Introduction

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Cryptococcosis is an opportunistic infection caused by the encapsulated yeastlike fungus Cryptococcus neoformans (Cne) (1). In humans, the infection is acquired by inhalation of the desiccated yeast or basidiospore, and results in a primary pulmonary infection that usually remains subclinical (2). However, the pulmonary infection may disseminate to cause cryptococcal meningitis, particularly in immunocompromised patients who have a defect in cell- mediated immunity (3). Studies in mice have demonstrated that immune BALB/c T-lymphocytes from spleen, or those from lung plus lung-associated lymph nodes (LALN), could transfer protection against pulmonary Cne to C.B-17 scid/ scid mice (4). Further investigation showed that both CD4⁺ and CD8⁺ T lymphocytes were required for clearance of a pulmonary infection (5), and that CD4⁺ T lymphocytes were essential in controlling subsequent dissemination of infection to the central nervous system (CNS) (6, 7).

In the study described here, we utilized an intratracheal infection model (8) to study the role of interleukin (IL)-12 and interferon- (IFN-) in the early development of a protective T-cell response to pulmonary Cne infection. We have previously demonstrated that C57BL/6 mice are susceptible to Cne infection and fail to clear the organism, whereas C.B-17 mice are resistant (9). In this previous work, C.B-17 mice, following early rapid fungal growth, showed a progressive reduction of Cne lung colony-forming unit (cfu) burden from day 7 to day 14 of the infection, and clearance in these mice correlated with secretion of IFN- and IL-2 from immune cells isolated from LALN on day 7 of infection (9). Furthermore, clearance correlated with inducible nitric oxide synthase (iNOS) expression in the lungs and increased nitrate excretion in the urine of infected mice (10). Most importantly, clearance was blocked by administering the iNOS inhibitor n-monomethyl L-arginine or by treatment with anti-IFN- antibody begun after immunity developed but just prior to the beginning of lung clearance (10). These data suggested that a Th1-type cytokine response, with subsequent nitric oxide (NO) production by IFN--activated macrophages, was necessary for clearance of the pulmonary infection.

Other infection models have shown the importance of IL-12 in driving IFN- production and the development of a Th1-type cytokine response. Treatment in vivo with neutralizing mAb specific for murine IL-12 has demonstrated that this cytokine is essential for a beneficial Th1 response against a variety of pathogens including Listeria monocytogenes (11), Leishmania major (12), Toxoplasma gondii (13), Mycobacterium avium (14, 15), and Candida albicans (16). Exogenous treatment with recombinant IL-12 (rIL-12) has been shown to be beneficial in reducing the numbers of viable organisms in mice infected intravenously with Mycobacterium avium (17) or Cryptococcus neoformans (18). IL-12 has also been shown to increase the efficacy of immunization against pulmonary respiratory syncytial virus (RSV) infection (19) and a Schistosoma mansoni infection of the lung (20). However, the role of IL-12 or IFN- in development of an early protective Th1 immune response to an opportunistic pulmonary fungal infection has not been addressed. To directly assess the role of IL-12 and IFN- in the development of a protective immune response to Cne in resistant C.B-17 mice, we administered neutralizing mAbs against either IL-12 or IFN- in vivo at the time of initiation of a pulmonary infection, and monitored the effect of this treatment on lung clearance and cytokine patterns in lung and LALN during infection.

	Materials and Methods

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Mice

Male and female C.B-17 mice were obtained from the University of New Mexico School of Medicine animal resources breeding colony. Mice were housed in cages with filter tops, under specific pathogen-free conditions. Sterile food and water were given ad libitum.

Cryptococcus Neoformans

Cne organisms were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Strain 52D (ATCC 24067) is a low-virulence, encapsulated, serotype-D organism, and strain J305 (ATCC 52816) is an avirulent, unencapsulated organism. Stock and working cultures of both organisms were prepared as previously described (8). Organisms for intratracheal infection (strain 52D) were inoculated from working slants in Sabouraud dextrose broth and grown with continuous shaking at room temperature for 28 to 40 h (late log phase). Aliquots were washed in sterile nonpyrogenic saline (Gibco BRL, Grand Island, NY), and diluted to 1 × 10⁵ organisms/ml. Strain J305 (for heat-killed cryptococcal [HKC] antigen preparation) was grown in Sabouraud dextrose broth as described earlier, washed, heat killed at 56°C for 1 h, and then diluted to 1 × 10⁸ HKC/ml in sterile nonpyrogenic saline. Aliquots were frozen at 70°C until used in in vitro culture. Strain J305 has been previously shown to stimulate antigen-specific proliferation of splenocytes from mice infected with strain 52D, but not of splenocytes from uninfected mice (5). Indeed, lymphocyte stimulation is optimized by using an unencapsulated Cne strain, because the capsule hinders uptake of the yeast preceding the antigen-processing step (21).

Intratracheal Infection

Mice 8 to 12 wk old were infected via intratracheal inoculation with 2 to 3.5 × 10³ cfu of Cne strain 52D in 50-µl aliquots as described previously (8). The first and last 50-µl aliquots from each syringe were collected to monitor initial and final cfu delivered.

Lung CFU

Lungs from animals treated as described in the text were harvested. All lung lobes from individual mice were homogenized in sterile water. Alternatively, an aliquot of enzymatically digested lung was collected. Single 50-µl aliquots of appropriate dilutions were plated on Sabouraud dextrose agar plates with chloramphenicol (Becton Dickinson, Cockeysville, MD), and were grown at room temperature for 48 to 72 h. Colonies were counted and the numbers multiplied by the dilution factor to obtain cfu/lung.

Monoclonal Antibodies for In Vitro Depletion

Blocking mAbs for murine IFN- (hybridoma XMG1.2) and murine IL-12 (hybridoma C17.15; a generous gift from Dr. Giorgio Trinchieri of the Wistar Institute, Philadelphia, PA) were produced in vitro by cell culture. The mAbs were precipitated from hybridoma supernatants with a 50% saturated ammonium sulfate preparation, resuspended, and extensively dialyzed against phosphate-buffered saline (PBS) (pH 7.0). The antibodies were then purified with GammaBind Plus Sepharose affinity columns (Pharmacia LKB Biotechnology, Piscataway, NJ). Eluted mAb was dialyzed against PBS (pH 7.0) and filter sterilized, and its concentration determined from its OD₂₈₀. Each mAb, or rat IgG (RIgG; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for control mice, was diluted to 2 mg/ml in sterile nonpyrogenic saline. On day 1 and day +1, C.B-17 mice were injected intraperitoneally with 0.5 ml (1 mg) of XMG1.2, C17.15, or RIgG, as described in the text, for a total dose of 2 mg/mouse, day 0 being the day of intratracheal Cne infection.

LALN, Lung-Cell Preparation, and Cytokine-Secretion Cultures

Single-cell suspensions of LALN and lung cells were prepared as previously described (9). LALN cells were cultured at 5 × 10⁶ cells/ml in complete (c)RPMI (RPMI 1640 medium [Cellgro, Mediatech, Inc., Herndon, VA]; 10% heat-inactivated fetal bovine serum [FBS; (Gibco BRL)]; 2 mM L-glutamine [Sigma Cell Culture, St. Louis, MO]; 100 U/ml penicillin-streptomycin [Sigma]; 1 mM minimal essential medium (MEM) sodium pyruvate [Gibco BRL]; 1× MEM nonessential amino acids [Gibco]; 2.5 × 10⁵ M 2-ME [-merceptoethanol] [Eastman Kodak Co., Rochester, NY]; and 2 µg/ml amphotericin B [Sigma]) in a volume of 160 µl in a 96-well flat-bottom tissue-culture plate (Costar, Cambridge, MA) in media alone, 5 × 10⁵ HKC/ml, or 5 µg/ ml concanavalin A (Con A; Sigma) and incubated at 37°C with 5% CO₂. Lung cells were cultured at 5 × 10⁶ cells/ml in a volume of 500 µl in 48-well flat-bottom tissue-culture plates (Costar) in media alone. LALN culture supernatants to be analyzed for IL-2, IL-10, and IL-12 were harvested at 24 h, or at 48 h if analyzed for IFN-, IL-4, and IL-5. Lung-cell-culture supernatants for all cytokines tested were harvested at 24 h. Supernatants were centrifuged to remove cells and debris, and were frozen at 85°C until analyzed for cytokines with enzyme-linked immunosorbent assays (ELISA).

Cytokine Assays

Cytokine ELISAs for IFN-, IL-2, IL-5, and IL-10 were performed as previously described (22, 23). For IL-4 and IL-12, after incubation with a biotinylated secondary mAb, plates were washed and blocked with 5% FBS/ PBST for 10 min at 37°C. After blocking, 75 µl of streptavidin-alkaline phosphatase (Jackson ImmunoResearch Inc.) in 1% FBS/PBS with Tween (PBST) was added and plates were incubated at room temperature for 1 h. Plates were washed, and 100 µl of 5 mM 104 phosphate substrate (p-nitrophenyl phosphate substrate [Sigma]) in 0.1 M 221 alkaline buffer solution (2-amino-2-methyl-1-propanol buffer [Sigma]) was added. Plates were developed at room temperature for 30 to 240 min and the OD measured at 405 nm (OD₄₀₅), using a VMAX microplate reader (Molecular Devices Co., Menlo Park, CA). Sample concentrations were calculated by comparison with a standard curve of either recombinant cytokine (rIFN- [Genentech, Inc., South San Francisco, CA], rIL-2 [Pharmingen, San Diego, CA], rIL-4 [R&D Systems, Minneapolis, MN], rIL-6 [Pharmingen], and rIL-12 [a generous gift from Dr. Maurice Gately of Hoffmann-LaRoche, Inc., Nutley, NJ]), or Con A-stimulated D10 Th2 cell line supernatant with known cytokine concentrations (IL-5 and IL-10). MAbs were prepared by affinity purification, and included R46A2 and biotin-XMG1.2 (IFN-), 11B11 and biotin-C213 (IL-4), TRFK5 and biotin-TRFK4 (IL-5), SXC2 and biotin-SXC1 (IL-10), and C17.15 and biotin-C15.6 (IL-12; a gift from Dr. Giorgio Trichieri). MAbs JES6-1A12 and biotin-JES6-5H4 (IL-2) were purchased from Pharmingen.

Cytospin Preparations and Differentials

Lung cells from individual mice were diluted to 1 × 10⁶ cells/ml, and duplicate cytocentrifuge preparations were made for each sample. Differential counts were made in a blinded manner on samples that were stained with Wright's stain. The total cell number of lung cells harvested from each animal (obtained by trypan blue enumeration) was multiplied by the percent of each cell type counted on the differential to obtain the total number of each cell type per mouse. Data presented are the mean total number of each cell type ± 1 SEM per treatment group in Figure 3.

View larger version (26K): [in this window] [in a new window]   Figure 3.   Day 14 lung-cell numbers from mAb-treated C.B-17 mice. Lungs from day 14 Cne-infected C.B-17 mice treated with control RIgG (open bars), anti-IFN- mAb (XMG1.2, hatched bars), or anti-IL-12 mAb (C.17.15, closed bars) were collagenase/ deoxyribonuclease (DNase) digested to prepare single-cell suspensions. Cytospin preparations were made from individual lungs and stained with a modified Wright's stain. A differential count was performed in duplicate on each sample at ×1,000 magnification, using oil immersion. Data presented are mean cell numbers ± 1 SEM, calculated from total cell number multiplied by the percent of that cell type in the differential count. Data are pooled from two experiments; n = 7 to 10 per group. * P  0.05 or ** P  0.001: significantly different from control C.B-17 mice.

Immunocytochemical Staining for iNOS Protein

Cytospin preparations of day 14 lung single-cell suspensions were made as described earlier. Sections were allowed to air dry, were fixed in ice-cold acetone for 2 min, and were frozen at 70°C until stained. Prior to staining, the cytospin preparations were brought to room temperature and rehydrated for 15 min in pH 7.4 PBS. The cytospin preparations were then pretreated with 20% normal goat serum in PBS (NGS/PBS) for 15 min to prevent nonspecific antibody interactions. Mouse anti-iNOS primary mAb (IgG_2a; Transduction Laboratories, Lexington, KY) or control mouse IgG_2a (DAKO, Carpinteria, CA) in NGS/PBS was added to the cytospin preparations, and they were incubated in a humidified chamber at room temperature. Slides were washed three times in PBS, the Fc fragment of horseradish peroxidase (HRP)-conjugated, affinity-purified goat antimouse IgG (Jackson ImmunoResearch) in NGS/PBS was added, and slides were incubated in a humidified chamber at room temperature. Slides were washed in PBS, washed with acetate buffer, and then developed with 3-amino-9-ethylcarbazole (AEC; Aldrich Chemicals, Milwaukee, WI) in acetate buffer. Cytospin preparations were counterstained with Gill's hematoxylin (No. 1; Fisher Scientific, Pittsburgh, PA). Slides were assessed for the percentage of positive-staining macrophages per 200 macrophages counted per slide. The specific staining percent, shown in Table 1, was calculated by subtracting the percent positive-staining macrophages on slides stained with control mouse IgG_2a (a measure of background endogenous peroxidase activity) from the percent positive-staining macrophages on slides stained with specific anti-iNOS antibody.

Statistics

Statistical analysis was performed on a VAX computer at the University of Texas Southwestern Medical Center, using the UTSTAT package (generated at University of Texas Southwestern Medical Center). All data are presented as the mean ± 1 SEM. Reported P values were obtained with Welsch's modified T test or Welch's modified analysis of variance (ANOVA), depending upon the number of groups being compared. In figures, values of P  0.05 but > 0.01 are designated with *, and values of P  0.01 are designated with **.

	Results

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Pulmonary Cne Clearance in C.B-17 Mice Treated with Antibodies to IL-12 or IFN-

We have previously shown that resistant C.B-17 mice develop a Th1 response in LALN and lung cells following Cne infection (9). Since IL-12 and IFN- have been shown to be essential in the development of Th1 responses to intravenous and subcutaneous infections in other murine models of infection, we wished to assess the role of those two cytokines in the development of a Th1 response to opportunistic pulmonary fungal infection. In order to directly test the role of these two cytokines in the development of a protective immune response, C.B-17 mice were treated with a rat antimouse IFN- mAb (XMG1.2), rat antimouse IL-12 (C17.15) mAb, or control RIgG in vivo during the initiation of a Cne lung infection. Figure 1 shows that all three treatment groups of C.B-17 mice demonstrated lung cfu numbers exceeding 1 × 10⁶/mouse at day 7. Between day 7 and day 14 of infection, control RIgG-treated mice began to clear the infection as indicated by decreased lung cfu numbers at day 14. However, both anti-IFN- mAb and anti-IL-12 mAb-treated C.B-17 mice were unable to resolve the infection and had significantly higher lung cfu numbers at day 14. 

View larger version (14K): [in this window] [in a new window]   Figure 1.   Lung Cne cfu burden (log10) in mAb-treated C.B-17 mice were treated intraperitoneally with 2 mg total (1 mg at day 1 and 1 mg at day +1) of either control RIgG (open triangle), anti-IFN- mAb (XMG1.2, open square), or anti-IL-12 mAb (C17.15, closed square). On day 7 and day 14, data shown are the means pooled from three experiments; n = 15 per group. **P  0.01: significantly different from control group.

Effect of Anti-IFN- and Anti-IL-12 mAb Treatment on LALN Cell Number and Cytokine Production in C.B-17 mice

The effect of anti-IFN- or anti-IL-12 on the Th1 response in LALN was assessed. LALN cellularity at day 7 in Cne-infected C.B-17 mice treated with RIgG (control), anti-IFN- mAb, or anti-IL-12 mAb was not significantly different in the three treatment groups (2.9 ± 1.1 × 10⁷, 3.5 ± 0.9 × 10⁷, and 3.1 ± 1.0 × 10⁷ LALN cells/mouse, respectively; P > 0.05 for all pairwise comparisons). LALN cell suspensions were cultured in vitro with medium, HKC, or Con A, and the supernatants were analyzed for cytokines with ELISA, as described in MATERIALS AND METHODS. LALN cell suspensions from all three treatment groups secreted similar amounts of IL-2 (Figure 2). In contrast, C.B-17 mice treated with anti-IL-12 mAb showed significantly less IFN- production in all three culture conditions than did RIgG-treated mice. Anti-IFN- treatment significantly reduced IFN- production by LALN cells stimulated with Con A. IL-12 production by LALN cells from C.B-17 mice treated with either anti-IL-12 or anti-IFN- and cultured in medium was significantly lower than that of controls. In vivo treatment with anti-IFN- and anti-IL-12 increased both IL-4 and IL-5 secretion by LALN cells in medium and HKC antigen cultures. IL-10 production by LALN cells from C.B-17 mice treated with anti-IFN- mAb that were cultured in medium, HKC, or Con A, was significantly greater than that in similar cultures of LALN cells from control RIgG C.B-17 mice, whereas anti-IL-12 mAb treatment did not affect production of IL-10.

View larger version (27K): [in this window] [in a new window]   Figure 2.   Cytokine production from day 7 LALN cells from mAb-treated C.B-17 mice. LALN cells from Cne-infected C.B-17 mice treated with control RIgG (open bars), anti-IFN- mAb (XMG1.2, hatched bars), or anti-IL-12 mAb (C17.15, closed bars) were cultured at 5 × 106/ml in medium, HKC (5 × 105/ml), or Con A (5 µg/ml) as indicated. Culture supernatants for IL-2, IL-10, and IL-12 analysis were harvested at 24 h, and for IFN-, IL-4, and IL-5 at 48 h. Supernatants were analyzed with ELISA for cytokine production, as described in MATERIALS AND METHODS. Data shown is mean ± 1 SEM; n = 7 to 10 mice per data point, pooled from two separate experiments. *P  0.05 or **P  0.01: significantly different from control C.B-17 mice. N.D. = not detected.

Effect of Anti-IFN- or Anti-IL-12 mAb Treatment on Lung Inflammatory Cell Recruitment and Activation

Previous studies have shown that NO can inhibit the growth of Cne in vitro (24), and activated macrophages have been shown to be the source of IFN--induced antimicrobial NO in the growth control of L. major (25), Mycobacterium tuberculosis (26), and Trypanosoma cruzi (27). Additionally, it has been shown that pulmonary clearance of Cne in resistant C.B-17 mice can be ablated by in vivo treatment with anti-IFN- given during the effector stage of the immune response ( day 7), which blocks induction of iNOS, or with an arginine analogue that blocks NO production without affecting expression of iNOS (10). Therefore, we were interested in determining whether the lack of clearance we observed in mice treated with anti-IFN- and anti-IL-12 mAbs was due to: (1) lack of recruitment of macrophages and/or polymorphonuclear leukocytes (PMN) to the lung; (2) lack of iNOS induction by lung macrophages; (3) switching of the pulmonary T-helper-cell response from Th1 to Th2; or (4) more than one of these events. To address these points, we analyzed cell influx, macrophage iNOS protein, and in vitro cytokine expression from day 14 lung cells. As shown in Figure 3, C.B-17 mice treated with either anti-IFN- mAb or anti-IL-12 mAb had significantly lower numbers of macrophages, lymphocytes, and neutrophils in Cne-infected lungs at day 14 than did RIgG-treated mice. In contrast, eosinophil numbers were significantly higher in both of the treatment groups than in control mice. When macrophages in day 14 lung cells were assessed for iNOS protein through immunocytochemistry, macrophages from both anti-IFN- mAb- and anti-IL-12 mAb-treated C.B-17 mice had significantly lower percentages of cells positive for iNOS expression than did macrophages from control RIgG-treated mice (Table ). Additionally, in vitro cultures of lung cells from day 14 C.B-17 mice treated with anti-IFN- mAb or anti-IL-12 mAb showed significantly less secretion of IFN- and IL-12 and significantly more secretion of IL-5 than did control mice (Figure 4). Although the levels of IL-4 secreted by day 14 lung-cell cultures from anti-IFN- mAb- or anti-IL-12 mAb-treated mice appeared higher than for control mice, no significant differences can be reported.

View larger version (32K): [in this window] [in a new window]   Figure 4.   Day 14 lung-cell cytokine secretion from mAb-treated C.B-17 mice. Lungs from day 14 Cne-infected C.B-17 mice treated with control RIgG (open bars), anti-IFN- mAb (XMG1.2, hatched bars), or anti-IL-12 mAb (C17.15, closed bars) were collagenase/ deoxyribonuclease (DNase) digested to prepare single-cell suspensions. Cells were cultured at 5 × 106 cells/ml for 24 h in vitro without additional stimulation. Culture supernatants were analyzed with ELISA for cytokines, as described in MATERIALS AND METHODS. Data shown are mean ± SEM; n = 7 to 10 per data point, pooled from two separate experiments. TNF- was not detectable in any samples tested (< 5 ng/ml), and IL-10 did not differ in the treatment groups (P = 0.471; data not shown). *P  0.05 or **P  0.01: significantly different from control C.B-17 mice. N.D. = not detected.

	Discussion

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The goal of this study was to directly assess the role of IL-12 and IFN- in the early development of a protective Th1 response to a pulmonary Cne infection. The major findings of the study are that: (1) IL-12 and IFN- are essential cytokines for initiating lung clearance of Cne in C.B-17 mice; and (2) pretreatment of C.B-17 mice with either an anti-IL-12 mAb or anti-IFN- mAb results in a shift to a Th2-like cytokine profile in LALN and lung, with development of pulmonary eosinophilia.

The role of IL-12 and IFN- in driving a Th1 immune response has been the focus of many recent publications. The role of these two cytokines in driving protective responses to pulmonary pathogens has been much less studied. Saunders and colleagues (1995) showed that depletion of endogenous IL-12 in Mycobacterium avium infections in mice greatly increased the bacterial burden that developed after intranasal infection (15). Exogenous IL-12 treatment has also been shown to decrease the burden of RSV (19) and Schistosoma mansoni (20) in the lung. With regard to Cne, Clemons and colleagues (1994), using an intravenous inoculation BALB/c model, showed that although exogenous rIL-12 was beneficial in decreasing cfu numbers in the brain and liver, it had no effect on spleen and lung cfu numbers (18). This result suggests that the role of IL-12, and perhaps even the requirement for an IFN--dependent clearance mechanism, differs with the route by which an organism infects the lung. More recently, Kawakami and associates (1996), using a different strain of mouse ([BALB/c × DBA/2] F1) and a higher dose (10⁵ cfu) of a more virulent Cne strain (killing mice within 4 to 6 wk), also studied the role of IL-12 in protection (28). This group found that intraperitoneal administration of IL-12 increased pulmonary fungal clearance, reduced dissemination to the brain, and decreased mortality.

Our studies confirm and extend the findings in these latter studies by showing that in a host fully capable of controlling a lung infection, antibodies to either IL-12 or IFN- will reduce lung clearance of Cne. Furthermore, our findings are associated not only with the early loss of a Th1 response in LALN, but demonstrate a shift to a Th2 response with significant pulmonary eosinophilia. These latter findings are consistent with the hypothesis that an altered cytokine milieu at the time of infection via the lung can result not only in the absence of a protective immune response, but in the development of an immune response that can lead to deleterious allergic responses. These data yield an important lesson about future vaccine development for pulmonary infectious organisms: that in order to protect against immediate hypersensitivity responses to infectious challenges following vaccination, IL-12 and/or IFN- could be incorporated into the vaccine.

Our studies underline the importance of the development of a Th1 immune response in resistant C.B-17 mice. However, the precise mechanism(s) that prevent susceptible C57BL/6 mice from mounting a protective Th1 response remain under investigation. Analysis of spontaneous cytokine production by bronchoalveolar lavage (BAL) cells from resistant C.B-17 and susceptible C57BL/6 mice has revealed the presence of significantly higher production of IFN- in Cne-resistant C.B-17 mice at day 7 and day 14, which are the days after infection when clearance begins (data not shown). Lovchik and coworkers (10) showed that cultured lung inflammatory cells from Cne-infected C.B-17 mice make NO, that IFN- is essential for the expression of iNOS messenger RNA (mRNA), and that in the absence of NO, mice cannot begin to clear a pulmonary Cne infection. Additional preliminary data demonstrate early production of tumor necrosis factor- (TNF-) by BAL cells from C.B-17 mice, but not those from C57BL/6 mice, suggesting that this cytokine may play a role during in vivo development of a Th1-like cytokine response. Others have also reported finding that TNF- may be as important in the initiation phase of an immune response as it is in the effector phase. TNF- has been previously shown to be essential for IL-12 production by Mycobacterium bovis-infected murine bone-marrow-derived macrophages in vitro (29), and IFN- priming of these macrophages was necessary to induce the IL-12 production, suggesting that all three cytokines cooperate to induce a Th1 response. Moreover, anti-TNF- treatment of resistant CBA/J mice prior to intratracheal infection with Cne has been shown to block lung clearance of Cne, increase dissemination of the organism to the spleen and brain, and eliminate a Cne-specific footpad delayed-type hypersensitivity (DTH) response (30). These data suggest that TNF-, IFN-, and IL-12 may cooperate in the afferent phase of a Th1 cytokine response in vivo, and that the absence of any one of these cytokines may result in default to a Th2-like response. Experiments are in progress to test this hypothesis in susceptible C57BL/6 mice.