The cytolytic activity of natural killer cells is not involved in the restriction of Mycobacterium avium growth

Manuela Flórido1, Margarida Correia-Neves1, Andrea M. Cooper2 and Rui Appelberg1

1 Laboratory of Microbiology and Immunology of Infection, Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal 2 Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA

Correspondence to: R. Appelberg; E-mail: rappelb{at}ibmc.up.pt
Transmitting editor: L. L. Lanier


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Severe combined immunodeficiency (SCID) mice were used to analyze the role of NK cells in resistance to Mycobacterium avium. The neutralization of IFN-{gamma} in these animals led to an exacerbation of the infection associated with a reduction in macrophage activation, suggesting a role for NK cells in innate immunity to mycobacteria. In contrast, administration of anti-asialo-GM1 polyclonal serum or mAb specific for Thy1.2 did not affect mycobacterial growth or macrophage activation despite causing the almost complete abrogation of the natural cytolysis of a tumor cell target. Treatment with anti-asialo-GM1-specific serum depleted only two-thirds of the Thy1.2+ spleen cells, and anti-Thy1.2 treatment allowed for the persistence of a small number of cells still exhibiting an NK cell marker recognized by mAb DX5 and able to express IFN-{gamma} as analyzed by flow cytometry. In vivo treatment of B6.SCID mice with anti-NK1.1 mAb again failed to affect resistance to infection and allowed for the persistence of 2–8% of IFN-{gamma}-producing cells, many of them still expressing the DX5 marker. In vitro depletion studies showed that removal of IFN-{gamma}-expressing cells required the combined action of anti-Thy1.2, anti-Ly49C and DX5 antibodies in the presence of complement. Our data show that resistance to M. avium mediated by NK cells is independent of their cytolytic activity, and that there is a marked phenotypic and functional heterogeneity of the NK cell lineage in vivo during infection.

Keywords: bacteria, cell-surface molecule, infectious immunity, in vivo animal model, NK cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NK cells exhibit different functions that may underlie their participation in the control of infection. They are able to lyse infected cells without any previous exposure to their antigens, a process that depends on the negative regulation of the cytolytic machinery by class I MHC molecules (1). NK cells are also able to secrete cytokines such as IFN-{gamma}, a major modulator of antimicrobial functions (3), after priming with other inducer molecules such as IL-12, -15 and -18 or oligonucleotides (2). The role played by NK cells in resistance to mycobacterial infections is not clear. Although it has been reported that depletion of NK cells causes an increase in susceptibility to Mycobacterium avium (4), we as well as others (5,6) have been unable to confirm this observation.

Using in vitro assays, it has been shown that NK cells interact with macrophages and that this leads to a reduction in mycobacterial proliferation (7). It is, however, unclear whether cytolysis of infected macrophages, production of cytokines such as IFN-{gamma} or another as-yet unidentified mechanism mediates this reduction. To address this question we have made use of the severe combined immunodeficiency (SCID) mouse. This mouse has no T cell compartment, but it is still capable of exhibiting detectable resistance to mycobacterial infection (8). M. avium-infected SCID mice were depleted of NK cells using a variety of NK cell-specific antibodies and the effect on bacterial growth determined. We show here that SCID mice express IFN-{gamma}-dependent protective mechanisms against mycobacterial infection and that depletion of naturally cytotoxic cells did not reduce this protection. That NK cells may still be mediating the observed protection was suggested by the observed heterogeneity of the NK cell population during infection.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female Fox Chase SCID mice were purchased from Bommice (Ry, Denmark). Female SCID mice on a C57Bl/6 background (B6.CB17-Prkdcscid/SzJ) were obtained from the Jackson Laboratory (Bar Harbor, ME). HSD nude mice were purchased from Instituto Gulbenkian de Ciência (Oeiras, Portugal). Animals were kept under standard hygiene conditions, in HEPA filter-top cages, fed commercial chow and given acidified drinking water ad libitum. Animals were used at 6–12 weeks of age.

Bacteria
M. avium strain 2447, smooth transparent morphotype, were grown to mid-log phase in Middlebrook 7H9 medium (Difco, Sparks, MD) containing 0.04% Tween 80 at 37°C. Bacteria were harvested by centrifugation, suspended into a small volume of saline and briefly sonicated to disrupt bacterial clumps. This suspension was diluted, frozen in aliquots and kept at –70°C until use. Before being used for inoculation, bacterial aliquots were thawed at 37°C and diluted in saline to the desired concentration.

Hybridomas and antibodies
The following hybridomas were used: XMG 1.2, secreting an anti-IFN-{gamma}-specific rat IgG1 (DNAX, Palo Alto, CA); 30H12, secreting an anti-Thy1.2-specific rat IgG2b (ATCC, Manassas, VA); M5, secreting an anti-Thy1.2-specific rat IgG2a (ATCC); 5E6, secreting an anti-Ly49C/I-specific rat Ig2a; DX5, secreting an anti-CD49b rat IgM (DNAX); PK136, secreting an anti-NK1.1-specific rat IgG2a (ATCC); and the isotype-matched controls GL113, secreting an anti-ß-galactosidase rat IgG1 (DNAX), and GL117, secreting an anti-ß-galactosidase rat IgG2b (DNAX). The hybridoma cell line 5E6 secretes an antibody that recognizes epitopes shared by the molecules Ly49C and Ly49I. Mice used in this study are on the BALB/c background and therefore do not express the Ly49I antigen (10); consequently, the cells detected by 5E6 in this study are referred to as Ly49C+. All hybridomas were cultured until reaching exponential growth in DMEM (Gibco, Paisley, UK) containing 10 mM of HEPES buffer (Gibco) and supplemented with 10% FCS (Gibco). The mAb were obtained from ascites of HSD nude mice inoculated i.p. with the corresponding hybridomas from exponential growth cultures and purified by Protein G–agarose affinity chromatography (Protein G column; Gibco) followed by saline dialysis. Rabbit anti-asialo-GM1 polyclonal serum was purchased from Wako (Richmond, VA). FITC-conjugated rat anti-mouse Thy1.2, FITC-conjugated rat anti-mouse DX5, FITC-conjugated rat anti-mouse Thy1.2, phycoerythrin-conjugated rat anti-mouse IFN-{gamma} and phycoerythrin-conjugated rat anti-mouse Ly49C mAb were purchased from PharMingen (San Diego, CA).

In vivo infection and depletions
Mice were infected i.v. with 106 c.f.u. of M. avium strain 2447. Fox Chase SCID mice were used in all experiments but one. These animals have a BALB/c background. One day after the infection, each experimental group of mice, consisting of five mice, was injected i.p. with the mAb or corresponding isotype-matched controls. Depletion was continued throughout the course of infection by injecting the mice with either 2.0 mg anti-IFN-{gamma} every 2 weeks or 0.4 mg anti-Thy1.2 antibodies twice a week or 50 µl anti-asialoGM1 serum every 5 days. In some experiments depletion of NK cells was achieved by treating each mouse with 0.4 mg of each of the anti-Thy1.2 antibodies (30H12 and M5) combined with 0.4 mg anti-Ly49C antibodies (5E6), twice a week. In one experiment, SCID mice on a B6 background were used and treated with 1 mg anti-NK1.1, twice a week. All the isotype-matched controls were administered at the same dosage and periodicity as the corresponding antibodies. The number of M. avium c.f.u. in the liver, spleen and lung of the infected mice was determined by plating serial dilutions of the tissue homogenates onto Middlebrook 7H10 agar medium (Difco). In one experiment (Fig. 5), SCID mice were infected i.v. with 106 c.f.u. of M. avium strain 2447 for 15 days and 24 h prior to being sacrificed mice were injected i.p. with either 0.4 mg of each of the anti-Thy1.2 mAb (30H12 and M5) or with 0.8 mg non-immune rat Ig as control.



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Fig. 5. Characterization of the activity of an anti-NK1.1 mAb. B6.CB17 SCID mice were infected for 40 days with M. avium and treated twice a week with either PK136 mAb or non-immune IgG. The analysis of surface markers (A) and intracellular staining of IFN-{gamma} (B) was performed as described in Fig. 4.

 
In vitro depletions
Single-cell suspensions were treated for 5 min at room temperature with a hemolytic solution (155 mM NH4Cl, 10 mM KHCO3, pH 7.2), washed and incubated with a mixture of the different anti-NK antibodies (30H12, M5, 5E6 and DX5) or with DX5 antibodies alone (10 µg of each antibody/107cells/ml) in the presence of rabbit complement (Serotec, Kidlington, UK) at a dilution of 1/30 for 45 min at 37°C. As a control, cells were incubated with complement alone. After extensive washing, cells were resuspended in DMEM tissue culture medium supplemented with 10% FCS.

Quantification of the NO2 production by peritoneal exudate cells (PEC)
PEC were obtained by washing the peritoneal cavity of mice with a small volume of saline followed by collection with a syringe fitted with a 19G caliber needle. Cells were resuspended in DMEM supplemented with 10% FCS and cultured at a density of 3 x 106 cells/ml/well in a 24-well tissue culture plate. After 2 h of incubation at 37°C in a CO2 atmosphere, non-adherent cells were removed and the remaining layer of adherent PEC were further cultured with fresh cell culture medium containing 1 µg/ml LPS (Sigma, St Louis, MO). Three days later, supernatants were collected, the concentration of NO2 determined using the Griess reagent, cells lysed by freezing/thawing cycles and the protein content in each well determined (9).

Cytotoxicity assay
Cells of the YAC-1 mouse lymphoma cell line (ATCC) were used as targets in this assay. Cells were harvested from an exponentially growing culture, labeled by incubation with 200 µCi 51Cr-labeled sodium dichromate (Amersham, Little Chalfont, UK) at 37°C for 2 h, washed 3 times and 5 x 103 cells were plated in a U-bottom 96-well cell culture plate. Different numbers of effector cells were added in triplicate. E:T ratios of 100:1, 50:1, 25:1 and 12.5:1 were used in the assay. After 4 h of culture at 37°C in a CO2 atmosphere, 100 µl of the supernatants was transferred to scintillation tubes and isotope release was measured in a scintillation counter. Spontaneous 51Cr release was measured by incubation of target cells in DMEM in the absence of effector cells and total release was measured by incubation of target cells with 10% of saponin (Merck, Darmstadt, Germany). Specific cytotoxicity was calculated as % lysis = (AB)/(C B) x 100, where A is the experimental release, B is the spontaneous release and C the total release.

FACS analysis
To evaluate the efficiency of the in vivo and the in vitro depletions, splenic cell suspensions from the infected mice were labeled with FITC- or phycoerythrin-conjugated antibodies specific for the NK cell markers (Thy1.2, DX5 and Ly49C) (dilution 1:100). For the analysis of IFN-{gamma} production, splenic cells were cultured in DMEM supplemented with 10% FCS at a density of ~1 x 106 cells/ml and incubated for 2 h at 37°C in the presence of phorbol myristate acetate (Sigma) plus ionomycin (Calbiochem, San Diego, CA) at a final concentration of 25 µg/ml each followed by an incubation of 2 h in the presence of 0.01 mg/ml Brefeldin A (Sigma). After extensive washing with ice-cold PBS, cells were labeled using FITC-conjugated anti-DX5 antibodies (dilution 1:100) and fixed for 20 min in a 2% paraformaldehyde solution. The fixed cells were then permeabilized for 10 min at room temperature with PBS/1%FCS/0.1%NaN3/0.5% saponin and incubated with 5 µg/ml phycoerythrin-conjugated anti-IFN-{gamma} mAb for 20 min at room temperature. The stained populations were analyzed in a Becton Dickinson (San Jose, CA) FACSorter using PCLysys II software.

RT-PCR analysis
Splenic cell suspensions were obtained from a pool of four non-infected control SCID mice and from a pool of four SCID mice infected i.v. with 106 c.f.u. M. avium 2447 SmT for 20 days. Total cells (5 x 106) from each pool were immediately mixed with a guanidinium thiocyanate lytic solution and frozen at –70°C. The remaining cells from each pooled cell suspension were then processed for MACS purification using anti-NK Cell (DX5) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, after incubation of the cells with an ammonium chloride 0.155 M/potassium bicarbonate 0.01 M solution to lyse erythrocytes, cells were washed and centrifuged through a 3-ml FCS layer, filtered through a 70-µm nylon mesh to remove clumps and dead cells, and incubated with DX5 beads at the concentration recommended by the manufacturer. DX5+ NK cells were obtained using a MidiMACS magnetic field and running the samples through a LS+ MACS Column, and resulted in a 90–92% pure DX5-expressing population of cells. The DX5+ cells were washed, pelleted, resuspended in the guanidinium thiocyanate lytic solution and frozen at –70°C. Total mRNA from the samples was obtained by phenol–chloroform extraction and stored at –70°C until further processed. Reverse transcription was performed using p(dT)12–18 oligonucleotides (Pharmacia Biotech, Uppsala, Sweden) and Superscript reverse transcriptase (Invitrogen, Carlsbad, CA) in the presence of 10 U RNase inhibitor (Invitrogen). cDNA was diluted and amplified with Taq polymerase (Invitrogen) in the presence of specific pairs of primers for the housekeeping gene coding for HPRT and for IFN-{gamma} in a Gene Amp PCR system 9600 (Perkin-Elmer, Foster City, CA). The sequences of the primers used were sense: GTT GGA TAC AGG CCA GAC TTT GTT G, anti-sense: GAT TCA ACT TGC GCT CAT CTT AGG C for HPRT and sense: AAC GCT ACA CAC TGC ATC TTG G, anti-sense: GAC TTC AAA GAG TCT GAG G for IFN-{gamma} (Genset, San Diego, CA). The amplification products were generated under conditions of linear correlation with the amount of cDNA, standardized for similar HPRT signals, and run in parallel with titrations of both HPRT and IFN-{gamma} signals from internal standards in a 1.4% agarose gel with ethidium bromide at a final concentration of 0.2 µg/ml. The gel was scanned using a Variable Mode Imager Typhoon 8600 and the image analyzed using Imagequant 5.1 software.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The effects of IFN-{gamma} depletion on M. avium proliferation in SCID mice were studied by treating M. avium-infected mice with either mAb specific for IFN-{gamma} or control antibodies with irrelevant specificity. As shown in Fig. 1(A), neutralization of IFN-{gamma} led to an exacerbation of the growth of M. avium as determined 40 days after infection in all three organs studied. In addition to the increased susceptibility, PEC from infected, IFN-{gamma}-depleted mice exhibited decreased nitric oxide production when cultured in vitro (Fig. 1B).



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Fig. 1. Neutralization of IFN-{gamma} exacerbates mycobacterial infection and reduces macrophage activation in SCID mice infected with M. avium. (A) Geometric means of the number of c.f.u. (± SD) of M. avium in the indicated organs of SCID mice infected for 40 days and given non-specific rat IgG (open columns) or anti-IFN-{gamma} mAb (hatched columns). (B) Production of nitrite by PEC isolated from the same mice as in (A) and cultured for 3 days with 1 µg LPS/ml. Statistically significant differences between the two groups are labeled **P < 0.01 according to Student’s t-test.

 
In a similar experiment, M. avium-infected SCID mice were treated with anti-asialo-GM1 rabbit polyclonal antiserum or a non-immune rabbit serum. The treatment, classically used in vivo to deplete NK cells, had no effect on either mycobacterial growth in the organs of the SCID mice or the state of activation of the PEC (Fig. 2A and B). In contrast, the anti-asialo-GM1 treatment depleted the NK activity against target YAC-1 cells by >90% (Fig. 2C). However, the depletion of Thy1.2+ cells was incomplete, with approximately one-third of the Thy1.2+ cells surviving the treatment (Fig. 2D).



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Fig. 2. Depletion of anti-asialo-GM1-sensitive cells did not exacerbate infection by M. avium nor reduce macrophage activation, but effectively depleted natural cytotoxicity against YAC-1 cells. (A) Geometric means of the number of c.f.u. (± SD) of M. avium in the indicated organs of SCID mice infected for 40 days and given non-immune rabbit serum (open columns) or anti-asialo-GM1 rabbit serum (hatched columns). (B) Production of nitrite by PEC isolated from the same mice as in (A) and cultured for 3 days with 1 µg LPS/ml. (C) Spontaneous lysis of labeled YAC-1 cells induced by spleen cells from control (open squares) and anti-asialo-GM1-treated (closed circles) mice from the same experiment. (D) Number of spleen cells staining for Thy1.2, but not for CD3, from the control (open column) or the anti-asialo-GM1-treated (hatched column). No statistically significant differences were found for data in (A), (B) or (D).

 
To address the role of these asialo-GM1-insensitive Thy1.2+ cells in protection, M. avium-infected SCID mice were treated with either anti-Thy1.2 mAb or control Ig with an irrelevant specificity. Mice were given 30H12 mAb (data not shown) or a combination of 30H12 and M5 mAb (Fig. 3). As in the case of the anti-asialo-GM1 treatment, virtually all the NK cytolytic activity was eliminated following anti-Thy1.2 treatments (Fig. 3B). Despite an effective depletion of cytolytic activity, there was a minimal effect on the control of bacterial growth (Fig. 3A). The efficacy of the depletion of the Thy1.2+ cells was analyzed by flow cytometry using a third mAb which showed no interference with the two antibodies used to deplete the cells and ranged from 77 to 96% in the five experiments performed with the use of anti-Thy1.2 antibodies. All experiments failed to substantially affect the mycobacterial growth, whilst treatments with anti-IFN-{gamma} performed in parallel in four out of the five experiments confirmed the exacerbation of mycobacterial growth reported above (data not shown). To determine which cells were lost during anti-Thy1.2 depletion, splenocytes from treated mice were analyzed by flow cytometry. Analysis of the cells remaining after treatment with the Thy1.2-specific reagents showed in a representative experiment that 44% of the DX5+ cells, 22% of the Thy1.2+ cells and 45% of the Ly49C+ cells survived the treatment (Fig. 3C). Although most of the Thy1.2+ cells were also DX5+, a significant proportion of the DX5+ cells were Thy1.2. The latter accounted for most of the cells surviving the anti-Thy1.2 treatment. When the DX5 monoclonal IgM was used in vivo it failed to lyse the target cells (not shown). In addition, combination of two anti-Thy1.2-specific mAb with one specific for Ly49C (hybridoma 5E6) failed to exacerbate mycobacterial growth further and allowed the survival of a significant proportion (27%) of DX5+ cells despite an almost complete abrogation of natural cytotoxicity (data not shown). Also, we treated SCID mice with a combination of anti-asialo-GM1 serum and anti-Thy1.2 mAb in vivo looking at the survival of DX5+ cells by FACS. No increase in depletion was found and, therefore, no further attempts were made to use this protocol during infection.



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Fig. 3. Depletion of Thy1.2+ cells had minor effects on mycobacterial growth despite effectively decreasing natural cytotoxicity against YAC-1 cells. Mice were given a combination of 30H12 and M5 mAb (hatched columns and closed squares) or the respective non-specific IgG (open columns and open squares) during the infection with M. avium. The geometric means of the number of c.f.u. (± SD) of M. avium in the indicated organs of SCID mice are shown for 40 days of infection (A). The spontaneous lysis of labeled YAC-1 cells induced by spleen cells from the same mice is shown in (B). Statistically significant differences between the two groups are labeled *P < 0.05 according to Student’s t-test. The effect on splenic NK cell populations is shown in (C). Panels show the number of spleen cells gated on Thy1.2 which are DX5+ or DX5 (left), the number of cells gated on DX5 which are Thy1.2+ or Thy1.2 (center) and the number of cells gated on Ly49C which are Thy1.2+ or Thy1.2 (right).

 
We then studied the expression of IFN-{gamma} by spleen cells from SCID mice infected for 15 days with M. avium. Figure 4(A) shows that control mice had a significant proportion of spleen cells, ranging from 19 to 25%, able to express IFN-{gamma}. Most of these cells stained positive for DX5. Similarly infected mice treated with anti-Thy1.2 antibodies still showed an important fraction of IFN-{gamma}-expressing cells that were for the most part DX5+. Since the DX5 mAb fails to deplete the target cells in vivo, we performed in vitro depletions using either DX5 alone or a combination of all NK cell-specific mAb available. As shown in Fig. 4(C and D), depletion of the DX5+ cells resulted in a decrease in the fraction of spleen cells expressing IFN-{gamma} from 21 to 13%. The combination of all four NK-specific antibodies resulted in an almost complete disappearance of the IFN-{gamma}-expressing cells.




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Fig. 4. Comparison of in vivo and in vitro depletion of NK cells. (A) Dot-plots representing typical analysis of control SCID mice (left) and SCID mice treated with 30H12 plus M5 antibodies (right) after staining spleen cells with antibodies specific for Thy1.2 and DX5. Mice were infected for 15 days with M. avium 2447. In vivo depletion of NK cells with anti-Thy1.2 mAb (30H12/M5) was performed by injecting the antibodies 1 day prior to harvesting the spleens. A control group received non-immune rat IgG. (B) Intracellular expression of IFN-{gamma} by spleen cells after in vitro stimulation as described in Methods, and staining for IFN-{gamma} and DX5. (C and D) Spleen cells from control mice (infected for 15 days with M. avium) were treated in vitro with DX5 antibodies combined or not with anti-Thy1.2 and anti-Ly49C mAb in the presence of rabbit complement, and analyzed for expression of surface markers (C) or for the ability to express intracellular IFN-{gamma} (D).

 
Another commonly used antibody able to deplete NK cells in vivo is the one targeting NK1.1 and secreted by hybridoma PK136. Since the NK1.1 is not expressed in a BALB/c background, we now used SCID mice in a C57Bl/6 background. The treatment of infected mice with two injections of PK136 twice a week failed to exacerbate the infection (data not shown). As previously found with the other treatments, a significant proportion of cells expressing Thy1.2, DX5 or both markers survived the treatment (Fig. 5A). Likewise, surviving cells were capable of expressing IFN-{gamma} after in vitro stimulation (Fig. 5B).

Since detection of IFN-{gamma} produced in vivo was not possible at the protein level due to the low amounts present in the cells we analyzed by RT-PCR for the presence of mRNA coding for this cytokine in total spleen cells and purified DX5+ cells from uninfected and infected SCID mice. Infection with M. avium for 20 days led to an increase in the mRNA coding for IFN-{gamma} detectable by RT-PCR (Fig. 6). Purification of cells expressing the DX5 epitope by magnetic cell sorting led to an increase in the signal in both cells from uninfected or infected animals, with a higher mRNA level found among cells of the latter group showing that IFN-{gamma} is being expressed in DX5+ cells in response to infection.



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Fig. 6. In vivo expression of IFN-{gamma} by total (1 and 2) or purified DX5+ (3 and 4) cells from uninfected SCID mice (1 and 3) or SCID mice infected for 20 days with M. avium 2447 (2 and 4). Scans of the agarose gels where the RT-PCR products were run are shown. The percentages of DX5+ cells in the samples were: 1, 26.5%; 2, 30.4%; 3, 89.5%; 4, 92.1%.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We report here that treatment of mice with anti-asialo-GM1 antibodies does not lead to an exacerbation of M. avium infection. This confirms observations previously reported by our laboratory (5) and others (6). We have extended this observation by demonstrating that depletion of NK cells expressing Thy1.2 or NK1.1 also fails to consistently reduce the resistance of the SCID mouse to the mycobacterial infection. It is noteworthy that both of these treatments were fully capable of eliminating natural cytolytic activity. Thus, these data strongly argue against a protective role for NK cells mediated via their cytolytic activity.

The absence of acquired immunity in the SCID mouse model allows for the protective role of the NK cell to be studied since NK cells are the dominant lymphocyte population in these mice. We report here the ability of the SCID mouse to mediate resistance to mycobacterial infection. This is clearly shown by the small, yet significant, increase in bacterial growth when IFN-{gamma} is neutralized. As we were unable to deplete all the NK cells in vivo, particularly those cells positive for DX5, it is certainly possible that these cells are the source of the protective cytokine. This assumption is corroborated by the cytometric data that showed most of the IFN-{gamma} being expressed by DX5+ cells. In these experiments, since no staining was demonstrable when cells were studied ex vivo, a strong activator of the NK cells was used instead of the infection itself. Thus, these data should be interpreted as evidence that cells with NK cell markers and which are able to secrete IFN-{gamma} survived the treatments. On the other hand, macrophages have been shown to secrete IFN-{gamma} (1113), and mice deficient in the common {gamma} chain of the receptors for IL-2, -4, -7, -9 and -15 are capable of producing IFN-{gamma} despite a major defect in NK cells as well as T and B lymphocytes (14). However, we found that the in vitro depletion of the cells exhibiting Thy1.2, Ly49C and DX5 resulted in an almost complete disappearance of IFN-{gamma}-expressing cells. Thus, we favor the view that IFN-{gamma} is being secreted by NK rather than other cell types. This interpretation is supported by the finding that ~90% pure DX5+ cells are enriched in IFN-{gamma} mRNA-expressing cells and that the expression of this cytokine as measured by RT-PCR is increased in response to infection, particularly in the DX5+ cell population. This issue may be solved once we find a way to completely deplete the NK cells in vivo, i.e. those that express the DX5 cell marker.

Our data point to the extensive heterogeneity of the NK cell population in terms of surface phenotype, as assessed by flow cytometry using antibodies for three distinct surface molecules. Heterogeneity has long been known to exist among NK cells. Thus, distinct reagents or treatments have been shown to differentially affect the cytolytic activity against different cell targets (15,16). The heterogeneity was also shown by others to exist at the level of the expression of surface markers. Thus, the expression of Thy1 varied according to the mouse strain studied: 50% of the cytolytic cells in C57Bl/6 were Thy1+ (17), whereas only one-third were positive in CBA and almost all were positive in BALB/c nude mice (18). In our work, the SCID mice used in all but one experiment have a BALB/c background and indeed most NK cells expressed the Thy1.2 marker. More recently, the molecular characterization of the NK cell receptors has uncovered the regulation of the expression of certain receptors in a way akin to repertoire selection leading to heterogeneous expression of these receptors (1). Our data, on the other hand, illustrate heterogeneity at a functional level, i.e. the ability to protect against infection versus the capacity to lyse target cells. Similar results were reported during toxoplasmosis, induced in SCID mice, where treatments with anti-asialo-GM1 sera and anti-Thy1.2 mAb had variable effects on the lytic activity as compared to the ability to secrete IFN-{gamma} (19). These authors showed that IFN-{gamma} may be produced by Thy1+ cells unable to mediate natural killing. Studying viral infections, Orange and Biron (20) found that neutralization of IL-12 led to the abrogation of priming of NK cells for IFN-{gamma} secretion, but had no effect on the cytotoxicity mediated by NK cells. NK cells can also be heterogeneous in the amounts of IFN-{gamma} they secrete (21), and IFN-{gamma} and cytotoxicity may be differentially regulated (22).

NK cells were named for their ability to spontaneously lyse tumor cell targets. Apparently, there may exist subsets among this cellular population which are not cytolytic. Although we cannot definitely incriminate NK cells as the source of enough IFN-{gamma} to induce mycobacterial growth control, were this to be the case there would be a subpopulation of these cells which are not NK, but rather are involved in natural help. It will be interesting to study in further detail whether the latter cells represent a new cell type which cannot functionally be defined as killer cells or whether they are precursors or progeny of the cytolytic subset.


    Acknowledgements
 
The authors are indebted to Drs J. Phillips, L. Lanier and M. Bennett for the gift of hybridomas. This work was supported by contracts AI-41922 from the NIH and P/SAU58/96 from the PRAXIS XXI program (Lisbon). M. F. received a PhD fellowship from PRAXIS XXI and M. C.-N. is the recipient of a post-doctoral fellowship from the Foundation for Science and Technology (Lisbon).


    Abbreviations
 
NK—natural killer

PEC—peritoneal exudate cell

SCID—severe combine immunodeficiency disease


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[CrossRef][ISI][Medline]
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  8. Appelberg, R., Castro, A. G., Pedrosa, J., Silva, R. A., Orme, I. M. and Minóprio, P. 1994. The role of gamma interferon and tumor necrosis factor-alpha during the T cell independent and dependent phases of Mycobacterium avium infection. Infect. Immun. 62:3962.[Abstract]
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