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*Pneumocystis Infections
The Journal of Immunology, 1998, 161: 360-366.
Copyright © 1998 by The American Association of Immunologists

Susceptibility to Pneumocystis carinii in Mice Is Dependent on Simultaneous Deletion of IFN-{gamma} and Type 1 and 2 TNF Receptor Genes1

Daniel G. Rudmann2,*, Angela M. Preston{dagger}, Mark W. Moore{ddagger} and James M. Beck3,{dagger},§

* Department of Pathology, Genentech, Inc., South San Francisco, CA 94066, and Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 47907; {dagger} Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109; {ddagger} Deltagen, San Francisco, CA 94131; and § Pulmonary Section, Department of Veterans Affairs Medical Center, Ann Arbor, MI 48105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pneumocystis carinii pneumonia is an important cause of morbidity and mortality in immunosuppressed patients, particularly HIV-infected individuals. An improved understanding of pulmonary host response, including the cytokines required for defense, could suggest novel immunotherapeutic approaches to this infection. The cytokines IFN-{gamma} and TNF have contributory roles in host defense against P. carinii, but their combined and interactive importance is unclear. To test the roles of these cytokines in defense against P. carinii directly, organisms were inoculated intratracheally into wild-type mice and into three groups of gene-deleted mice: those lacking genes for IFN-{gamma} (IFN-{gamma}-/-), for TNF receptors 1 and 2 (TNFR-/-), and for both IFN-{gamma} and TNFR (TNFR-IFN-{gamma}-/-). Four weeks after P. carinii inoculation, lungs of the wild-type, IFN-{gamma}-/-, and TNFR-/- mice demonstrated clearance of P. carinii and only mild inflammation. However, TNFR-IFN-{gamma}-/- mice demonstrated severe P. carinii infection and lung inflammation. Our findings demonstrate conclusively that deletion of either IFN-{gamma} or TNF activity alone does not block clearance of P. carinii. However, simultaneous deletion of IFN-{gamma} and TNF receptor genes results in susceptibility to P. carinii. Rather than focusing exclusively on individual cytokines, our data suggest that immunotherapy targeted at maximizing both the IFN-{gamma} and TNF responses to P. carinii may be required to augment host defense against this important opportunistic pathogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
P;44qneumocystis carinii is a frequent cause of serious, opportunistic pneumonia in immunosuppressed patients, particularly those infected with HIV (1, 2). While there are well-established therapeutic and prophylactic modalities for P. carinii pneumonia, none are completely effective (2). An attractive therapeutic approach for immunosuppressed patients with P. carinii pneumonia would be selective augmentation of host responses (3, 4). Lymphocyte- and macrophage-derived cytokines, important in the host response to P. carinii, may have therapeutic potential in P. carinii pneumonia (4, 5, 6, 7, 8, 9, 10). Two of these cytokines, IFN-{gamma} and TNF, modulate resistance to numerous pathogens and have multiple effects on leukocyte and parenchymal cell populations in the lung (11, 12, 13, 14). While several studies demonstrate that IFN-{gamma} and TNF contribute to host defense against P. carinii pneumonia (4, 5, 7, 15, 16), the combined and interactive roles of these cytokines in host defense have not been tested directly.

IFN-{gamma}, produced predominantly by CD4+ T cells and NK cells, is an important activator of macrophages and Th1 lymphocytes, but its role in host defense against P. carinii is controversial (4, 5, 7). Rat spleen cells cultured with P. carinii Ags produce increased amounts of IFN-{gamma} (17). Aerosolized IFN-{gamma} reduces histologic lesions of P. carinii pneumonia in mice selectively depleted of CD4+ T cells (4) and, in vitro, IFN-{gamma} potentiates the killing activity of macrophages against P. carinii, possibly by increasing nitric oxide production (10, 18). In other experimental systems, however, neutralization of IFN-{gamma} with mAbs does not alter the course of P. carinii infection in splenocyte-reconstituted scid mice (5). Additionally, mice in which the IFN-{gamma} gene has been deleted are not susceptible to P. carinii, but reconstitution of scid mice with splenocytes from these IFN-{gamma} knock-out mice results in increased pulmonary inflammation (7). Taken together, these data demonstrate that the role of IFN-{gamma} in host defense against P. carinii is complex and remains mechanistically unclear.

TNF is a potent proinflammatory cytokine that mediates its various effects by engaging two membrane receptors, designated TNFR1 and TNFR2 (12, 14, 19). While TNF is predominantly produced by macrophages, it can be synthesized and secreted from virtually all cell types (12, 14). In AIDS patients infected with P. carinii, increased concentrations of TNF in bronchoalveolar lavage (BAL)4 are associated with decreased parasite burdens (20). Additionally, alveolar macrophages from P. carinii-infected patients produce increased concentrations of TNF (20). However, this TNF elaboration is insufficient to clear P. carinii from these patients’ lungs, and direct infection of macrophages with HIV may impair TNF production (21). In experimental animal models, the major surface glycoprotein of P. carinii induces TNF elaboration by rat T cells and macrophages (22). TNF concentrations in sera and lavage fluids are increased in P. carinii-infected mice compared with uninfected mice, and alveolar macrophages from infected mice produce more TNF than macrophages from uninfected mice (23). In P. carinii-infected scid mice reconstituted with splenocytes, neutralization of TNF with mAbs results in persistent P. carinii pneumonia (5, 15). While these data indicate that TNF is an important component of host defense against P. carinii, direct examination of the role of TNF using mice incapable of responding to TNF has not been accomplished.

To determine the combined roles of IFN-{gamma} and TNF in host defense against P. carinii, we examined whether the concurrent ablation of genes for IFN-{gamma} and TNF receptors would increase the susceptibility of mice to P. carinii. We hypothesized that both IFN-{gamma} and TNF, working in concert, would be required for an effective host response to P. carinii. To test this hypothesis directly, P. carinii organisms were inoculated intratracheally into wild-type (wt) mice and into the following gene-deleted mice: those deficient in IFN-{gamma} (IFN-{gamma}-/-), in TNF receptors 1 and 2 (TNFR-/-), and in both IFN-{gamma} and TNF receptors 1 and 2 (TNFR-IFN-{gamma}-/-). We found that mice with either IFN-{gamma} or TNF signaling cleared the P. carinii inoculum. However, immunosuppression associated with the combined loss of IFN-{gamma} and TNF signaling results in marked susceptibility to P. carinii.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Cytokine- and cytokine receptor-deficient mice. IFN-{gamma}-/- mice were obtained from a colony generated at Genentech as previously described (11). Mice deficient in TNFR1 and TNFR2 were generated by backcrossing TNFR1-/- and TNFR2-/- mice, which were also characterized previously (24, 25). Mice deficient in IFN-{gamma}, TNFR1, and TNFR2 were produced by backcrossing the IFN-{gamma}-/- and TNFR-/- mice. The mouse genotypes were confirmed by analysis of tail DNA, using gene-specific primers or probes for PCR and Southern blots (25). All genetically deficient mice were of mixed C57BL/6 and 129/SV background and were homozygous for the specific gene deletion(s). Wt mice derived from littermates of each group of mice were used as controls.

The phenotypes of the IFN-{gamma}-/-, TNFR1-/-, and TNFR2-/- knock-out mice were described previously (11, 24, 25, 26, 27). The TNFR-/-, TNFR-IFN-{gamma}-/-, and wt mice have comparable baseline serum chemistry values for liver, kidney, and gastrointestinal function, and there are no significant differences between groups in complete blood counts or in bone marrow cytology. T cell ontogeny is normal in TNFR-/- mice, and thymus, lymph nodes, and spleens are grossly comparable in TNFR-/-, TNFR-IFN-{gamma}-/-, and wt mice. In contrast, intestinal Peyer’s patches are not identified grossly and are hypoplastic histologically in both TNFR-/- and TNFR-IFN-{gamma}-/- mice. Also, lymph node cortices are thin and lack primary or secondary cortical follicles histologically. Confirmed by histologic evaluation, B220 immunohistochemistry, and peanut agglutinin lectin histochemistry, both naive and immunized TNFR-/- and naive TNFR-IFN-{gamma}-/- fail to form germinal centers in the spleen and peripheral lymphoid tissues. Despite the lack of germinal centers, the splenic white pulp has distinct B and T cell areas, and isotype switching of B cells occurs in TNFR-/- mice. Mice necropsied at various ages have no gross or histologic evidence of spontaneous disease, including P. carinii pneumonia, indicated by examination of lung tissue stained with hematoxylin and eosin (H&E).

CD4-depleted mice. As a positive control group, BALB/c mice (Taconic Laboratories, Germantown, NY) were depleted of CD4+ lymphocytes by treatment with a mAb directed against the murine CD4 epitope (GK 1.5, American Type Culture Collection, Manassas, VA) (28). Depletion of CD4 lymphocytes renders mice susceptible to P. carinii as described previously (29).

P. carinii-infected athymic mice. P. carinii-infected athymic mice (nu/nu on a BALB/c background; Taconic Laboratories) were used as a source for P. carinii in all experiments (29). To insure that the inflammatory response in mice of C57BL/6 and 129/SV background was not caused by alloantigen, uninfected athymic mice were used as a source of sham inoculum for several mice in each of the gene-deleted and wt groups.

Animal housing and disease surveillance. All mice were housed in microisolator caging under laminar flow conditions and maintained in barrier-protected rooms. Sentinel animals were regularly monitored for common rodent diseases. Variously aged gene-deleted and wt mice were always histologically negative for spontaneous P. carinii infection. All procedures were approved by the Animal Care Subcommittee, Department of Veterans Affairs Medical Center (Ann Arbor, MI).

Intratracheal inoculation with P. carinii organisms

Mice were inoculated intratracheally with P. carinii harvested from chronically infected athymic mice as previously described (29). We performed intratracheal inoculations under direct vision to ensure that each mouse received an identical inoculum of P. carinii organisms, delivered to the alveolar space, at an identical time point. Briefly, athymic mouse lungs were frozen at -20° C for 2 h, homogenized mechanically, filtered, and centrifuged. Smears were stained with modified Giemsa stain for quantitation of P. carinii organisms, and touch preparations of lung were examined with Gram’s stain to exclude tissue contaminated with bacteria. Groups of 6- to 8-wk-old wt, CD4-depleted, IFN-{gamma}-/-, TNFR-/-, and TNFR-IFN-{gamma}-/- mice were anesthetized with pentobarbital. For intratracheal inoculation, the trachea was exposed using a midline neck incision and a blunted needle was passed through the mouth into the mid-trachea under direct vision. A polyethylene catheter was passed through this needle and 0.1 ml of inoculum (2 x 105 P. carinii) was injected, followed by 0.4 ml of air. The incision was sutured, and mice were allowed to recover in a prone position.

To assure that residual host material from the lungs of the athymic mice did not incite inflammatory responses, sham inoculations of wt and gene-deleted mice were also performed. The lung homogenates used for P. carinii inoculation contained P. carinii organisms as well as residual particulate manner, including cell fragments, from the lungs of the donor mice. Additional wt mice and mice from each gene-deleted group were inoculated with homogenates from uninfected athymic mice. These homogenates, prepared in parallel with the homogenates from infected athymic mice, contained comparable particulate material but demonstrated no P. carinii organisms in smears stained with GMS stain or with modified Giemsa stain.

BAL and cytology

Four weeks after intratracheal inoculation, mice were exsanguinated during pentobarbital anesthesia. The 4-wk time point was selected based on previous work from our laboratory in examining the development of infection in CD4-depleted mice and clearance of infection in immunologically intact mice (29). A polyethylene catheter was inserted into the trachea, and the lungs were lavaged to a total volume of 11 ml using 0.5 ml aliquots of warmed calcium- and magnesium-free PBS containing 0.6 mM EDTA (29). The BAL was centrifuged at 500 x g for 10 min at 4° C, the pellets were washed twice in PBS, and the cells were enumerated using a hemocytometer. For flow cytometry, cells were resuspended and stained for 30 min with fluorescein- or phycoerythrin-conjugated Abs directed against CD3, CD4, or CD8, or with irrelevant, isotype-matched control Abs (PharMingen, San Diego, CA) (29). After centrifugation, cells were resuspended in PBS with 2% paraformaldehyde, stored overnight at 4° C in the dark, and analyzed on a FACScan the following morning. For differential counting, 105 cells per lavage sample were washed onto nitrocellulose filters with 5-µm pores (Millipore, South San Francisco, CA). Filters were mounted onto glass slides, fixed overnight in 10% buffered formalin, and stained with H&E. Blinded differential counts were performed on at least 200 cells per slide (29).

Cytokine analysis and histopathology

Cytokine analyses and histopathologic examinations were performed on lungs obtained from the same mice for each experiment. For cytokine analysis, the right bronchus was ligated, and the right lung was removed, weighed, and homogenized in 1 ml of PBS containing 0.05% Triton (Sigma, St. Louis, MO). After centrifugation for 5 min at 10,000 x g, the supernatants were filtered through sterile 1.2-µm syringe filters (Whatman, Clifton, NJ) and were frozen at -80° C until analyzed.

For scoring of infection and inflammation, the left lung was inflated with 10% buffered formalin, fixed overnight, and embedded in paraffin. Five-micrometer histologic sections were stained with H&E and GMS and were graded blindly for extent of P. carinii infection (Table IGoA) and pulmonary inflammation (Table IGoB), using modifications of semiquantitative scales previously described and validated (29, 30). These scales are based upon grading of the entire lung surface area present on the slide rather than numbers of microscopic fields. Our laboratory and others have demonstrated that histologic grading of infection correlates closely with organism counts from homogenized lungs (30, 31).


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  		Table I. P. carinii infection and pulmonary inflammation score criteria

 
Cytokine ELISA

Lung homogenates stored at -80° C were assayed for IL-2, IL-4, IL-5, IL-10, p40 IL-12, TNF, and IFN-{gamma} using standard sandwich ELISA procedures (32, 33). All incubations were performed on a shaker platform at room temperature, and plates were washed three to nine times using an automatic washer between all steps. Briefly, dilutions of standard and sample were added in duplicate to high affinity 96-well flat-bottom plates (Nalge Nunc, Rochester, NY) that had been coated for 12 to 24 h with 0.5 to 1.0 µg/ml of anti-murine IL-2, IL-4, IL-5, IL-10, p40 IL-12 (PharMingen), TNF (Endogen, Woburn, MA), or IFN-{gamma} (Genentech; hybridoma 22.01) and blocked with 1% BSA in PBS for at least 1 h. Sample and standard incubation was 2 to 4 h. Except for the IFN-{gamma} ELISA, the detection Abs were all biotinylated and used at 1 µg/ml. The detection Ab for IFN-{gamma} was affinity-purified rabbit anti-murine IFN-{gamma} antiserum (Genentech, lot 18138-73). Streptavidin-horseradish peroxidase (Zymed, San Francisco, CA) was used at 1:6000 for 30 to 60 min for all cytokines except IFN-{gamma}, for which horseradish peroxidase-donkey anti-rabbit Ig (Amersham, Arlington Heights, IL) was used at 1:5000 for 30 min. Plates were developed with O-phenylenediamine dihydrochloride substrate (OPD; Sigma, St. Louis, MO; 5 mg OPD, 12.5 ml PBS, and 5 µl 30% H2O2) for 15 to 30 min, and the reaction was stopped with 4 N H2SO4. Plates were read with a spectrophotometer at 490 and 405 nm. Measured concentrations of the cytokines in lung homogenates (400 pg/ml to 12 ng/ml) were well above the sensitivities of the individual ELISAs (31.25 pg/ml for IL-2, IL-4, and IL-5; 125 pg/ml for IL-10 and IL-12; 62.5 pg/ml for TNF and IFN-{gamma}). The lung cytokine concentrations were calculated against standard curves with known concentrations of recombinant cytokines and were expressed as picograms of cytokine per microgram of wet lung weight.

Statistical methods

Lavage cell counts and cytokine ELISAs were compared using a one-way ANOVA with Fisher’s follow-up testing (34). Inflammation and infection scores between experimental groups were compared using the Kruskal-Wallis test with unpaired Mann-Whitney follow-up testing, and a correlation coefficient was calculated to compare inflammation scores and infection scores (34). Testing was performed with StatView software (Abacus Concepts, Berkeley, CA). Significance was accepted at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Simultaneous deletion of TNFR1, TNFR2, and IFN-{gamma} is required for establishment of P. carinii pneumonia

Four weeks after intratracheal inoculation, the lungs of sham-inoculated mice demonstrated no evidence of P. carinii infection (Fig. 1Go). Because data obtained from all sham-inoculated groups of mice were comparable (wt, IFN-{gamma}-/-, TNFR-/-, and TNFR-IFN-{gamma}-/- mice), these data were pooled and expressed as a single value in Figure 1Go and subsequent figures. Four weeks after P. carinii inoculation, wt, IFN-{gamma}-/-, and TNFR-/- mice demonstrated no evidence of P. carinii infection. In contrast, the lungs of CD4- depleted mice and TNFR-IFN-{gamma}-/- mice demonstrated severe infection. Thus, simultaneous deletion of IFN-{gamma} and TNFR genes rendered mice susceptible to P. carinii, whereas deletion of the individual genes did not interfere with clearance of P. carinii by 4 wk after challenge.



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  		FIGURE 1. Intensity of infection 4 wk after intratracheal inoculation. Lungs from mice were scored blindly for intensity of infection according the criteria in Table IGoA. Sham-inoculated mice (intratracheal inoculation with lung homogenate from uninfected athymic mice) did not demonstrate P. carinii pneumonia. All other groups of mice received an intratracheal inoculation of P. carinii obtained from infected athymic mice. Wild-type, IFN-{gamma}-/-, and TNFR-/- mice cleared the P. carinii inoculum. In contrast, CD4-depleted mice and TNFR-IFN-{gamma}-/- mice demonstrated intense infection (*p < 0.05 compared with sham-inoculated mice by Kruskal-Wallis test). Bars represent medians from 16 to 18 mice per group, except the CD4-depleted group (n = 9), in two separate experiments.

 
Simultaneous deletion of IFN-{gamma}, TNFR1, and TNFR2 in mice challenged with P. carinii results in severe pulmonary inflammation

To quantify the inflammatory response in mice 4 wk after P. carinii inoculation, we measured lavage cell counts in mice (Fig. 2Go). The lavages from sham-inoculated mice contained ~2 x 105 cells, and >95% of these cells were macrophages (Fig. 2Go, A and D). Numbers of leukocytes in lavages from wt, IFN-{gamma}-/-, TNFR-/-, and CD4-depleted mice were 5- to 10-fold higher than lavages from sham-inoculated mice (Fig. 2Go, A–D). In contrast, numbers of leukocytes in lavages from the TNFR-IFN-{gamma}-/- mice were ~75-fold higher than lavages from sham-inoculated mice and 10-fold higher than lavages from wt, IFN-{gamma}-/-, TNFR-/-, and CD4 -depleted mice (Fig. 2GoA). As compared with wt, TNFR-/-, IFN-{gamma}-/-, and CD4-depleted mice, the TNFR-IFN-{gamma}-/- mice demonstrated significant increases in eosinophil and lymphocyte numbers (Fig. 2Go, B and C). Therefore, the TNFR-IFN-{gamma}-/- mice were susceptible to P. carinii infection despite the development of severe pulmonary inflammation, indicated by increased numbers of lymphocytes and eosinophils.



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  		FIGURE 2. Cell counts in bronchoalveolar lavages 4 wk after intratracheal inoculation. Compared with all other groups, lavages from TNFR-IFN-{gamma}-/- mice contained significantly increased numbers of total leukocytes (A), eosinophils (B), and lymphocytes (C; *p < 0.05 compared with sham-inoculated mice by ANOVA). Bars represent means ± SEM for 12 mice per group, except wt (n = 24) and sham-inoculated (n = 9) groups, in two separate experiments.

 
To better characterize the T cell response in mice 4 wk after P. carinii inoculation, we examined the phenotype of lavage T cells by flow cytometry. Compared with wt mice, percentages of CD3+, CD4+, and CD8+ cells were not significantly altered in lavages from IFN-{gamma}-/-, TNFR-/-, or TNFR-IFN-{gamma}-/- mice (data not shown). Because lavages from TNFR-IFN-{gamma}-/- mice contained significantly increased numbers of lymphocytes, however, the absolute numbers of each lymphocyte subset were increased in TNFR-IFN-{gamma}-/- mice compared with wt, IFN-{gamma}-/-, and TNFR-/- mice (p < 0.0001).

Simultaneous deletion of IFN-{gamma}, TNFR1, and TNFR2 results in altered histologic characteristics of lung after P. carinii challenge

To evaluate differences in the morphologic characteristics of the pulmonary inflammatory response in mice 4 wk after P. carinii inoculation, histologic lesions were graded (Table IGoB). Sham-inoculated mice demonstrated no pulmonary inflammation (Figs. 3Go and 4A). Wild-type, IFN-{gamma}-/-, and TNFR-/- mice demonstrated minimal pulmonary inflammation characterized by mild lymphocytic cuffing of vessels and airways, airway epithelial hyperplasia, and alveolar histiocytosis (Figs. 3Go and 4Go, B–D). In contrast, CD4- depleted mice and TNFR-IFN-{gamma}-/- mice had significantly more intense pulmonary inflammation (Figs. 3Go and 4Go, E and F). Lesions in CD4 depleted mice were characterized by moderate perivascular and peribronchiolar lymphocytic inflammation; neutrophilic, eosinophilic, and histiocytic alveolitis; and airway hyperplasia. In TNFR-IFN-{gamma}-/- mice, inflammation was often severe and was characterized histologically by dense perivascular and peribronchial lymphocyte infiltrates and by alveolar exudate composed of macrophages, multinucleated giant cells, and eosinophils (Fig. 4GoF). Grading of intensity of P. carinii infection correlated significantly with intensity of histologic inflammation (r = 0.644, p < 0.0001). Additionally, the airway epithelium was severely hyperplastic, the alveolar septa were thickened, and there were variable amounts of type 2 pneumocyte hypertrophy.



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  		FIGURE 3. Inflammation scores 4 wk after intratracheal inoculation. Lungs from mice were scored blindly for intensity of inflammation according to the criteria in Table IGoB. TNFR-IFN-{gamma}-/- and CD4-depleted mice demonstrated significantly increased inflammation scores compared with wt, sham-inoculated, IFN-{gamma}-/-, or TNFR-/- mice (*p < 0.05 by Kruskal-Wallis test). Bars represent medians from 16 to 18 mice per group, except the CD4-depleted group (n = 9), in two separate experiments.

 


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  		FIGURE 4. Histologic changes in lungs of mice 4 wk after intratracheal inoculation. Representative sections shown from sham-inoculated mice (A) and mice inoculated with P. carinii: wt mice (B), IFN-{gamma}-/- mice (C), TNFR-/- mice (D), CD4-depleted mice (E), and TNFR-IFN-{gamma}-/- mice (F). See Results for discussion of characteristic histologic features. H&E stain; original magnification, x200.

 
Simultaneous deletion of IFN-{gamma}, TNFR1, and TNFR2 in mice challenged with P. carinii results in increased lung cytokine levels

We tested whether the increased susceptibility of the TNFR-IFN-{gamma}-/- mice at 4 wk after P. carinii inoculation was associated with defective elaboration of other lung cytokines. Using sandwich ELISAs, concentrations of IL-2, IL-4, IL-5, IL-10, and p40 IL-12 were measured in lung homogenates from individual sham-inoculated, wt, and TNFR-IFN-{gamma}-/- mice. At 4 wk after intratracheal inoculation, concentrations of all of measured cytokines were elevated in lung homogenates from TNFR-IFN-{gamma}-/- mice as compared with lung homogenates from sham-inoculated or wt mice (Fig. 5Go). ELISAs were also performed to measure antigenic IFN-{gamma} and TNF concentrations in lung homogenates from sham-inoculated, wt, and all gene-deleted groups. Four weeks after intratracheal inoculation, IFN-{gamma} was below the level of detection in lung homogenates from these groups of mice (data not shown). In contrast, concentrations of TNF were significantly increased in lung homogenates from wt and all gene-deleted mice as compared with concentrations in lung homogenates from sham-inoculated mice (Fig. 6Go).



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  		FIGURE 5. Cytokine concentrations in lung homogenates 4 wk after intratracheal inoculation. Lung tissue from individual mice was removed, weighed, and homogenized, and cytokine concentrations were measured in duplicate by ELISA in comparison with recombinant standards. Lung homogenates from TNFR-IFN-{gamma}-/- mice demonstrated significantly increased concentrations of cytokines compared with lung homogenates from sham-inoculated and wt mice (*p < 0.05 by ANOVA). Bars represent means ± SEM from 12 mice per group, except the wt group (n = 24).

 


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  		FIGURE 6. Antigenic TNF concentrations in lung homogenates 4 wk after intratracheal inoculation. Lung tissue from individual mice was removed, weighed, and homogenized, and TNF concentrations were measured in duplicate by ELISA in comparison with recombinant standard. Lung homogenates from wt, IFN-{gamma}-/-, TNFR-/-, and TNFR-IFN-{gamma}-/- mice demonstrated significantly increased concentrations of TNF compared with homogenates from sham-inoculated mice (*p < 0.05 by ANOVA). Bars represent means ± SEM from 12 mice per group, except the wt group (n = 24).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
While the individual roles of IFN-{gamma} and TNF in host defense against P. carinii pneumonia have been examined experimentally, the combined and interactive roles of these cytokines in host defense had not been tested directly in immunologically relevant animal models. We have demonstrated for the first time that immunosuppression induced by the combined loss of IFN-{gamma} and TNF signaling results in marked susceptibility to P. carinii. IFN-{gamma}-/- mice are able to clear the P. carinii inoculum completely, demonstrating that ablation of this individual cytokine does not confer susceptibility to P. carinii. TNFR-/- mice are also able to clear the inoculum, indicating that complete loss of TNF signaling does not preclude effective host defense. In contrast, the lungs of TNFR-IFN-{gamma}-/- mice demonstrate severe pneumonia. The TNFR-IFN-{gamma}-/- mice are unable to mount effective defense against this organism, but their lungs contain increased numbers of inflammatory cells, demonstrate intense histologic inflammation, and contain increased concentrations of other cytokines. Because these additional inflammatory events cannot restore defense, combined IFN-{gamma} and TNF signaling are essential elements of host defense against P. carinii.

Using knock-out mice, we have provided data that significantly advance the understanding of the combined importance of IFN-{gamma} and TNF in the host response to P. carinii. Most previous investigations examining host defense against pathogens focused on deletions of individual cytokine genes. However, the complexity and redundancy of cytokine pathways strongly suggests that single cytokine responses are unlikely to control susceptibility or defense (35). Rather, interactions between cytokines, either synergistic or antagonistic, determine the host’s response to challenge with pathogens. In these experiments, only the concurrent ablation of genes for IFN-{gamma} and TNF receptors increased the susceptibility of mice to P. carinii. Thus, mice in which multiple cytokine genes have been deleted provide a powerful tool for testing hypotheses focused on interactions among cytokines.

The finding that IFN-{gamma}-/- mice are not susceptible to P. carinii pneumonia is significant, because it further clarifies the controversial role of this important cytokine in host defense. Our results confirm and extend a recent report demonstrating that IFN-{gamma}-/- mice are not susceptible to P. carinii infection (7). After P. carinii inoculation, we measured no significant differences in intensity of inflammation or in lavage cell counts in the lungs of IFN-{gamma}-/- mice compared with wt mice, but concentrations of TNF in lung homogenates were increased. These data suggest that when TNF is available for host response, IFN-{gamma} is not required and only minimal inflammatory responses are necessary to clear P. carinii. Our data differ from those obtained by reconstituting scid mice with splenocytes from IFN-{gamma}-/- mice, which demonstrated prolonged inflammation during clearance (7). While these two models produced discrepant data concerning the role of IFN-{gamma} in control of inflammation, both models agree that IFN-{gamma} alone does not control host defense against P. carinii. Our data extend these observations by demonstrating that the concerted actions of IFN-{gamma} and TNF are required for defense against this pathogen.

We also determined that TNFR-/- mice are not susceptible to P. carinii, despite complete lack of TNF signaling. Although numerous studies, from our and other laboratories, have indicated that TNF is important in the host response to P. carinii (5, 9, 20, 23, 36, 37, 38, 39), the role of this important cytokine has not previously been tested in knock-out mice. We previously demonstrated that alveolar macrophages obtained from CD4-depleted mice during P. carinii pneumonia do not elaborate TNF spontaneously (23). However, these alveolar macrophages possess an enhanced capacity for TNF generation, demonstrated by TNF release after LPS stimulation. Therefore, we suggested that the lungs of CD4-depleted mice lack critical signals for TNF elaboration. Our data complement those obtained in a different experimental model, which examined the clearance of established, environmentally acquired P. carinii pneumonia in scid mice (5). In those experiments, reconstitution of scid mice with immunocompetent splenocytes cleared infection, but the addition of neutralizing Abs directed against TNF prevented clearance. Taken together, the results of both studies are significant because they suggest that TNF has different roles in P. carinii pneumonia depending on the stage of infection. When IFN-{gamma} is present, TNF may be more important in clearance of established infection than in prevention of induced infection.

To identify mechanisms for the susceptibility of TNFR-IFN-{gamma}-/- mice to P. carinii, we examined whether accumulation of inflammatory cells or production of other cytokines might be altered in the lungs of TNFR-IFN-{gamma}-/- mice. The enhanced susceptibility of CD4-depleted (28, 40), CD4- and CD8-depleted (30), TCR-{alpha}ß-deficient (41), B cell-deficient (42), and CD40 ligand-deficient (43) mice to P. carinii emphasizes the importance of both T and B lymphocyte populations in host defense against this organism. Accordingly, we measured the numbers and phenotypes of lavaged lymphocytes to determine whether the lungs of TNFR-IFN-{gamma}-/- mice demonstrated impaired recruitment of lymphocytes. However, numbers of lymphocytes were increased in lavages from TNFR-IFN-{gamma}-/- mice in comparison with lavages from other groups of mice. Furthermore, percentages of CD3+, CD4+, and CD8+ cells were not altered in the TNFR-IFN-{gamma}-/- mice. Quantitatively, then, ample numbers of lymphocyte effectors were available for defense in the lungs of TNFR-IFN-{gamma}-/- mice, but these lymphocytes were insufficient for defense in the absence of both IFN-{gamma} and TNF.

Alternatively, recruitment or activation of alveolar macrophages could be defective in the TNFR-IFN-{gamma}-/- mice. TNF and IFN-{gamma} are potent macrophage activators, and macrophages are important in the in vitro and in vivo clearance of P. carinii (44, 45, 46, 47). When compared with lavages from sham-inoculated mice, macrophage numbers were not increased significantly in the lavages from wt, IFN-{gamma}-/-, TNFR-/-, or TNFR-IFN-{gamma}-/- mice. Histologically, however, the lungs of TNFR-IFN-{gamma}-/- mice demonstrated large macrophages with foamy cytoplasm and frequent multinucleation. Although quantitatively unaltered, a likely explanation for the susceptibility of TNFR-IFN-{gamma}-/- mice to P. carinii is abnormal function of their alveolar macrophages. In vitro, P. carinii induces nitric oxide production and an oxidative burst in rat alveolar macrophages (44, 48), and blocking nitric oxide production eliminates ex vivo killing of P. carinii in the mouse (33). Both IFN-{gamma} and TNF individually stimulate macrophage secretion of nitric oxide and superoxide, and macrophages from IFN-{gamma}-/- mice demonstrate impaired nitric oxide production (11, 12). In response to P. carinii challenge, therefore, the alveolar macrophages in the TNFR-IFN-{gamma}-/- mice likely have deficiencies related to both nitric oxide and superoxide production. Additionally, alterations in receptor-mediated functions of alveolar macrophages may occur in the TNFR-IFN-{gamma}-/- mice. For example, decreased macrophage expression of receptors for mannose (46, 49) or fibronectin (50) may result in the inability of these mice to clear P. carinii organisms.

To determine whether the increased susceptibility in the TNFR-IFN-{gamma}-/- mice was associated with an attenuation of the production of other cytokines, we measured IL-2, IL-4, IL-5, IL-10, and p40 IL-12 in lung homogenates 4 wk after intratracheal inoculation. When adjusted for lung weight, all cytokines were increased in the TNFR-IFN-{gamma}-/- mice as compared with wt and sham-inoculated mice. Thus, despite the immunosuppressed state of the TNFR-IFN-{gamma}-/- mice, these mice are able to produce increased amounts of other cytokines. These data demonstrate that increases in other cytokines are insufficient for defense when both IFN-{gamma} and TNF are absent. When measured 4 wk after inoculation, IFN-{gamma} concentrations were not elevated in the lungs of sham-inoculated, wt, or TNFR-/- mice. Therefore, IFN-{gamma} may function in host defense in these mice at earlier time points after intratracheal inoculation. Compared with sham-inoculated mice, all gene-deleted mice demonstrated increases in lung homogenate concentrations of antigenic TNF. Therefore, production of TNF is a key component of host defense against P. carinii (8, 23), and it can confer resistance to P. carinii when IFN-{gamma} is absent.

In conclusion, our data emphasize the critical importance of both IFN-{gamma} and TNF in the host response to P. carinii. Simultaneously eliminating IFN-{gamma} and TNF signaling in mice challenged with P. carinii results in severe pneumonia, while ablation of the individual cytokines does not confer susceptibility to P. carinii. These data have important therapeutic and prophylactic implications for cytokine-based approaches to immunotherapy. Rather than focusing exclusively on individual cytokines, our data suggest that immunotherapy targeted at maximizing both the IFN-{gamma} and TNF responses to P. carinii may be required to augment host defense against this important opportunistic pathogen.


   Acknowledgments
 
We thank Drs. Harm HogenEsch, Alan Rebar, Tim Terrell, and Dan Tumas for their guidance and support during the first author’s tenure in the collaborative Purdue University and Genentech Pathology Fellow Program; and Drs. Jeffrey L. Curtis and Margaret R. Gyetko for helpful review of the manuscript. We acknowledge the expert technical assistance of John Wagner, Joanne Sonstein, Tim Pollack, Fran Wolber, and members of the Departments of Pathology and Immunology at Genentech.


   Footnotes
 
1 This work was supported by Genentech, Inc. (D.G.R.), U.S. Public Health Service Research Grants R01 HL-57011 and R01 HL-59823 (J.M.B.), and Merit Review Funds and a Career Development Award from the Medical Research Service, U.S. Department of Veterans Affairs (J.M.B.). Back

2 Current address: The DuPont Merck Pharmaceutical Company, Stine-Haskell Research Laboratories, 1094 Elkton Road, Newark, DE 19714. Back

3 Address correspondence and reprint requests to Dr. James M. Beck, Pulmonary and Critical Care Medicine (111G), Department of Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105-2303. E-mail address: Back

4 Abbreviations used in this paper: BAL, bronchoalveolar lavage; wt, wild-type; IFN-{gamma}-/-, IFN-{gamma} knock-out mice; TNFR-/-, TNFR 1 and 2 knock-out mice; GMS, Gomori methenamine silver; H&E, hematoxylin and eosin. Back

Received for publication December 10, 1997. Accepted for publication March 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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