Activated Pulmonary Macrophages Are Insufficient for Resistance against Pneumocystis carinii

Abstract

CD4⁺ T cells are pivotal for elimination of Pneumocystis carinii from infected lungs, and alveolar macrophages are considered the main effector cells clearing the infected host of P. carinii organisms. To investigate this issue, several mutant mouse strains were used in a previously established experimental setup which facilitates natural acquisition of disease through inhalation of airborne fungal organisms. Mutant mice deficient in major histocompatibility complex class II molecules (Aβ^−/−), T-cell receptor αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) were naturally susceptible to P. carinii, whereas mouse mutants lacking the gamma interferon (IFN-γ) receptor (IFN-γ-R^−/−) or tumor necrosis factor alpha (TNF-α) type I receptor (p55) (TNF-α-RI^−/−) resisted disease acquisition. Analysis of pulmonary cytokine patterns and free radical expression revealed the presence of superoxide, nitric oxide, and interleukin-1 (IL-1) mRNA and elevated levels of IFN-γ, TNF-α, and IL-12 in diseased TCRβ^−/− and RAG-1^−/− mice. Pulmonary macrophages of all diseased mouse mutants expressed scavenger and mannose receptors. Morbid Aβ^−/− mutants displayed significant NO levels and IL-1 mRNA only, whereas heterozygous controls did not exhibit any signs of disease. Interestingly, neither IFN-γ nor TNF-α appeared to be essential for resisting natural infection with P. carinii, nor were these cytokines sufficient for mediating resistance during established disease in the absence of CD4⁺ T lymphocytes. Taken together, the results indicated that an activated phagocyte system, as evidenced by cytokine and NO secretion, in diseased mutants was apparently operative but did not suffice for parasite clearance in the absence of CD4⁺ TCRαβ cells. Therefore, additional pathways, possibly involving interactions of inflammatory cytokines with CD4⁺ T lymphocytes, must contribute to successful resistance against P. carinii.

Immunocompromised patients, especially those suffering from AIDS, are at elevated risk of acquiring Pneumocystis carinii pneumonia (PCP), a major cause of premature mortality among AIDS patients (8, 35, 53). Various studies have emphasized that CD4⁺ T lymphocytes play a pivotal role in the orchestration of resistance to P. carinii (22, 43, 45), an opportunistic fungus, but the mechanisms underlying protection remain a conundrum. Pulmonary macrophages are considered the main effector cells in clearing the immunocompetent host from invading P. carinii organisms (25). It seems conceivable, therefore, that macrophage-activating functions mediated by CD4⁺ T cells are central to resistance. Impaired gamma interferon (IFN-γ) production by T cells from AIDS patients is thought to enhance susceptibility to P. carinii (34, 41). This notion is supported by reports that application of exogenous IFN-γ ameliorates disease in experimental animal models (2, 45). In contrast, in vivo neutralization of IFN-γ in spleen cell-reconstituted severe combined immunodeficiency (SCID) mice by a specific monoclonal antibody (MAb) does not affect parasite clearance (5). Further studies point to a critical role of tumor necrosis factor alpha (TNF-α) (5) and interleukin-1 (IL-1) (6) in maintaining an immunocompetent state. Both cytokines are mainly produced by macrophages and induce inflammatory responses (4, 10, 26). Overall, these findings support involvement of macrophage-derived cytokines in successful host resistance against P. carinii.

To analyze in more depth the role of inflammatory and Th1/Th2-related pulmonary defense mechanisms in control of aerogenically acquired PCP, we took advantage of naturally susceptible gene disruption mutant mice lacking major histocompatibility complex (MHC) class II molecules (and therefore conventional CD4⁺ T cells) (Aβ^−/−), T-cell receptor (TCR) αβ cells (TCRβ^−/−), or all mature T and B lymphocytes (RAG-1^−/−) (19). We further exploited mice deficient in the IFN-γ receptor (IFN-γ-R^−/−) or the TNF-α type I receptor (p55) (TNF-α-RI^−/−) to analyze their capacity to cope with aerogenic P. carinii organisms.

Bronchoalveolar lavage (BAL) cells of healthy and diseased mice were investigated for expression of the proinflammatory cytokines IL-1, TNF-α, IFN-γ, and IL-12, as well as IL-4, IL-5, and IL-10. The latter three cytokines counteract IFN-γ- and IL-12-mediated responses but participate in protection against certain extracellular pathogens (9). Moreover, production of superoxide (SO) and nitric oxide (NO), putative effector molecules of antimicrobial defense, was taken as a further indicator of macrophage activation. Contact with foreign material induces a rapid respiratory burst in professional phagocytes which results in SO production as a first line of defense. SO has been implicated in destruction of P. carinii (31), whereas NO produced by IFN-γ-stimulated macrophages encountering pathogens (4, 18, 30) does not appear to participate in control of P. carinii infection (47). Of further interest was the role of macrophage-expressed mannose receptors (MR) and scavenger receptors (SR). MR were previously found crucial for mediating P. carinii internalization (11, 37). The relevance of SR with respect to PCP has not been evaluated, but they are mainly expressed by tissue macrophages (36) and nonspecifically bind a large array of molecules, including surface molecules of microorganisms (39). Receptors with such broad pattern reactivity may be involved in direct differentiation of self from non-self, and recent data suggest that not only MR but also SR aid pattern recognition by macrophages and subsequent internalization of invading pathogens (27).

We found that BAL cells from P. carinii-diseased RAG-1^−/− and TCRβ^−/− mutants secreted elevated IFN-γ, TNF-α, IL-12, NO, and SO levels and expressed IL-1 mRNA. In contrast, cells from morbid Aβ^−/− mice produced IL-1 mRNA and high levels of NO only, whereas all other parameters were low to absent in these mutants. SR were expressed on pulmonary macrophages of all diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants, whereas MR were also expressed by macrophages of healthy animals. Yet, the apparently activated phagocyte system in the lung, most pronounced in morbid TCRβ^−/− and RAG-1^−/− mutant mice, was insufficient for protection against natural P. carinii infection. Elevated levels of IFN-γ and TNF-α in morbid mutants (not in Aβ^−/− mice) and the naturally resistant status of IFN-γ-R^−/− and TNF-α-RI^−/− mice further argue not only for independence from IFN-γ and TNF-α. Our findings indicate that CD4⁺ αβ T lymphocytes prevent and clear infection with P. carinii by mechanisms distinct from, or in addition to, pulmonary macrophage activation.

(This study is part of the Ph.D. thesis of R. Hanano.)

MATERIALS AND METHODS

Mice.

Breeding pairs of TCRβ^−/−, RAG-1^−/−, Aβ^−/−, and IFN-γ-R^−/− mutant mice were kindly provided by S. Tonegawa, P. Mombaerts (Massachusetts Institute of Technology, Boston, Mass.), D. Mathis (INSERM, Strasbourg, France), and M. Aguet (ISREC, Lausanne, Switzerland). TNF-α-RI^−/− mutant mice were thankfully obtained from H. Bluethmann (Hoffmann-La Roche, Basel, Switzerland) (7, 24, 32, 33, 42). All animals were maintained at the animal facilities of the University of Ulm under specific-pathogen-free conditions and were of 129 × C57BL/6 background. Aβ^−/− mutants were from the 12th backcross to C57BL/6 mice upwards; TCRβ^−/− and RAG-1^−/− mutants were from the 5th backcross upwards. IFN-γ-R^−/− mice were from the first and second backcrosses to C57BL/6 mice, and TNF-α-RI^−/− mice were not backcrossed. Homozygous Aβ^−/−, TCRβ^−/−, and RAG-1^−/− mutants were identified by screening blood samples by fluorocytometry for absence of MHC class II molecules (and reduced CD4⁺ T-cell numbers), of TCRαβ lymphocytes, or of all T and B cells, respectively, using appropriate MAbs. Mutant IFN-γ-R^−/− and TNF-α-RI^−/− strains were characterized by molecular biological techniques as described previously (24, 42). Heterozygous littermates of homozygous TCRβ^−/−, RAG-1^−/−, and Aβ^−/− mutants from the same backcrosses were used as controls. Healthy mutants from our breeding colonies served as further controls.

Experimental setup.

Preweighed mutant mice as well as heterozygous littermates were cohoused in an isolator under specific-pathogen-free conditions with P. carinii-diseased mutants, as described in detail elsewhere (20). Susceptible mutant mouse strains acquired PCP naturally through inhalation of airborne parasites. After loss of 20% of the initial body weight, animals had attained the moribund state and were sacrificed for analysis. Different individual mice were used for histology, BAL, or PCR. For each method at least three animals of each mutant strain were used, and at least two replicates were performed. Animals were selected at random for the various analyses. Sampling en bloc from any mutant strain for a single method was avoided as not to bias data due to potential environmental fluctuations within the isolator.

BAL and cell culture.

Lungs of sacrificed mice were perfused thoroughly with sterile phosphate-buffered saline (PBS). Subsequently, BAL was performed with four volumes of 0.7 ml of Dulbecco modified Eagle medium (DMEM; Gibco Laboratories, Grand Island, N.Y.) supplemented with 10% fetal calf serum (FCS; PAA Labor- und Forschungsgesellschaft, Linz, Austria), penicillin (100 U/ml), streptomycin (100 μg/ml; Gibco), and amphotericin B (1.5 μg/ml; Sigma Chemical Co., St. Louis, Mo.). Viability of cells was consistently >99%, as determined by trypan blue exclusion. Cell numbers were determined, and BAL fluids were transferred to flat-bottom 96-well plates (Nunc, Roskilde, Denmark) at a volume of 100 μl with 10⁵ cells/well unless otherwise specified. Plates were incubated at 37°C in a 10% CO₂ atmosphere for 24 h. Thereafter, supernatants were removed and frozen at −20°C until used for cytokine analysis. BAL cells of healthy mice were incubated at 5 × 10⁴ cells/well in DMEM supplemented with 10% FCS. Cells were either incubated with medium alone or stimulated with 2.5 × 10⁵ Mycobacterium bovis BCG (DSM, Braunschweig, Germany) to induce TNF-α production or with 100 μg of lipopolysaccharide (LPS; Difco Laboratories, Detroit, Mich.) to stimulate IL-12 secretion. Cells from diseased mice were treated similarly and also with 1 μg of concanavalin A (ConA)/well to provoke IFN-γ production.

Lung tissue sections, cytospins of BAL cells, and histology.

Lungs of euthanized mice were displayed, cannulated, and gently inflated with sterile PBS. Lungs were removed, snap-frozen in liquid nitrogen, and stored at −70°C. Sections of 7 μm were prepared by using a Frigocut 2800 (Reichert-Jung, Heidelberg, Germany), dried at room temperature, fixed in acetone for 10 min, and stored at −20°C. Lung sections of all mouse mutants were checked for the presence of P. carinii sporangia by staining with silver methenamine (Sigma). General tissue morphology was investigated by using hematoxylin-eosin staining, and periodic acid-Schiff staining was used to detect foamy honeycomb material. Cytospins of BAL cell aliquots (prepared with a Cytospin 3 [Shandon Scientific Ltd., Runcorn, England]) and cryostat sections were analyzed for expression of inducible NO synthase (iNOS), using a polyclonal rabbit antibody (Ab) directed against this enzyme (Biomol, Plymouth Meeting, Pa.), SR type I/II (clone 2F8, rat immunoglobulin G2b [IgG2b]; Serotec, Wiesbaden, Germany), and MR by binding to fluorescein isothiocyanate (FITC)-conjugated mannopyranosyl-phenyl isothiocyanate (Sigma). Alkaline phosphatase (AP)-conjugated secondary Abs were used to label the primary Ab, followed by nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP; Dako, Glostrup, Denmark) to evolve an insoluble dark blue precipitate or by NBT-BCIP-iodine nitrotetrazolium violet (NBT-BCIP-INT; Dako) to obtain brown staining.

Fluorocytometry.

NK cells in BAL fluids were detected by a biotinylated MAb (clone PK136; American Type Culture Collection, Rockville, Md.). An FITC-conjugated anti-CD3 MAb (clone 145-2C11; kindly provided by J. Bluestone, Ben May Institute, Chicago, Ill.) served for counterstaining. Strepavidin-phycoerythrin (Gibco) was added to label the biotinylated MAb with fluorescence. For each sample, 10,000 cells were taken up and the lymphocyte-rich region was analyzed by using a FACScan with Lysis II software (Becton Dickinson, Mountain View, Calif.).

TNF-α bioassay.

TNF-α-sensitive L929 cells were seeded into 96-well plates at a concentration of 2 × 10⁴/well in 50 μl of DMEM supplemented with 10% FCS and incubated at 37°C (10% CO₂) for 24 h. Supernatant was discarded and replaced by medium supplemented with 3 μg of actinomycin D (Sigma) per ml. Standard TNF-α concentrations (Genzyme Corporation, Cambridge, Mass.) or experimental supernatants (50 μl) were added and incubated at 37°C for a further 24 h. To control for TNF-β secretion, selected samples were additionally preincubated with an excess of anti-TNF-α polyclonal Ab (Genzyme). Afterwards, the mixture was supplemented with 20 μl of 2.5 mg of thiazolylblue (Sigma) per ml in PBS and incubated at 37°C for 4 h. Cells were lysed with 10% sodium dodecyl sulfate in 0.01 M HCl and incubated overnight. Analysis was performed at 570 nm with reference to 690 nm, using an enzyme-linked immunosorbent assay (ELISA) reader (Spectramax 250; Molecular Devices, Sunnyvale, Calif.).

Cytokine ELISA.

Supernatants of BAL cell cultures were assayed for IL-4, IL-5, IL-10, IL-12, and IFN-γ by using standard sandwich ELISA techniques. For IL-4, we used two specific rat MAbs: BVD4-1D11 (rat IgG2b; Dianova, Hamburg, Germany) for coating and biotinylated BVD6-24G4 (rat IgG1; kindly provided by R. L. Coffman, DNAX, Palo Alto, Calif.) for detection. Murine recombinant IL-4 (rIL-4) was purchased from Genzyme and had a specific activity of 10⁷ U/ml. The detection limit was 5 pg/ml. To analyze IL-5, MAb TRFK5 (rat IgG1; Pharmingen, San Diego, Calif.), biotinylated TRFK4 (rat IgG2a; Pharmingen), and murine rIL-5 (specific activity, 8 × 10⁶ U/mg; Pharmingen) were used. The detection limit was 10 pg/ml. For IL-10, MAb JES5-2A5.7 (rat IgG1; DNAX) and biotinylated MAb SXC-1 (rat IgM; Pharmingen) were used. Murine rIL-10 (specific activity, 5 × 10⁵ U/mg of protein) was kindly provided by A. Sher and I. Oswald (National Institutes of Health, Bethesda, Md.). The detection limit was 1 U/ml. IL-12 (p40) was assayed with MAb C15.6.7 (rat IgG1) and biotinylated C17.8 (rat IgG2a), kindly provided by G. Trinchieri (The Wistar Institute, Philadelphia, Pa.). Murine rIL-12, with a specific activity of 5.6 × 10⁶ U/mg of protein, was a kind gift from S. Wolf (Genetics Institute, Cambridge, Mass.). The detection limit was 50 pg/ml. For IFN-γ ELISA, we used two rat mAbs: R4-6A2 and biotinylated AN18-17.24 (kindly provided by J. Langhorne, Max Planck Institute for Immunobiology, Freiburg, Germany). Murine rIFN-γ, with a specific activity of 10⁷ U/mg of protein, was a kind gift of G. Adolf (Ernst-Boehringer Institut für Arzneimittelforschung, Vienna, Austria). The detection limit was 0.1 U/ml.

IL-1 RT-PCR.

For detection of cytokine mRNA in BAL cells, total RNA of cells was extracted, reverse transcribed, and amplified with 35 cycles, using specific primers following a standard protocol (12). The following sense and antisense primers were used: for IL-1α, 463-486 (3′-AAGTTT-GTCATGAATGATTCCCTC-5′) and 705-725 (3′-GTCTCACTACCTGTGATGAGT-5′) (Stratagene, La Jolla, Calif.); for IL-1β, 330-350 (5′-CAGGATGAGGACATGAGCACC-3′) and 756-776 (5′-CTCTGCAGACTCAAACTCCAC-3′) (Stratagene). Primers for β-actin, 206-227 (5′-TGTGATGGTGGGAATGGGTCAG-3′) and 698-719 (5′-TTTGATGTCACGCACGATTTCC-3′) (Stratagene), were used to control for reverse transcription-PCR (RT-PCR) efficiency. PCR products were resolved on 1.5% agarose.

Detection of NO and SO.

NO in supernatants of cell cultures was measured by the Griess test (17). Some wells were supplemented with superoxide dismutase (SOD) to eliminate SO anions (29). In parallel, the respiratory burst of alveolar macrophages was determined by chemiluminescence. After centrifugation of BAL fluids at 700 × g and 4°C for 10 min, the pellet was taken up in DMEM without FCS and phenyl red (Seromed, Berlin, Germany) and supplemented with 2% HEPES (Seromed), and 5 × 10⁴ cells in 100 μl were seeded into 96-well flat-bottom microtiter plates (Nunc). Subsequently, 50 μl of N-methylacridinium nitrate (500 μg/ml; Sigma) and zymosan A (100 μg/ml; Sigma) were added. Controls received DMEM-HEPES instead of zymosan A. Measurements were made immediately after addition of zymosan A over 1.5 h in cycles of 90 s, using a MicroLumat LB 96 P and WinGlow software (EG&G Gerthold, Bad Wildbad, Germany). Respiratory burst was quantified by measurement of light emission (relative light units per second).

PCR analysis for detection of P. carinii-specific DNA.

To track minor P. carinii accumulations in lungs not detectable by silver methenamine staining, PCR was performed with the oligonucleotide primers pAZ102-E (5′-GATGGCTGTTTCCAAGCCCA-3′) and pZA102-H (5′-GTGTACGTTGCAAAGTACTC-3′) according to an established protocol (52). These primers amplify part of the gene encoding the large subunit of P. carinii-specific mitochondrial rRNA. To achieve higher sensitivity while at the same time verifying the specificity of the amplified products, PCR products were blotted and hybridized with ³²P-labeled linearized plasmid pBS-PC, which contains a fragment of the P. carinii-specific mitochondrial RNA gene as previously described (20).

Statistical analysis.

Differences were analyzed by the Student t test, and variances were determined by analysis of variance (ANOVA) followed by the Duncan test.

RESULTS

Detection of P. carinii in lung tissues.

As reported previously (20), RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants consistently developed PCP when cohoused with diseased mice within approximately 3 months, and none of these mutants survived periods beyond 4.5 months. In contrast, TNF-α-RI^−/− and IFN-γ-R^−/− mutants never acquired disease under these conditions, even when confined to isolators with diseased animals for 10 to 15 months. Like control mice, these mutants never showed any symptoms and readily gained weight. In support of this observation, lungs of IFN-γ-R^−/− and TNF-α-RI^−/− mutants were devoid of any P. carinii organisms, as determined by silver methenamine staining of pulmonary tissues (Fig. 1A), in contrast to diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutants (Fig. 1B and C). P. carinii-specific PCR analysis of lung tissues from parasite-exposed mutants verified these findings (Fig. 1D). These data argue against an essential role of IFN-γ and TNF-α in prevention of natural acquisition of PCP. The possibility that the susceptibility of the mouse mutants used was affected by different stages of backcrossing was excluded, because heterozygous littermates, having genetic backgrounds comparable to those of the homozygous mutants used, never acquired disease under these conditions.

FIG. 1.

P. carinii detection by histology and PCR. P. carinii organisms were undetectable by silver methenamine staining of lung sections from parasite-exposed healthy mutants (A, represented by IFN-γ-R^−/−), in contrast to morbid mutant mice (B, represented by TCRβ^−/−). Magnification of the diseased lung section is shown to distinguish stained sporangia (C). Whole lung digests were used for detection of P. carinii by PCR, products of which were blotted on nitrocellulose and hybridized with a P. carinii-specific gene fragment (D). Parasitized lung tissues of TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutant mice gave positive signals, whereas tissues from P. carinii-exposed heterozygous control mice (+/−) and from exposed IFN-γ-R^−/− or TNF-α-RI^−/− mutants did not reveal a detectable PCR signal. Bars, 50 μm.

Surface expression of SR and MR by pulmonary macrophages of diseased mice.

SR expression was profoundly induced in lungs of all diseased mutants (Fig. 2A) but was not detectable in healthy mice (Fig. 2B). Immunohistologic stainings of cytospins revealed this receptor type to be expressed by macrophages only (Fig. 2C). In contrast, MR were constitutively expressed by macrophages and apparently also by granulocytes from diseased and healthy mouse mutants (Fig. 2D and E).

FIG. 2.

Expression of SR and MR in lung tissues and BAL cells of diseased mouse mutants. Cells in lung sections of diseased mutants (A, represented by TCRβ^−/−) exhibited profound SR expression, as determined by immunohistology, whereas none were detected in lungs of healthy animals (B, represented by TCRβ^−/−). Histochemical staining of cytospins of BAL cells from diseased mice identified expression of SR to be restricted to alveolar macrophages (C, represented by TCRβ^−/−). Labeling cytospins of BAL cells with FITC-conjugated mannopyranosyl-phenyl isothiocyanate from diseased (D, represented by TCRβ^−/−) and healthy (E, represented by TCRβ^−/−) mice illustrates constitutive MR expression by macrophages and granulocytes. (F) Unstained cells to control for green autofluorescence. Panels A to C represent AP-conjugated anti-SR type I/II MAb developed with NBT-BCIP and counterstained with nuclear fast red. Bars, 50 μm.

Expression of iNOS by pulmonary macrophages of diseased animals.

We have previously described phagocyte accumulation in lungs of diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutant mice (20). In mice, activated macrophages express iNOS, and NO is considered a major effector molecule in antimicrobial defense (30). Lung sections of diseased RAG-1^−/−, TCRβ^−/−, and Aβ^−/− mutant mice reacted with a specific polyclonal Ab against iNOS (Fig. 3A), whereas lung sections from control mice (healthy homozygous and heterozygous mutants) (Fig. 3B) as well as from P. carinii-exposed TNF-α-RI^−/− and IFN-γ-R^−/− mutants were consistently negative (Fig. 3C). Immunohistology with the same Ab of cytospins of BAL cells from diseased mutants revealed exclusive expression of iNOS in macrophages, as determined by cell morphology (Fig. 3D), whereas none of the numerous granulocytes reacted with this Ab.

FIG. 3.

Detection of iNOS expression in lung sections and cytospins of BAL cells by immunohistology. Lung sections of diseased TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutant mice consistently stained positively with a polyclonal Ab against iNOS (A, represented by Aβ^−/−). Lung sections from P. carinii-exposed IFN-γ-R^−/− (B) and TNF-α-RI^−/− (C) mutant mice were iNOS negative. Staining of cytospin preparations of BAL cells derived from parasitized mutant mice revealed that iNOS expression was restricted to alveolar macrophages (D, represented by Aβ^−/−). Arrow, multinucleated giant cell. (A to C) AP-conjugated anti-iNOS polyclonal Ab developed with NBT-BCIP and counterstained with nuclear fast red; (D) AP-conjugated anti-iNOS Ab developed with NBT-BCIP-INT and counterstained with hematoxylin. Bars, 50 μm.

NO production by BAL cells.

To determine NO production by BAL cells directly, supernatants of cultured cells were analyzed for nitrite after 24 h of culture. NO production was detected in all diseased mouse strains, levels of which varied considerably within each strain (Fig. 4A). Variance of NO release among different mouse strains was statistically not significant (P = 0.1 by ANOVA). In contrast, NO levels were undetectable in BAL cells from nonparasitized mutant mice as well as from parasite-exposed IFN-γ-R^−/− and TNF-α-RI^−/− mutants (Fig. 4A). Addition of SOD to lavage cell cultures from morbid TCRβ^−/− and RAG-1^−/− mutant mice considerably increased nitrite levels (Fig. 4B), whereas NO concentrations produced by BAL cells from Aβ^−/− mutants remained virtually unchanged. SOD catalyzes the reaction of SO with protons to form oxygen molecules and hydrogen peroxide (29). Hence, SO, which readily reacts with NO to form peroxynitrite (23), is rapidly eliminated from the cellular system by SOD, rescuing NO radicals from being scavenged (29). Thus, differences of NO levels in cultures with, relative to cultures without, SOD indicate SO production. According to this method, morbid TCRβ^−/− and RAG-1^−/−, but not Aβ^−/−, mutant mice secreted SO. Obtained SO values varied significantly between mutant strains (P < 0.05 by ANOVA), with significant differences between TCRβ^−/− and RAG-1^−/− BAL cells in comparison to Aβ^−/− mutants (Duncan test). The issue of SO production was further examined in the next experiment.

FIG. 4.

NO production by BAL cells in the presence or absence of SOD. BAL cells from diseased TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutants produced high levels of NO, whereas cells from nonparasitized mice did not (A). NO production by pulmonary cell cultures with (black bars) and without (white bars) SOD were determined (B). Corresponding cultures with and without SOD were performed with cells from the same mice. SOD eliminates secreted SO, therefore preventing SO from scavenging NO. Differences of NO production with and without SOD of each mutant strain are therefore used as an indication of SO secretion by the BAL cells. Shown are means of duplicates of three mice per mutant strain; error bars represent standard deviations. Results refer to 10⁵ pulmonary cells. h, healthy; d, diseased.

Capacities of BAL cells from healthy and diseased mutants to secrete SO.

Freshly isolated BAL cells from healthy TCRβ^−/−, RAG-1^−/−, Aβ^−/−, and C57BL/6 mice were stimulated with zymosan A, and the respiratory burst was measured. BAL cells from all mice failed to produce a burst in the absence of zymosan A, whereas potent respiratory activity was observed after zymosan stimulation (Fig. 5A). It could be argued that in contrast to BAL cells from TCRβ^−/− and RAG-1^−/− mice, cells from diseased Aβ^−/− mutants were exhausted of constitutive SO production. To address this issue, BAL cells from morbid Aβ^−/− and TCRβ^−/− mutants were stimulated with zymosan A. Cells from both mutant mice produced a respiratory burst, albeit of different intensities (Fig. 5B). Cellular exhaustion of diseased Aβ^−/− mouse-derived BAL cells, therefore, appears unlikely.

FIG. 5.

Respiratory burst of BAL cells from healthy and P. carinii-parasitized mutant mice. (A) Respiratory burst by BAL cells from healthy mice stimulated with zymosan A. Cells from mouse strains without zymosan A did not show any activity. Results are representative of duplicates using 5 × 10⁴ BAL cells. (B) Respiratory burst by BAL cells from diseased TCRβ^−/− and Aβ^−/− mutants with and without zymosan A. Results are representative of duplicates using 10⁵ BAL cells. RLU, relative light units.

BAL cell cultures of diseased mutants produce proinflammatory cytokines IFN-γ and IL-12 but not IL-4, IL-5, and IL-10.

Cells from morbid RAG-1^−/− and TCRβ^−/− mutants produced elevated concentrations of IFN-γ, TNF-α, and IL-12 compared to their healthy counterparts (Fig. 6). High variations among mice of the same mutant strain were observed, although all animals were apparently moribund at the time of cytokine determination. We considered it important to assess whether TNF values determined by the bioassay corresponded to TNF-α, TNF-β, or both, since the cell line used for TNF detection responds to both. Therefore, randomly selected samples from each mutant strain were tested in the presence of a neutralizing Ab directed against TNF-α. Addition of this Ab abolished the activity, suggesting that TNF-α rather than TNF-β was the responsible cytokine. Cytokine production by BAL cells from diseased Aβ^−/− mice was lower than in TCRβ^−/− and RAG-1^−/− mutants (Fig. 6). Cytokine production did not correlate with the pulmonary P. carinii burden in individual mice (data not shown). Even after parasite exposure for more than 10 months, IFN-γ-R^−/−, TNF-α-RI^−/−, or C57BL/6 mice never expressed elevated levels of IL-12, IFN-γ, or TNF-α. IL-4, but not IL-10 or IL-5, was only occasionally detected in small amounts (up to 8 pg/ml) in diseased Aβ^−/− mutants. Additionally, IL-1β and IL-1α mRNAs were monitored by RT-PCR. Healthy TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutants were consistently devoid of these messages (Fig. 7), whereas constitutive coexpression of IL-1α and IL-1β mRNAs was demonstrable in BAL cells of all three diseased mutant strains (Fig. 7).

FIG. 6.

Constitutive IFN-γ (A), TNF-α (B), and IL-12 (C) production by 10⁵ BAL cells from healthy and diseased mouse mutants with indicated deficiencies. Each data point corresponds to one mouse, for which at least two replicates were performed. Average values are indicated by horizontal bars. Asterisks indicate significant differences (Student t test, P < 0.05) between cytokine levels attained by healthy and diseased mice of each mutant strain. Statistics are not applicable in panel A, since healthy animals do not produce any IFN-γ. h, healthy; d, diseased.

FIG. 7.

IL-1 mRNA of BAL cells from healthy and diseased mutants. IL-1β and IL-1α mRNAs were expressed by BAL cells from diseased TCRβ^−/− (T), Aβ^−/− (A), and RAG-1^−/− (R) mutants but not by their healthy counterparts. β-Actin controls verify abundant total RNA content in all probes used. h, healthy; d, diseased. m, size markers.

Cytokine production by BAL cells from healthy and diseased mutant mice after LPS, M. bovis BCG, and ConA stimulation.

To assess whether the reduced cytokine production in diseased Aβ^−/− mutants compared to TCRβ^−/− and RAG-1^−/− mutant mice was due to an intrinsic defect, cells from healthy and diseased animals were stimulated with LPS, M. bovis BCG, or ConA, and levels of secretion of IL-12, TNF-α, and IFN-γ were determined. In healthy mice, pulmonary cells from all mouse strains produced marked concentrations of IL-12 and TNF-α (Table 1). ConA stimulation was omitted, because pulmonary T cells were virtually absent in nondiseased mutants. Cells from IFN-γ-R^−/− and TNF-α-RI^−/− mutants and C57BL/6 mice were derived from P. carinii-exposed animals. Upon stimulation of BAL cells from diseased TCRβ^−/− and Aβ^−/− mutants with the same agents, cytokine production was consistently higher than in nonstimulated cells. Thus, depressed cytokine production by morbid Aβ^−/− mutants was apparently not attributable to an intrinsic defect; rather, P. carinii infection in this mutant strain failed to induce secretion of these cytokines. Explicit reasons for this deficiency in the absence of surface-expressed MHC class II molecules remain to be defined.

TABLE 1.

Cytokine production by alveolar macrophages from healthy and diseased mice after stimulation

Mouse strain	Mean concn ± SDa
	TNF-α (pg/ml)		IL-12 (pg/ml)		IFN-γ (U/ml)
	Medium	BCGb	Medium	LPSc	Medium	ConAd
C57BL/6	7.3 ± 4.7	415 ± 243	350 ± 60	6,680 ± 1,870	NDf	ND
TCRβ−/−	7.2 ± 5.0	490 ± 460	1,250 ± 490	5,420 ± 2,370	ND	ND
	90 ± 27e	560 ± 10e	6,120 ± 1,240e	8,210 ± 2,020e	10.7 ± 3.2e	34 ± 4.6e
Aβ−/−	7.2 ± 7.1	950 ± 280	530 ± 250	5,000 ± 1,790	ND	ND
	20 ± 7.8e	260 ± 40e	1,870 ± 425e	3,920 ± 275e	3.8 ± 1.2e	21 ± 3.3e
RAG-1−/−	8.0 ± 7.7	1,460 ± 360	850 ± 300	3,170 ± 2,020	ND	ND
IFN-γ-R−/−	8.6 ± 5.8	190 ± 90	350 ± 210	12,500 ± 5,200	ND	ND
TNF-α-RI−/−	9.7 ± 5.9	490 ± 460	500 ± 170	35,200 ± 570	ND	ND

^aResults for 10⁵ cells; at least two replicates per mutant strain. 

^bStimulation with 2.5 × 10⁵ M. bovis BCG/well for 24 h. 

^cStimulation with 100 μg of LPS/well for 24 h. 

^dStimulation with 1 μg of ConA/well for 24 h. 

^eCells from diseased mice; at least two replicates per mutant strain. 

^fND, not determined. 

Fluorocytometric identification of NK cells in BAL fluids from P. carinii-diseased mouse mutants.

Parasitized RAG-1^−/− and TCRβ^−/− mouse-derived BAL cells produced similar amounts of IFN-γ. Hence, the production of this cytokine could be mainly attributed to NK cells, because in RAG-1^−/− mutants this is the only cell type known to be capable of producing IFN-γ. Determination of NK cell proportions in BAL fluids of all three diseased mutants revealed that 0.6 to 2% of total BAL cells encompassed this cell population (Fig. 8). Hence, lower IFN-γ expression in the Aβ^−/− mutants than in TCRβ^−/− and RAG-1^−/− mice does not correspond to different numbers of NK cells in the diseased lung. We are aware that the presence of other IFN-γ-producing cells in Aβ^−/− and TCRβ^−/− mutants is likely. Of note is the apparent presence of CD3⁺ NK cells in BAL fluids of Aβ^−/− mutants, which were not present in every individual Aβ^−/− mouse. The identification of such NK⁺ T cells is in agreement with several previous studies (3) but was not further investigated.

FIG. 8.

NK1.1 cells in BAL fluids of diseased mutant mice. BAL cells from diseased TCRβ^−/− (A), Aβ^−/− (B), and RAG-1^−/− (C) mutants were stained with FITC-conjugated anti-NK1.1 and phycoerythrin-conjugated anti-CD3 MAb and analyzed by fluorocytometry. Illustrated are cells within the lymphocyte gate. NK1.1 single-positive cells comprise 0.6 to 2% of total BAL cells for each mutant strain.

DISCUSSION

Immunodeficiency is a prerequisite for disease manifestation of P. carinii infection both in humans and in experimental animal models. Previous studies generally used active infection of immunocompromised mice and rats with high inocula of P. carinii organisms. Experiments with spleen cell-reconstituted SCID mice in which endogenous cytokines were neutralized by Ab treatments, or in which animals received cytokines, implicate numerous cytokines in successful defense against P. carinii (2, 5, 6, 45). Through the use of gene deletion mutant mice, we investigated the pulmonary immune responses of susceptible mutants suffering from naturally acquired PCP. We chose an experimental setup which facilitates natural transmission of the P. carinii organism, rather than active infection. This approach allows gradual development of disease and immune response similar to natural infection of humans. Studies using active infection generally apply inocula in the order of 10⁷ P. carinii pathogens. Even if one considers that only a small proportion of P. carinii survive in the lung, we consider it likely that such inocula exceed numbers transmitted by infected animals at a given time. Moreover, we consider constant exposure to infected animals different from single inoculation.

By using several gene deletion mutant mice, namely, TCRβ^−/−, Aβ^−/−, RAG-1^−/−, IFN-γ-R^−/−, and TNF-α-RI^−/− mutants, we investigated various capacities of pulmonary macrophages in diseased and resistant P. carinii-exposed mice. TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutants have been characterized as naturally susceptible to P. carinii (20). The findings reported here suggest that in the absence of CD4⁺ TCRαβ cells, conventional macrophage activation, as assessed by secretion of IL-12, TNF-α, and free NO and SO radicals, and IL-1 mRNA expression fail to prevent the development of naturally acquired disease. These features are generally considered to indicate macrophage activation. Consistent with this view, enlarged and multinucleated cells have been observed in P. carinii-diseased mice (1, 19). We therefore consider it most likely that pulmonary macrophages had achieved a state of activation in gene deletion mutant mice suffering from PCP.

A previous study considered the importance of IFN-γ and TNF-α for P. carinii resistance (5). The resistant status of IFN-γ-R^−/− and TNF-α-RI^−/− mutants described here, therefore, raises the question about the extent to which these two cytokines are involved in host resistance. Treatment of diseased SCID mice with antibodies against IFN-γ concomitantly with or after reconstitution of mice with spleen cells did not impair P. carinii clearance (5). Our data verify these results and findings of a further report (14) which was published while this report was in preparation. It was shown that IFN-γ participates in clearance of P. carinii but is not essential. Clearance mechanisms ultimately executed by pulmonary macrophages, therefore, appear to be IFN-γ independent, as also suggested by the relatively high level IFN-γ production in lungs of diseased RAG-1^−/− and TCRβ^−/− mutants. Furthermore, the relatively low levels of IFN-γ produced by BAL cells of Aβ^−/− mutants, but similar parasite burdens in Aβ^−/− and TCRβ^−/− mutants and elevated burdens in RAG-1^−/− mice (19, 20), argue against a correlation between IFN-γ production and fungal colonization. Since IL-12 promotes IFN-γ production in CD4⁺ T cells as well as NK cells (28, 50), elevated levels of this cytokine appear redundant for resolution of PCP. However, whether IL-12 plays a role in clearing manifested disease in the presence of CD4⁺ T cells remains to be determined.

Anti-TNF-α antibodies were found to abrogate defense mechanisms in diseased SCID mice when administered comcomitantly with spleen cell reconstitution (5). Mice did convalesce, however, if antibody treatment was delayed by 6 days (5). Since TNF-α titers in lung homogenates did not vary significantly at or after reconstitution (5), it appears likely that TNF-α-mediated parasite clearance in established disease is associated with the presence of CD4⁺ T cells but not with a time delay in production of this cytokine. These results suggest a central role of TNF-α for initiating CD4⁺ T-cell-mediated resistance mechanisms. Yet in our experiments, TNF-α-RI^−/− mutant mice did not acquire disease under conditions under which TCRβ^−/−, RAG-1^−/−, and Aβ^−/− mutants did. At least two explanations can be offered for the apparent discrepancy between our findings and the above-described report (5). First, it is possible that TNF-α, which is produced by TNF-α-RI^−/− mutants (13, 42) (Table 1), confers protection through the TNF-α type II receptor (p75), at least in the absence of p55. TNF-α-RI^−/− mutant mice severely suffer from infection with Listeria monocytogenes (42). However, L. monocytogenes is an intracellular bacterium whereas P. carinii is an extracellular fungus, and mechanisms of resistance to these two pathogen types may vary significantly. Further studies using appropriate mouse mutants or selective blockage of TNF-α receptors will be necessary to clarify this issue. Alternatively, TNF-α may be essential for clearance of established disease in the presence of CD4⁺ T lymphocytes, as shown previously (5), but not for resisting disease acquisition due to low but steady aerogenic parasite numbers. Thus, differential protective mechanisms may be responsible for convalescence from manifested disease and for resisting fungal acquisition.

Similar to TNF-α depletion, blockage of IL-1 receptors of P. carinii-parasitized SCID mice interferes with parasite clearance when applied directly after spleen cell reconstitution but not at later time points (6). Since IL-1α and IL-1β mRNAs were detected in BAL cells of diseased TCRβ^−/−, Aβ^−/−, and RAG-1^−/− mutants, expression of this cytokine group apparently did not confer resistance to P. carinii in the absence of CD4⁺ T cells. Like TNF-α, therefore, IL-1 appears to be involved in the initiation of CD4⁺ T-lymphocyte-dependent clearance functions. Once appropriate mechanisms have been induced, continued availability of IL-1 seems to be dispensable.

The production of NO, which exhibits profound antimicrobial effects (30), also appears insufficient for fighting P. carinii. It was previously noted that the fungus alone failed to induce NO synthesis in vitro but did so in combination with IFN-γ (48, 49). On average, pulmonary cells of diseased TCRβ^−/−, RAG-1^−/−, and Aβ^−/− mutants secreted high levels of NO, despite lower IFN-γ levels in Aβ^−/− mutants, yet succumbed to disease. Although parasite numbers in lungs of moribund RAG-1^−/− mice were higher than in lungs of TCRβ^−/− and Aβ^−/− mice (19, 20), similar levels of NO were produced in these three mutants. Hence, we conclude that reactive nitrogen intermediates are of no or little relevance for parasite clearance.

Positively associated with parasite clearance has been the expression of MR on macrophages, which bind glycoprotein A expressed on the surface of P. carinii (11, 37). Due to the constitutive expression of this receptor type on naive pulmonary macrophages, however, clearance of P. carinii is not expected to depend exclusively on MR expression. Whether MR expression was upregulated in diseased mice was not determined. Further macrophage receptors may be involved. It has been shown previously, for example, that surfactant protein D, secreted by type II pneumocytes and nonciliated bronchiolar cells (51), also binds to glycoprotein A, thereby enhancing binding of P. carinii to alveolar macrophages (38). This binding is not inhibited by α-mannan, which blocks MR (38). Since surfactant protein D contains short collagenous domains (40), which are bound with high affinity by SR (27), it is conceivable that this receptor type, in addition to MR, is involved in phagocytosing P. carinii. Indeed, pulmonary macrophages derived from diseased mutants exhibited profound upregulation of SR. More functional studies will be necessary for evaluating a definite role of this receptor type for P. carinii resistance.

Taken together, our data reveal that pulmonary macrophages which appear activated in terms of the described parameters are insufficient for prevention and cure of PCP in the absence of CD4⁺ T lymphocytes. Histological analysis of lung sections from mice with early stages of fungal infection revealed phagocyte infiltrations only at sites of parasitic accumulation (20), demonstrating directed migration of these cells into corresponding compartments. In advanced disease, P. carinii organisms are disseminated throughout the alveolar compartment of the lung and large numbers of macrophages have accumulated accordingly (20). Therefore, macrophages receive appropriate signals for migration, seemingly facilitating close proximity to P. carinii organisms. We cannot formally exclude, however, that material shed by large numbers of P. carinii may interfere with macrophage effector functions. An additional immune parameter influencing parasite clearance by macrophages is the presence of opsonizing Abs. Reductions in parasite numbers have been noted after in vivo application of (i) a MAb against specific epitopes of P. carinii (15, 16) and (ii) hyperimmune serum, provided that high doses were administered daily (44). Furthermore, in contrast to naive immunocompetent mice, T-cell depletion of immunized animals did not affect resistance to parasite challenge (21). Protection was considered to be mediated by Abs. Lack of P. carinii-specific Abs, therefore, could have profound effects on resistance to PCP. Specific Ab production in Aβ^−/− and TCRβ^−/− mutants (RAG-1^−/− mutants do not have mature B cells) is currently being investigated. In light of a previous report (5), the apparent requirement of CD4⁺ T cells for the largely macrophage derived cytokines IL-1 and TNF-α to mediate clearance of established PCP is of interest. These cytokines may either act on T cells directly, interact with T-cell-derived products, or be involved in some stages of cell-cell interactions between CD4⁺ T cells and macrophages which probably occur via the CD40-CD154 system to ultimately induce convalescence of the host.