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The Journal of Immunology, 2004, 172: 2407-2415.
Copyright © 2004 by The American Association of Immunologists

Macrophages, CD4+ or CD8+ Cells Are Each Sufficient for Protection against Chlamydia pneumoniae Infection through their Ability to Secrete IFN-{gamma}1

Antonio Gigliotti Rothfuchs2,*, Maria Regina Kreuger2,{dagger}, Hans Wigzell* and Martin E. Rottenberg3,*

* Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden; and {dagger} Centro de Ciências da Saúde, Universidade do Vale do Itajai, Itajai, Brasil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
By using a T, B, or NK cell-deficient mouse strain (recombinase-activating gene (RAG)-1-/-/common cytokine receptor {gamma}-chain ({gamma}CR)), and T and B cell and IFN-{gamma}-deficient (RAG-1-/-/IFN-{gamma}-/-) mice, we have studied the generation of immunity against infection by Chlamydia pneumoniae. We found that IFN-{gamma} secreted by innate-cell populations protect against C. pneumoniae infection. However, NK cells were not needed for such IFN-{gamma}-dependent innate immune protection. Inoculation of wild type, but not IFN-{gamma}-/- bone marrow-derived macrophages protected RAG-1-/-/IFN-{gamma}-/- mice against C. pneumoniae infection. In line, pulmonary macrophages from RAG-1-/- C. pneumoniae-infected mice expressed IFN-{gamma} mRNA. Reconstitution of RAG-1-/-/{gamma}cR-/- or RAG-1-/-/IFN-{gamma}-/- mice with CD4+ or CD8+ cells by i.v. transfer of FACS sorted wild type spleen cells (SC) increased resistance to C. pneumoniae infection. On the contrary, no protection was observed upon transfer of IFN-{gamma}-/- CD4+ or IFN-{gamma}-/- CD8+ SC. T cell-dependent protection against C. pneumoniae was weaker when IFN-{gamma}R-/- CD4+ or IFN-{gamma}R-/- CD8+ SC were inoculated into RAG-1-/-/IFN-{gamma}-/- mice. Thus both nonlymphoid and T cell-derived IFN-{gamma} can play a central and complementary role in protection against C. pneumoniae. IFN-{gamma} secreted by nonlymphoid cells was not required for T cell-mediated protection against C. pneumoniae; however, IFN-{gamma} regulated T cell protective functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infection with Chlamydia pneumoniae, a Gram-negative obligate intracellular bacterium, will occur at least once in >50% of the human population world wide, causing, e.g., pneumonia, sinusitis, and bronchitis. In addition, the spectrum of C. pneumoniae infection has been suggested to extend to atherosclerosis and its clinical manifestations (1, 2).

A mouse model of infection has been used to study the immunological mechanisms of immunity against C. pneumoniae and other chlamydial species. In this model of infection, IFN-{gamma} appears to be essential in protection as demonstrated by the enhanced bacterial levels in IFN-{gamma}-/-, IFN-{gamma}R-/- or mice treated with anti-IFN-{gamma} Abs compared with controls (3, 4, 5, 6, 7). NK cells have been shown to participate in the resistance against a number of bacterial and protozoal infections through their ability to secrete IFN-{gamma}. However, their involvement in the control of chlamydial infection is not clear. NK cells have been shown to participate in control of Chlamydia trachomatis mouse pneumonitis strain (8). In contrast, the resistance of SCID mice to C. trachomatis or of recombination-activating gene-1-/- (RAG-1)4 mice to C. pneumoniae was not altered after eliminating NK cells using neutralizing Abs (9, 10).

Many reports indicate that macrophages can produce IFN-{gamma} (11, 12, 13, 14, 15, 16, 17, 18). IFN-{gamma} is produced by macrophages in response to stimulation with bacteria, IL-12, IL-12 and IL-18, or with IFN-{gamma} itself. Mouse bone marrow-derived macrophages (BMM) express IFN-{gamma} mRNA and protein after infection with C. pneumoniae (19). Moreover, such secretion of IFN-{gamma} controls chlamydial growth in infected macrophages in an autocrine/paracrine manner (19). However, whether IFN-{gamma} secretion by macrophages plays a role in the outcome of infection with C. pneumoniae or other pathogens in vivo is not known.

It is possible that innate IFN-{gamma} will prime Th1 development through its ability to confer a competent IL-12 response to the T cells (20, 21, 22, 23), and by negatively regulating the growth of Th2 cells (22). Thus, T cells conditioned in vivo in the absence of a source of exogenous IFN-{gamma} might display defective Th1 development.

A role for CD4+ and CD8+ T cells in resistance to primary chlamydial infection is likely since mice depleted of CD4+ and CD8+ T cells by Ab administration, and CD4-/-, CD8-/-, {beta}2-microglobulin-/-, or {beta}2-microglobulin-/-/TAP1-/- mice all exhibit exacerbated infection (5, 6, 24, 25, 26, 27). Both CD8+ and CD4+ T cells produce IFN-{gamma} in response to chlamydial infection, and are probably complementary in warranting protective levels of this cytokine (reviewed in Ref. 28). However, a critical assessment on whether IFN-{gamma} secreted by T cells plays any role in protection during a primary chlamydial infection is lacking. In fact, although IFN-{gamma} plays a critical role in defense against Listeria monocytogenes, IFN-{gamma} secretion by T cells does not seem to be necessary for protection (29).

In this study, we show here by using T, B, and NK cell-deficient and T and B cell, and IFN-{gamma}-deficient mouse strains that macrophages, CD4+ and CD8+ cells are each sufficient to play a role in in vivo protection against C. pneumoniae through their ability to secrete IFN-{gamma}.


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

Mutant mouse strains without IFN-{gamma}R (30), IFN-{gamma} (31), RAG-1 (32), and cytokine receptor {gamma}-chain ({gamma}cR) (33) were generated by homologous recombination in embryonic stem cells. RAG-1-/-/{gamma}cR-/- mice have been previously described (34). All mice used underwent 8–9 backcrosses with C57BL/6 mice, which were used as controls. Mice were bred and kept under specific pathogen-free conditions, and were maintained in isolation under negative pressure during experiments. They were used between 6 and 10 wk of age. All animal studies included were approved by the Stockholm’s Region Animal Welfare Review Board (Stockholm, Sweden).

Generation of RAG-1-/-/IFN-{gamma}-/- mice

RAG-1-/- were crossed with animals homozygous for IFN-{gamma} deficiency. The progeny was intercrossed and F2 mice were screened for homozygosity of the disrupted IFN-{gamma} gene by PCR analysis of tail DNA lysates. The presence of the introduced neomycin construct and the absence of the wild type (WT) gene in both alleles could be detected by amplification with the following primers: sense IFN-{gamma}: 5'-GA AGT AAG TGG AAG GGC CCA G-3'; anti-sense IFN-{gamma}: 5'-AGG GAA ACT GGG AGA GGA GAA A-3'; neomycin: 5'-CCT GCG TGC AAT CCA TCT TG-3'.

RAG-1-/- homozygosity was determined by absence of IgG3 in sera and of peripheral blood CD4+ cells in F2 mice.

Infection and infectivity assay

Mycoplasma-free C. pneumoniae isolate Kajaani 6 was propagated in HL cells. Infected cells were sonicated, cell remnants were removed by centrifugation and the bacteria were stored in small aliquots in sucrose-phosphate-glutamate solution at -70°C until used. The infectivity as measured by inclusion forming units (IFU) of bacterial preparation was determined in HL cell culture.

For infection, mice were mildly sedated with isofluorane and inoculated intranasally with 106 IFU diluted in 40 µl of PBS. Animals were sacrificed at different times after infection and right lungs, hearts, or spleens removed, minced, and mechanically homogenized in 2 ml of sucrose-phosphate-glutamate solution. Homogenates were centrifuged for 6 min, 500 x g to remove coarse tissue debris. Lysates were then diluted 10- and 100-fold in DMEM containing 5% FCS and 2.5 µg/ml streptomycin. The infectious titter was assayed by culturing 500 µl of duplicate dilutions of the lysates on confluent HL cells grown on round 13 mm2 coverslides in a shell vial. Inoculated cells were centrifuged for 1 h at 500 x g. Thereafter, supernatants were removed and DMEM containing 0.5 µg/ml cycloheximide and 2.5 µg/ml streptomycin was added. Cells were incubated at 35°C for 72 h, fixed with methanol, and stained with a FITC-conjugated Chlamydia genus-specific mAb (BioRad, Hercules, CA). Inclusion bodies were counted by fluorescence microscopy. The infectivity was expressed as IFU per organ.

Competitive RT-PCR assay

The accumulation of IFN-{gamma}, inducible NO synthase (iNOS), and {beta}-actin mRNA in freshly extracted left lungs from infected mice was measured by competitive PCR assays as previously described (35). The primer sequences for the amplification of the cDNA, are indicated in Table I. Competitor fragments with a different length but using the same primers as the target DNA (Table I) were constructed by using composite primers as described (36). A 580 bp BamH1/EcoRI fragment of v-erbB (Clontech, Palo Alto, CA) was used as template for construction of all competitors using composite primers recognizing sequences both from the target gene and from this fragment. Competitors were amplified by PCR, purified (Qiagen, Studio City, CA), and quantified in a spectrophotometer. Noncompetitive PCR was also performed for IL-12 p40.


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  		Table I. PCR primers used in this study and length of the amplified gene and competitor fragments. Only a noncompetitive PCR was used for IL-12p40

 
Three-fold serial dilutions of the competitor were amplified in the presence of a constant amount of cDNA. Reactions were conducted for 28–45 cycles in a thermal cycler (PerkinElmer Cetus, Shelton, CT) using an annealing step at 60°C (except 65°C for IL-12).

Pathology

Left lungs from C. pneumoniae-infected mice were fixed in 4% formalin and processed for conventional histopathological examination after paraffin embedding. Sagittal sections were cut at 4 µm, deparaffinized and stained with H&E. A single-blind microscopic evaluation of two sets of serial sections from each organ was performed on precoded slides.

Positive selection of CD4+ and CD8+ cells

CD4+- and CD8+-cell suspensions were prepared from spleens from uninfected WT, IFN-{gamma}-/-, or IFN-{gamma}R-/- mice. Briefly, spleens were removed, minced, and mechanically homogenized. The spleen cell (SC) suspensions obtained were washed once with RPMI 1640 medium containing 5% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (RPMI-5% FCS). RBC were removed by hypotonic shock using 0.8% NH4Cl, and the remaining cells were washed twice in cold RPMI-5% FCS. Cells were filtered through a nylon wool column, and nylon wool nonadherent SC were resuspended in ice-cold RPMI (107 cells/ml, ~4 x 108 cells per experiment) and incubated with either 0.5 µg/ml FITC-labeled rat anti-mouse CD4 (GK1.5), or PE-labeled rat anti-mouse CD8{alpha} (53–6.7) for 45 min on ice. Both mAbs were purchased from BD PharMingen (San Diego, CA). Cells were then washed once and resuspended at 5 x 106 cells/ml in PBS and CD4+ and CD8+ cells were sorted by using a FACSVantage sorter (BD Biosciences, Mountain View, CA). Positive cells were washed and resuspended in RPMI containing 1% FCS at 2.5 x 107 cells/ml. To control the phenotype of the positive selected cells, 5 x 105 cells were stained with either fluorochrome-labeled anti-CD3, anti-{alpha}{beta} TCR, anti-CD19, anti-CD8, or anti-CD4, before FACS analysis using a FACScan apparatus (data not shown). Contaminations of B cells or other subpopulation of T cells were not detected.

RAG-1-/-/{gamma}cR-/- or RAG-1-/-/IFN-{gamma} -/- mice were inoculated in the tail vein with 5 x 106 cells. Twenty-four days after cell inoculation, mice were infected with C. pneumoniae as described above. To confirm the efficiency and specificity of lymphoid reconstitution, splenocytes from cell-transferred animals were stained with anti-CD4, anti-CD8, anti-{alpha}{beta}TCR, anti-CD19, or anti-CD3, and analyzed by FACS.

Isolation of mononuclear cells from bronchoalveolar lavage fluids

Cells were obtained after bronchoalveolar lavage with PBS containing 0.1% EDTA. Mononuclear cells were isolated using a one-step gradient separation method with Nycoprep (Nycomed Pharma, Oslo, Norway) according to the instructions of the manufacturer. Cells were washed and diluted at 106 cells/ml in DMEM-5% FCS, and incubated for 72 h at 37°C. Supernatants were then collected and IFN-{gamma} levels were measured by ELISA performed as indicated by the manufacturer (R&D Systems, Oxon, U.K.).

Isolation of F4/80+, CD14+ pulmonary mononuclear cells

Lungs from control and C. pneumoniae-infected RAG-1-/- mice were obtained. The pulmonary vasculature was perfused, lungs were aseptically removed, pooled, and subjected to collagenase treatment for 1 h at 37°C in RPMI 1640 containing 2 mM glutamine, 5% FCS and 25 µg/ml streptomycin. Digested lung pieces were then mechanically homogenized and filtered through a 100 µm cell-strainer. Cells were washed three times and then counted by trypan blue exclusion. Single-cell suspensions were isolated using a one-step gradient separation method with Nycoprep (Nycomed Pharma). Cells were then subjected to total RNA extraction with RNAzol B solution according to the instructions of the manufacturer (Tel-Test, Friendswood, TX). Alternatively, 106 lung cells were blocked with 10% normal goat serum, and incubated with either 1/20-diluted FITC-conjugated rat anti-mouse F4/80 (Caltag Laboratories, Burlingame, CA) or 1/10-diluted PE-conjugated rat anti-mouse CD14 (BD PharMingen) in PBS for 30 min on ice. Cells were washed twice and sorted by FACS. Cells were gated for F4/80+ or CD14+ populations in relation to isotype control-stained cells. Approximately 1 x 106, F4/80+, and CD14+ cells were collected, respectively, and total RNA was extracted using RNAzol B.

Generation of bone marrow derived macrophages

BMMs were obtained as previously described (19). Briefly, mice were euthanized, and the femur and tibia of the hind legs were dissected. Bone marrow cavities were flushed with 5 ml cold, sterile PBS. The bone marrow cells were washed and resuspended in DMEM containing glucose and supplemented with 10% FCS, 30% L929 cell-conditioned medium (as a source of M-CSF), 100 µg/ml streptomycin, and 100 U/ml penicillin. Bone marrow cells (1.2 x 107 cells; 2 x 106 cells/ml) were plated in 6-well plates and incubated for 7 days at 37°C, 5% CO2. Before use, BMM cultures were washed vigorously to remove nonadherent cells. Cells were harvested, pooled, and counted by trypan blue exclusion.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of innate immune cell-secreted IFN-{gamma} in protection against C. pneumoniae

To investigate the protective role of IFN-{gamma} in the innate resistance to C. pneumoniae RAG-1-/-/IFN-{gamma}-/- mice were generated. RAG-1-/-/IFN-{gamma}-/- mice showed dramatically increased bacterial load in lungs during infection with C. pneumoniae as compared with RAG-1-/- controls (Fig. 1A), in agreement with observations of increased susceptibility to C. pneumoniae of RAG-1-/-/IFN-{gamma}R-/- mice (10). RAG-1-/-/IFN-{gamma}-/- mice also showed higher C. pneumoniae levels in spleen, heart (Fig. 1B), and liver (data not shown) than RAG-1-/- controls, indicating that innate IFN-{gamma} controls dissemination of C. pneumoniae from the lung. All RAG-1-/-/IFN-{gamma}-/- mice died within a month after infection (Fig. 1A), whereas all RAG-1-/- controls survived.



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  		FIGURE 1. Role of innate immune cell-secreted IFN-{gamma} in protection against C. pneumoniae. A and B, Course of C. pneumoniae infection in RAG-1-/- (B) and RAG-1-/-/IFN-{gamma}-/- (B) (10–13 mice per time point and group). Mice were sacrificed at the indicated time points after intranasal infection with 106 C. pneumoniae. The mean of the log10-transformed IFU titers per lung is depicted. Bars indicate SEM. All RAG-1-/-/IFN-{gamma}-/- died before 28 days after infection. *, Differences vs RAG-1-/- mice are significant (p < 0.05, Student t test). B, Mean log10 IFU of C. pneumoniae in hearts and spleens of RAG-1-/- and RAG-1-/-/IFN-{gamma}-/- (10–12 mice per group) measured 15 days after intranasal infection with C. pneumoniae are depicted. Bars indicate the SEM. Differences vs infected RAG-1-/- mice are significant (p < 0.05, Student t test). C, Course of C. pneumoniae infection in RAG-1-/- and RAG-1-/-/{gamma}cR-/- mice (10–12 mice per time point and group). Mice were sacrificed at the indicated time points after intranasal infection with 106 C. pneumoniae. The mean of the log10-transformed IFU titers per lung is depicted. Bars indicate SEM. Differences between C. pneumoniae levels are not significant (p > 0.05, Student t test). D, Total RNA was obtained from lungs of C. pneumoniae-infected RAG-1-/- and RAG-1-/-/{gamma}cR-/- mice and transcribed into cDNA. Equal aliquots of cDNA from four individual mice per group were amplified with IFN-{gamma} or {beta}-actin primers in the presence of 3-fold serial dilutions of the respective competitors. The mean moles of IFN-{gamma} per mole of {beta}-actin mRNA are depicted. Differences between groups are not significant. No amplification was detected in samples from uninfected RAG-1-/-/{gamma}cR-/- mice. E, IFN-{gamma} in supernatants of cultures of bronchoalveolar lavage mononuclear cells from mice at the indicated days after infection with C. pneumoniae, measured by ELISA. Results are mean IFN-{gamma} levels of four individual mice. Differences between RAG-1-/- and RAG-1-/-/{gamma}cR-/- groups are not significant. Few mononuclear cells were present in the BAL of uninfected controls, which hampered the test on these cells.

 
Because NK cells are a main innate-immune source of IFN-{gamma}, we compared the susceptibility of T, B, and NK cell deficient RAG-1-/-/{gamma}cR-/- with that of T and B cell deficient RAG-1-/- mice (37) to analyze the role of NK cells in protection against C. pneumoniae. Lungs from RAG-1-/-/{gamma}cR-/- and RAG-1-/- mice showed similar levels of C. pneumoniae and of IFN-{gamma} mRNA and protein during infection (Fig. 1, C–E). Thus, NK cells are not needed for innate resistance against C. pneumoniae.

Role of macrophage-derived IFN-{gamma} in protection against C. pneumoniae

Macrophages may play a protective role during the in vivo infection with C. pneumoniae through their ability to release IFN-{gamma}. Increased levels of IFN-{gamma} mRNA could indeed be detected in CD14+ and F4/80+ macrophages isolated by FACS sorting from unicellular suspensions generated by collagenase digestion of infected lungs (Fig. 2A). These cells also showed increased levels of IL-12 p40 mRNA. Next, WT or IFN-{gamma}-/- BMM were inoculated i.v. into RAG-1-/-/IFN-{gamma}-/- recipient mice, which were subsequently infected with C. pneumoniae. Transfer of WT but not of IFN-{gamma}-/- BMM strikingly reduced the numbers of C. pneumoniae in lungs and hearts (Fig. 2, B and C). Altogether, our data suggest that macrophage-derived IFN-{gamma} secretion plays a protective role against infection with C. pneumoniae.



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  		FIGURE 2. Role of macrophage-derived IFN-{gamma} in protection against C. pneumoniae. A, Total RNA was isolated from lungs, total mononuclear pulmonary cells, or FACS sorted F4/80+ and CD14+ mononuclear, pulmonary cells from individual RAG-1-/- mice at 0 or 20 days after infection with C. pneumoniae, and transcribed into cDNA. Aliquots of cDNA were amplified with IFN-{gamma}, IL-12 p40 or {beta}-actin primers. Three individuals were analyzed for each condition rendering similar results. B and C, 2 x 107 WT or IFN-{gamma}-/- BMM (10–12 mice per group) were inoculated i.v. into RAG-1-/-/IFN-{gamma}-/- mice. A control group was left untreated One day after transfer, mice were infected with C. pneumoniae. Mice were sacrificed 15 days after infection and the number of IFU in individual lungs (B) and hearts (C) was determined. The mean of the log10-transformed IFU titers per lung or heart is depicted. Bars indicate SEM. *, Differences vs untreated group are significant (p < 0.05, Student t test).

 
T cell-secreted IFN-{gamma} plays a protective role during infection with C. pneumoniae

The role of CD4+ or CD8+ cells in protection against C. pneumoniae was next analyzed. For this purpose, 5 x 106 CD4+ or CD8+ SC, obtained after positive isolation by FACS sorting, were inoculated i.v. into RAG-1-/-/{gamma}cR-/- naive mice. Mice were infected intranasally with C. pneumoniae 24 days after cell transfer, and sacrificed 24 days after infection. At this time point, spleens from mice transferred with CD8+ or CD4+ SC showed presence of these populations respectively albeit in lower numbers than C57BL/6 SC controls (data not shown). Mice transferred with WT and IFN-{gamma}-/- CD8+ SC or CD4 SC showed similar numbers of {alpha}{beta}-TCR+, CD8+, or CD4+ cells, respectively. Presence of CD19+ B cells, or the cells was not detected in mice transferred with CD8+ SC. No CD8+ or CD19+ cells were detected after inoculation of CD4+ SC (data not shown).

Inoculation of either WT CD4+ or WT CD8+ SC caused a 50- to 100-fold lower C. pneumoniae level in lungs when measured 24 days after bacterial inoculation, as compared with nontransferred controls (Fig. 3, A and B). On the contrary, no reduction in bacterial levels was observed when IFN-{gamma}-/- CD4+ or IFN-{gamma}-/- CD8+ SC were inoculated. This indicates that IFN-{gamma} is required for T cell-mediated protection, and either subset is sufficient to grant protection to the immunodeficient recipient mice (Fig. 3, A and B). Similar results were observed when bacterial inoculation was performed 7 days after SC transfer, or by using CD4+ or CD8+ SC positively selected using magnetic beads coated with the respective Abs (data not shown). Diminished bacterial numbers upon transfer with WT CD4+ or WT CD8+ SC were not observed when measured 7 and 14 days after infection (Fig. 3, C and D).



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  		FIGURE 3. T cell-secreted IFN-{gamma} plays a protective role during infection with C. pneumoniae. A and B, A total of 5 x 106 WT or IFN-{gamma}-/- CD4+ (A) WT or IFN-{gamma}-/- CD8+ (B) SC were inoculated i.v. into RAG-1-/-/{gamma}cR-/- mice (10–12 individual mice per group). A control group was left untreated. Twenty-four days after inoculation, mice were infected intranasally with 106 C. pneumoniae. Mice were sacrificed 24 days after infection and the number of IFU in individual lungs was determined. The mean of the log10-transformed IFU titers per lung is depicted. Bars indicate SEM. *, Differences vs untreated group are significant (p < 0.05 Student t test). C and D, A total of 5 x 106 WT CD4+ (C) or WT CD8+ (D) SC were inoculated i.v. into RAG-1-/-/{gamma}cR-/- mice. A control group was left untreated. Twenty-four days after inoculation, mice were infected intranasally with 106 C. pneumoniae. The number of C. pneumoniae in individual lungs was measured at the indicated time points after infection. The mean of the log10-transformed IFU titers per lung is depicted. Bars indicate SEM.

 
Perivascular and peribronchiolar mononuclear cell infiltrates were observed in lungs from CD4+ or CD8+ SC-reconstituted RAG-1-/-/{gamma}cR-/- mice but not in nonreconstituted controls. Nonreconstituted RAG-1-/-/{gamma}cR-/- showed large areas of lung consolidation, while groups of mice inoculated with WT CD4+ or WT CD8+ SC displayed a normal lung architecture, similar to that of C57BL/6 mice at the same time point after infection (Fig. 4). Mice inoculated with IFN-{gamma}-/- CD4+ or IFN-{gamma}-/- CD8+ SC showed mononuclear perivascular and peribronchiolar infiltrates but also large areas of pulmonary consolidation (Fig. 4). Altogether, our data suggest a vital and nonredundant role of IFN-{gamma} secreted from nonlymphoid and CD4+ or CD8+ cells in control of pulmonary infection with C. pneumoniae.



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  		FIGURE 4. Lung histopathology of WT or IFN-{gamma}-/- SC- reconstituted RAG-1-/-/{gamma}cR-/- mice. H&E staining of paraffin lung sections from RAG-1-/-/{gamma}cR-/- mice inoculated with WT or IFN-{gamma}-/- CD4+, or WT or IFN-{gamma}-/- CD8+ SC and infected intranasally with C. pneumoniae 24 days before sacrifice as described in legend to Fig. 4. Magnification, x50. Note presence of perivascular and peribronchiolar prominant infiltration of inflammatory monouclear cells in lungs from C. pneumoniae infected CD4+ or CD8+ SC-reconstituted RAG-1-/-/{gamma}cR-/- mice (E–I) but not in nonreconstituted, infected (D) or uninfected (A) controls. Such cellular infiltrates are also observed in infected IFN-{gamma}R-/- (B) and C57BL/6 (C) mice. Nonreconstituted RAG-1-/-/{gamma}cR-/- mice showed large areas of lung consolidation (D), while mice inoculated with WT CD4+ (E) or WT CD8+ (F) SC displayed a normal lung architecture, similar to that of C57BL/6 mice (C). RAG-1-/-/{gamma}cR-/- mice inoculated with IFN-{gamma}-/- CD4+ (G–H) or IFN-{gamma}-/- CD8+ (I) SC also displayed large areas of pulmonary consolidation.

 
Lungs from RAG-1-/-/{gamma}cR-/- mice reconstituted with WT CD4+ or WT CD8+ SC showed higher levels of IFN-{gamma} and iNOS mRNA than those inoculated with IFN-{gamma}-/- CD4+ or IFN-{gamma}-/- CD8+ SC (Fig. 5, A and B). Of importance, iNOS mRNA is positively regulated by IFN-{gamma} and accounts at least in part for IFN-{gamma}-mediated protection (10).



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  		FIGURE 5. Cytokine mRNA accumulation in lungs from RAG-1-/-/{gamma}CR-/- inoculated with WT or IFN-{gamma}-/- CD4+, or with WT or IFN-{gamma}-/- CD8+ SC during infection with C. pneumoniae. A and B, A total of 5 x 106 WT or IFN-{gamma}-/- CD4+, or WT or IFN-{gamma}-/- CD8+ SC were inoculated i.v. into RAG-1-/-/{gamma}cR-/- mice (four individual mice per group). Twenty-four days after inoculation, mice were infected intranasally with C. pneumoniae. Total RNA was obtained from lungs from mice after 24 days of infection and transcribed into cDNA. Equal aliquots of cDNA from individual mice per group were then amplified with IFN-{gamma} (A), iNOS (B), or {beta}-actin primers in the presence of three-fold serial dilutions of the respective competitors. The moles of cytokine per mole of {beta}-actin mRNA are depicted. *, Differences in IFN-{gamma} and iNOS mRNA levels in lungs of mice inoculated with WT CD4+ or WT CD8+ SC vs those inoculated with IFN-{gamma}-/- CD4+ or IFN-{gamma}-/- CD8+ SC are significant (p < 0.05 Student t test).

 
T cell-secreted IFN-{gamma} is sufficient to confer protection against C. pneumoniae

Whether IFN-{gamma} secreted by CD4+ or CD8+ cells is by itself sufficient to confer protection against C. pneumoniae was then tested. For this purpose CD4+ or CD8+ SC were inoculated i.v. into RAG-1-/-/IFN-{gamma}-/- mice, which were infected with C. pneumoniae 24 days after cell transfer. WT CD4+ or WT CD8+ cells are here the only source of IFN-{gamma}. Inoculation of either WT CD4+ or WT CD8+ SC conferred protection to RAG-1-/-/IFN-{gamma}-/- mice against C. pneumoniae as measured as a 100- to 1000-fold lower bacterial levels in lungs 21 days after infection (Fig. 6, A and B). On the contrary, no protection was observed in mice inoculated with IFN-{gamma}-/- CD4+ or IFN-{gamma}-/- CD8+ SC (Fig. 6, a and b).



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  		FIGURE 6. T cell-secreted IFN-{gamma} is sufficient to confer protection against C. pneumoniae. A and B, A total of 5 x 106 WT or IFN-{gamma}-/- CD4+ (A) or WT or IFN-{gamma}-/- CD8+ (B) SC were inoculated i.v. into RAG-1-/-/IFN-{gamma}-/- mice (10–12 individual mice per group). A control group was left untreated. Twenty-four days after cell inoculation, mice were infected intranasally with C. pneumoniae. Mice were sacrificed 21 days after infection and the number of IFU in individual lungs was determined. The mean of the log10-transformed IFU titers per lung is depicted. Bars indicate SEM. *, Differences vs untreated group are significant (p < 0.05 Student’s t test).

 
Finally, whether IFN-{gamma} regulation of T cell function played a role in protection against C. pneumoniae was studied. WT or IFN-{gamma}R-/- CD4+, or WT or IFN-{gamma}R-/- CD8+ SC were inoculated into RAG-1-/-/IFN-{gamma}-/- mice, which were infected 24 days after. In such systems, inoculated IFN-{gamma}R-/- T cells can secrete but not respond to IFN-{gamma}. The opposite is true for the cells of the recipient mice. WT T cells can of course produce and respond to IFN-{gamma}. Mice transferred with IFN-{gamma}R-/- CD4+ or IFN-{gamma}R-/- CD8+ SC showed lower numbers of C. pneumoniae in lungs as compared with nontransferred, infected controls, but showed higher numbers compared with those inoculated with WT CD4+ or WT CD8+ SC (Fig. 7, A and B). We asked then if IFN-{gamma} is implicated in an autocrine loop, regulating secretion of IFN-{gamma} by T cells. Lungs from RAG-1-/-/IFN-{gamma}-/- mice reconstituted with WT or IFN-{gamma}R-/- CD4+ or WT or IFN-{gamma}R-/- CD8+ SC contained similar IFN-{gamma} mRNA levels than those inoculated with WT SC. Thus, IFN-{gamma} apparently does not modulate its own secretion in CD4+ or CD8+ cells (Fig. 7, C and D). In summary, IFN-{gamma} probably protects against infection with C. pneumoniae by affecting the physiology of nonlymphoid effectors, a protection which is further expanded if the T cells themselves also can respond to their own IFN-{gamma}.



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  		FIGURE 7. Role of IFN-{gamma} regulation of T cell function in protection against C. pneumoniae. A and B, A total of 5 x 106 WT or IFN-{gamma}R-/- CD4+ (A) or WT or IFN-{gamma}R-/- CD8+ (B) SC were inoculated i.v. into RAG-1-/-/IFN-{gamma}-/- mice (10–12 individual mice per group). A control group was left untreated. Twenty-four days after inoculation, mice were infected with C. pneumoniae. Mice were sacrificed 21 days after infection and the number of IFU in individual lungs was determined. The mean of the log10-transformed IFU titers per lung is depicted. Bars indicate SEM. *, Differences vs untreated and infected group are significant (p < 0.05 Student t test). {Delta}, Differences between the groups treated with WT SC vs IFN-{gamma}R-/- SC-transferred groups are significant (p < 0.05, Student t test). C and D, IFN-{gamma} mRNA accumulation in lungs from RAG-1-/-/{gamma}cR -/- inoculated with WT or IFN-{gamma}R-/- CD4+, or with WT or IFN-{gamma}R-/- CD8+ SC during infection with C. pneumoniae. A total of 5 x 106 WT or IFN-{gamma}R-/- CD4+, or WT or IFN-{gamma}R-/- CD8+ SC were inoculated i.v. into RAG-1-/-/IFN-{gamma}-/- mice (4–5 individual mice per group). Twenty-four days after cell inoculation, mice were infected intranasally with C. pneumoniae. Nonreconstituted, control RAG-1-/-/IFN-{gamma}-/- mice were also infected. Total RNA was obtained from lungs from mice after 21 days of infection and transcribed into cDNA. C, Equal aliquots of cDNA from individual mice per group were then amplified with IFN-{gamma} or {beta}-actin primers in the presence (D) or absence (C) of 3-fold serial dilutions of the respective competitors. The moles of cytokine per mole of {beta}-actin mRNA are depicted. Bars indicate the SEM. Differences between groups are not significant. Note that the lower bands on the negative photograph (C) are primer-dimers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our observation that RAG-1-/-/{gamma}cR-/- mice can control primary C. pneumoniae infection as efficiently as RAG-1-/- mice contradicts the view that NK cells always play a major role in the innate immune defense by providing a rapid source of IFN-{gamma}. Together with the increased susceptibility of RAG-1-/-/IFN-{gamma}R-/- (9) or RAG-1-/-/

IFN-{gamma}-/- as compared with RAG-1-/- controls, this implies that an immune-cell population other than NK cells account for the secretion of protective levels of IFN-{gamma} during the innate immune response to C. pneumoniae.

We showed in this study that IFN-{gamma} transcripts were present in pulmonary macrophages from C. pneumoniae-infected mice. IFN-{gamma} secretion by macrophages has also been shown during in vivo infection with Mycobacterium tuberculosis or Salmonella typhimurium (12, 38). Moreover, the IFN-{gamma} response during primary Salmonella infection was dominated by the macrophage population (38). However, a causal role of macrophage-derived IFN-{gamma} in protection against in vivo intracellular bacterial infections has not previously been determined. Murine BMM express the IFN-{gamma} gene at the mRNA and protein level after in vitro infection with C. pneumoniae and this controls chlamydial growth (19). We could now show that inoculation of WT macrophages into RAG-1-/-/IFN-{gamma}-/- mice reduce C. pneumoniae load in lungs and dissemination into the heart. These results strongly suggest that macrophage populations protect against chlamydial infection in the in vivo infection via IFN-{gamma} release, and extend the role of this cell population in the control of intracellular infections. In this experimental model, macrophage-derived IFN-{gamma} may serve to directly enhance the bactericidal mechanisms of infected cells. Macrophage-derived IFN-{gamma} may also facilitate Ag presentation, and/or influence the Th polarization of the immune response in the immunocompetent host. The role of innate-cell populations other than macrophages in IFN-{gamma}-mediated protection remains to be investigated. Of interest, a role for macrophage-derived IFN-{gamma} in the development of glomerulonephritis was recently suggested (39).

We also found that selective repopulation of mice with CD4+ or CD8+ T cells will enhance immune protection against infection with C. pneumoniae. These data indicate that CD4+ cells are not needed for CD8+-mediated protection, and that CD4+ T cells can mediate anti-chlamydial protection in a CD8- and Ab-independent manner. Arguments that contaminating NK cells in the transplant are responsible for the anti-C. pneumoniae immunity seem unlikely since NK cells were not found in CD4+ or CD8+ cell-reconstituted RAG-1-/-/{gamma}cR-/- mice at the time of sacrifice (data not shown). Thus, protective T cell immunity against C. pneumoniae can be generated in the absence of NK cells. In agreement, mice lacking NK cells have been shown to develop Th1 immunity and control the infection of another intracellular pathogen, Leishmania major (40).

IFN-{gamma} derived from T cells and nonlymphoid cells can both play important and complementary roles in the control of chlamydial infection, as revealed by the IFN-{gamma}-dependent protection afforded by WT CD4+ or WT CD8+ cells inoculated into RAG-1-/-/{gamma}c R-/- mice, which possess IFN-{gamma}-secreting nonlymphoid cells. Moreover, whereas the protective effect of macrophage- or nonlymphoid cell-derived IFN-{gamma} was observed 7–14 days after infection, IFN-{gamma}-mediated protection after T cell transfer was only observed 3 wk after infection. In line with this, differences in C. pneumoniae levels between RAG-1-/- and WT mice were only observed 3 wk after infection (10). Thus, differences in the kinetics of production of innate vs T cell-derived IFN-{gamma} probably account for the additive protective effect.

Because naive T cells do not produce IFN-{gamma} until some time after activation, it is likely that IFN-{gamma} derived from innate immune cells primes Th1 development through its ability to mediate IL-12R{beta}2 (22) and IL-18R{alpha}-chain (41) expression and IL-12 secretion (20, 21). Such studies lead to the prediction that T cells conditioned in vivo in the absence of a source of IFN-{gamma} might display defective Th1 development. However, IFN-{gamma} secreted by nonlymphoid cell populations was not required for IFN-{gamma}-dependent control of C. pneumoniae. In agreement, IFN-{gamma} derived from CD4+ T cells was sufficient to mediate Th1 development during infection with L. major (42). In contrast, although IFN-{gamma}-dependent protection occurred when T cells were unable to respond to IFN-{gamma}, the weaker protection conferred by IFN-{gamma}R-/- CD4+ or IFN-{gamma}R-/- CD8+ SC suggest that IFN-{gamma} not only activates protective mechanisms of nonlymphoid cells but also modulates the protective response of T cells themselves. As mentioned above, regulation of IL-12 responses by T cells could account for this effect. The transcription factor T-bet has been reported to play an important role in CD4+ Th1 differentiation, by promoting both IL-12R{beta}2 expression and IFN-{gamma} production (43, 44). IFN-{gamma} gene regulation might also involve an autocrine loop, whereby IFN-{gamma} induces the transcription factor T-bet that in turn promotes IFN-{gamma} production (45). The later seems not to be responsible for diminished resistance conferred by IFN-{gamma}R-/- T cells since, when inoculated into RAG-1-/-/IFN-{gamma}-/- mice, IFN-{gamma}R-/- and WT T cells contained similar levels of IFN-{gamma} mRNA.

In summary, we have demonstrated that in vivo secretion of IFN-{gamma} by macrophages, CD4+ or CD8+ cells is sufficient to confer various levels of control against C. pneumoniae infection. Nonlymphoid- and T cell-derived IFN-{gamma}-mediated protection appear to be nonredundant. IFN-{gamma} secretion by T cells did not depend on the IFN-{gamma} secreted by nonlymphoid, innate cells. In contrast, IFN-{gamma} can regulate the protective activity of CD4+ and CD8+ T cells.


   Acknowledgments
 
We thank Berit Olsson for her excellent technical assistance.


   Footnotes
 
1 This work was supported by the European Community QLK2-CT-2002-00846 Grant, the Karolinska Institute, The Swedish Health Insurance Company AFA, The Swedish Cancer Society, and The Swedish Research Council, Sweden. Back

2 A.G.R. and M.R.K. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Martin E. Rottenberg, Microbiology and Tumorbiology Center, Karolinska Institute, S17177 Stockholm, Sweden. E-mail address: Martin.Rottenberg{at}mtc.ki.se Back

4 Abbreviations used in this paper: BMM, bone marrow-derived macrophages; RAG-1, recombination-activating gene-1; {gamma}cR, common cytokine receptor {gamma} chain; IFU, inclusion forming units; iNOS, inducible NO synthase; WT, wild type; SC, spleen cell. Back

Received for publication August 8, 2003. Accepted for publication December 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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