective than the anti-IFN-γ MAb in the inhibition and expression of anti-Listeria memory immunity.

FIG. 4.

A comparison of the abilities of the R4-6A2 anti-IFN-γ MAb and a rabbit anti-IFN-γ IgG preparation to inhibit the expression of memory anti-Listeria immunity. BALB/c mice rendered Listeria immune by i.g. inoculation of 2 × 10⁸ L. monocytogenes 30 days earlier were injected intraperitoneally with 10⁵ NU of anti-IFN-γ MAb, 2.5 × 10⁴ NU of a rabbit polyclonal anti-IFN-γ IgG, or control rabbit IgG. Four hours later, all treated Listeria immune mice and nonimmune mice were challenged with an i.g. inoculum of 7.2 × 10⁸ CFU of L. monocytogenes. Five days later, organ CFU (means ± standard deviations) were enumerated. Means lacking standard deviations indicate that bacterial CFU were below detection limits for one or more organs from an experimental group of mice. Dashed horizontal lines represent the detection limits of the assay. The asterisk indicates a significant difference from the value for the control mice at a P value of <0.05.

The results of the experiments presented in this study clearly establish roles for TNF and IFN-γ in anti-Listeria resistance mechanisms that are brought into play during primary and secondary infections caused by listeriae translocating from the gut lumen. Moreover, the results of these experiments not only establish the importance of IFN-γ in anti-Listeria resistance during enteric infection in immunocompetent and immunoincompetent hosts but also reveal that the IFN-γ-mediated effect occurs in the infected organs after the TNF-mediated effect. In addition, TNF- and IFN-γ-mediated effects in antibacterial resistance occurred, respectively, at corresponding times during both a primary infection in naive mice and a secondary infection in Listeria-immune mice. In contrast to our findings, Nishikawa et al. (29) reported that IFN-γ-mediated effects occurred during a primary enteric Listeria infection at the same time as TNF-mediated effects.

IFN-γ is produced by natural killer cells during the first 24 h of listeriosis in mice (10). However, based on the results of the present experiments with anti-IFN-γ MAb to neutralize IFN-γ in Listeria-infected immunocompetent or immunoincompetent hosts, the anti-Listeria effect mediated by this cytokine is not evident until after the third day of infection. This observation raises the question of whether IFN-γ is involved in the implementation and/or expression of the anti-Listeria resistance mechanism(s). Since the IFN-γ-mediated effect occurs at a time when macrophages populate infectious foci, it seems reasonable to assume that the IFN-γ is influencing the macrophages’ listericidal activity (4, 9). However, it is also possible that IFN-γ functions in events that result in the focusing of monocytes/macrophages at sites of inflammation. With regards to such a possibility, IFN-γ alone, or in combination with other cytokines, induces the expression of certain beta chemokines (C-C) which can function to focus monocytes to sites of infection (7, 33).

In addition to its effects on innate antibacterial resistance, IFN-γ has actions that could be important in the generation and expression of specifically acquired T-cell-mediated antibacterial immunity (2, 19, 21, 39, 40). As to possible roles for IFN-γ in the expression of T-cell-mediated immunity, this cytokine is capable of augmenting the activity of specifically sensitized CD8⁺ cytolytic T cells either directly, by enhancing the activity of these cells (3), or indirectly, by increasing the expression of major histocompatibility complex class I expression on infected target cells (11). However, it is also important that Harty and Bevan (12) have reported that CD8⁺ T cells capable of adoptively transferring anti-Listeria immunity are generated during Listeria infection in IFN-γ gene knockout mice and that Harty et al. (13) found that anti-Listeria CD8⁺ T cells can protect the host in an IFN-γ-independent manner. These findings seem to suggest that IFN-γ may not be important in the mediation of anti-Listeria resistance by CD8⁺ T cells but do not exclude the possibility that IFN-γ is important in the mediation of anti-Listeria resistance by other phenotypically distinct T cells which have been reported to be protective against this intracellular pathogen (22, 23, 34).

Following the generation of a primary anti-Listeria immune response, the numbers of T cells capable of adoptively transferring immunity rapidly decline. However, a state of long-lived memory immunity ensues. The T cells that are responsible for immunological memory are both physiologically and phenotypically distinct from those that mediate resistance during a primary Listeria infection (30, 31). In order to establish the importance of IFN-γ in anti-Listeria T-cell-mediated memory immunity, Listeria immune mice were treated with an anti-IFN-γ MAb preparation and challenged i.g. with L. monocytogenes. It was found that treatment with an anti-IFN-γ MAb had little or no effect on the expression of Listeria memory immunity. In view of both this finding and the knowledge that the anti-IFN-γ MAb exacerbated a primary Listeria infection in either immunocompetent mice (Fig. 1) or immunoincompetent SCID mice (Fig. 2), it seems reasonable to conclude that IFN-γ does not function in the expression of anti-Listeria memory immunity. However, the exacerbation of listeriosis during a secondary infection in immune mice treated with an anti-IFN-γ PAb establishes the importance of this cytokine in the expression of memory immunity. Of interest was the finding that while the anti-IFN-γ PAb treatment caused an increase in listerial CFU in all organs examined, the extent of the increase was not as great as the increase in CFU in the organs of naive mice (Fig. 4) and anti-IFN-γ MAb-treated mice during a primary infection (Fig. 1). These findings indicate that both IFN-γ-dependent and IFN-γ-independent resistance mechanisms serve to resolve the secondary infection. This conclusion is similar to that reached by Samsom et al. (36), who decided that IFN-γ played only a minor role in the expression of anti-Listeria immunity against a secondary infection initiated by intravenous inoculation of bacteria. This conclusion was based on results showing that in anti-IFN-γ MAb-treated memory immune mice, the liver bacterial CFU were only ∼1 log₁₀ higher than in control memory immune mice.

The apparent contradiction between the capacities of the anti-IFN-γ MAb and anti-IFN-γ PAb to interfere with the expression of anti-Listeria memory immunity may be explained in several ways. First, the antibodies may differ in their affinities for IFN-γ. Second, it is possible that these anti-IFN-γ preparations exhibit different specificities for distinct molecular domains on the IFN-γ molecule. Indeed, anti-IFN-γ MAb preparations have been reported to differ in their abilities to inhibit certain IFN-γ activities. These findings have been interpreted to mean that different IFN-γ activities are mediated by distinct functional domains (6, 37).