The Purinergic P2X7 Receptor Is Not Required for Control of Pulmonary Mycobacterium tuberculosis Infection
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感染与免疫杂志 2005年第5期
Department of Medicine, Division of Infectious Disease, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
ABSTRACT
The importance in vivo of P2X7 receptors in control of virulent Mycobacterium tuberculosis was examined in a low-dose aerosol infection mouse model. P2X7–/– mice controlled infection in lungs as well as wild-type mice, suggesting that the P2X7 receptor is not required for control of pulmonary M. tuberculosis infection.
TEXT
Signaling through the purinergic P2X7 receptor by mM concentrations of extracellular ATP has been shown to induce apoptosis and lead to killing of mycobacteria in infected human and murine macrophages (4, 6, 10). The mechanism of this Mycobacterium tuberculosis growth reduction is due to increased phagosome-lysosome fusion after signaling of the P2X7 receptor (3, 5, 15). Previously, we created an in vitro human system where lysed cells provided bystander ATP in the local microenvironment of mycobacterium-infected monocytes. No evidence of mycobacterial killing was observed in spite of achieving significant local concentrations of ATP (2). It is not clear whether sufficient extracellular ATP concentrations can be achieved in vivo or if there are modulating factors in vivo that cannot be recreated with in vitro models. As a result, we wanted to determine if ATP signaling of the P2X7 receptor plays a role in control of mycobacteria in vivo.
Control of M. tuberculosis after aerosol infection in P2X7–/– mice. P2X7–/– mice backcrossed six generations to C57BL/6 (Pfizer) mice and wild-type C57BL/6 mice (Charles River) received low-dose (50 CFU) aerosol infection with virulent M. tuberculosis Erdman as previously described (14). At various time points postinfection, mice were sacrificed to determine bacillary burdens in the lungs and draining mediastinal lymph node (LN) (13). In two independent experiments, there were no differences in total lung CFU from 14 to 100 days postinfection between P2X7–/– and control wild-type mice (Fig. 1A). A third experiment examined very early time points, and no difference in CFU in the lungs at day 5 or 10 was detected (data not shown). Bacillary burdens were also determined in the mediastinal LN in three experiments. There was no significant difference in numbers of M. tuberculosis CFU between the P2X7–/– mice and wild-type controls (Fig. 1B). This suggests that the P2X7 receptor is not required for the control of pulmonary M. tuberculosis infection.
Mononuclear cells in naive and infected mice. Initial experiments were performed to determine whether the baseline number and composition of mononuclear cells were similar in P2X7–/– and control mice. There were no significant differences in absolute numbers of cells or CD4+-, CD8+-, CD11c+-, or CD11b+-expressing cells in lungs or spleen between naive P2X7–/– and control mice (data not shown).
After aerosol M. tuberculosis infection, there were significantly higher total numbers of CD4+ T cells at weeks 4 (P = 0.05) and 11 (P = 0.005) in the lungs of P2X7–/– mice (Fig. 2A). CD8+ T-cell numbers in the lungs were similar in both groups after infection (Fig. 2B). There were no statistically significant differences in the percentages of CD11c+ and CD11b+ cells in lungs and mediastinal LN after infection in two experiments (data not shown).
Several experiments were performed on infected P2X7–/– and wild-type mice, using Annexin V and 7-aminoactinomycin D (7-AAD) staining, that did not show consistently different levels of apoptosis in either the T cells, dendritic cells (DC), or macrophages (data not shown).
Antigen-specific responses in lung and lymph node after infection. After infection, T-cell responses were assessed using two methods. First, T-cell responses from lungs and mediastinal LN were analyzed by intracytoplasmic gamma interferon (IFN-) staining after anti-CD3 and anti-CD28 stimulation. We have shown previously that this correlates well with T-cell responses of previously activated CD44high T cells (13). Total numbers of CD4+ T cells producing IFN- in the lungs of P2X7–/– mice were significantly higher at weeks 4 (P = 0.02) and 11 (P = 0.02) after infection, with a trend also at weeks 6 and 14 (Fig. 2C). Similarly, total numbers of CD8+ T cells producing IFN- in the P2X7–/– lungs were higher at weeks 4 (P = 0.02) and 11 (P = 0.007) (Fig. 2D).
To confirm that these responses were M. tuberculosis specific, IFN- enzyme-linked immunospot analysis was performed on the lungs and LN of infected animals in two separate experiments, using DC with and without M. tuberculosis infection to restimulate the T cells as previously described (7). In the lungs, there was no significant difference in M. tuberculosis-specific IFN--secreting T cells between the groups (Fig. 2E). There were significantly higher numbers of M. tuberculosis-specific IFN--secreting T cells in the LN of P2X7–/– mice at 2 and 11 weeks postinfection (P < 0.05). Otherwise, there was a trend at all time points tested for higher M. tuberculosis-specific IFN--secreting-T-cell frequencies in P2X7–/– mice.
In vitro priming and antigen presentation. We sought to determine a mechanism for the increased numbers of antigen-specific T cells in P2X7–/– mice. Antigen presentation and priming functions were examined. Bone marrow-derived macrophages (BMM) were activated with IFN- and infected in vitro with M. tuberculosis. Antigen processing and presentation by BMM was determined using a M. tuberculosis antigen 85B-specific T-cell hybridoma (11). T-cell responses were identical after stimulation with either P2X7–/– or wild-type BMM (Fig. 3A), demonstrating no difference in the intrinsic ability of P2X7–/– macrophages to present M. tuberculosis antigens.
The ability of P2X7–/– CD11c+ antigen-presenting cells (APC) to prime naive T cells was then determined. CD11c+ APC, primarily DC, were purified by immunomagnetic beads (Miltenyi) from spleens of naive P2X7–/– and wild-type mice. T cells from naive ovalbumin (OVA)-specific T-cell transgenic mice (1) were incubated with OVA antigen and CD11c+ APC. An [3H]thymidine (ICN, Costa Mesa, CA) incorporation lymphoproliferation assay was performed. P2X7–/– CD11c+ APC were similar to wild-type APC in their ability to prime naive T cells (Fig. 3B).
Overall, our studies demonstrate that the P2X7 receptor is not required in mice to contain M. tuberculosis infection. There was a modest but statistically significant increase in the number of IFN--secreting CD4+ T cells in lungs of M. tuberculosis-infected P2X7–/– mice. The etiology of this increase is not clear from our studies. It is not related to increased efficiency of P2X7–/– APC in presenting antigen or in priming naive T cells. In addition, we were unable to detect consistent differences in apoptosis between the groups to account for the difference in T cells.
Polymorphisms in the human P2X7 gene have not been associated with increased risk of tuberculosis (8, 9, 12). Our previous in vitro work with human macrophages exposed to ATP from degranulating T cells or lysed bystander cells did not induce P2X7-mediated control of mycobacterial growth (2). These human data, along with the data presented in this in vivo P2X7–/– mouse study, bring into question the importance of the P2X7 receptor in the control of mycobacterial disease. Perhaps the observation that high-dose extracellular ATP induces killing of mycobacteria through increases in phagolysomal fusion is just an interesting in vitro phenomenon that does not have a role in mycobacterial disease.
REFERENCES
1. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34-40.
2. Canaday, D. H., R. Beigi, R. F. Silver, C. V. Harding, W. H. Boom, and G. R. Dubyak. 2002. ATP and control of intracellular growth of mycobacteria by T cells. Infect. Immun. 70:6456-6459.
3. Fairbairn, I. P., C. B. Stober, D. S. Kumararatne, and D. A. Lammas. 2001. Atp-mediated killing of intracellular mycobacteria by macrophages is a p2x(7)-dependent process inducing bacterial death by phagosome-lysosome fusion. J. Immunol. 167:3300-3307.
4. Kusner, D. J., and J. Adams. 2000. ATP-induced killing of virulent Mycobacterium tuberculosis within human macrophages requires phospholipase D. J. Immunol. 164:379-388.
5. Kusner, D. J., and J. A. Barton. 2001. ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion. J. Immunol. 167:3308-3315.
6. Lammas, D. A., C. Stober, C. J. Harvey, N. Kendrick, S. Panchalingam, and D. S. Kumararatne. 1997. ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors. Immunity 7:433-444.
7. Lazarevic, V., A. J. Myers, C. A. Scanga, and J. L. Flynn. 2003. CD40, but not CD40L, is required for the optimal priming of T cells and control of aerosol M. tuberculosis infection. Immunity 19:823-835.
8. Li, C. M., S. J. Campbell, D. S. Kumararatne, R. Bellamy, C. Ruwende, K. P. McAdam, A. V. Hill, and D. A. Lammas. 2002. Association of a polymorphism in the P2X7 gene with tuberculosis in a Gambian population. J. Infect. Dis. 186:1458-1462.
9. Li, C. M., S. J. Campbell, D. S. Kumararatne, A. V. Hill, and D. A. Lammas. 2002. Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter. FEBS Lett. 531:127-131.
10. Molloy, A., P. Laochumroonvorapong, and G. Kaplan. 1994. Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guerin. J. Exp. Med. 180:1499-1509.
11. Noss, E. H., C. V. Harding, and W. H. Boom. 2000. Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages. Cell Immunol. 201:63-74.
12. Saunders, B. M., S. L. Fernando, R. Sluyter, W. J. Britton, and J. S. Wiley. 2003. A loss-of-function polymorphism in the human P2X7 receptor abolishes ATP-mediated killing of mycobacteria. J. Immunol. 171:5442-5446.
13. Serbina, N. V., and J. L. Flynn. 1999. Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect. Immun. 67:3980-3988.
14. Serbina, N. V., C. C. Liu, C. A. Scanga, and J. L. Flynn. 2000. CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages. J. Immunol. 165:353-363.
15. Stober, C. B., D. A. Lammas, C. M. Li, D. S. Kumararatne, S. L. Lightman, and C. A. McArdle. 2001. ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guerin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes. J. Immunol. 166:6276-6286.
16. Zhi-Jun, Y., N. Sriranganathan, T. Vaught, S. K. Arastu, and S. A. Ahmed. 1997. A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies. J. Immunol. Methods 210:25-39.(Amy J. Myers, Brandon Eil)
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
ABSTRACT
The importance in vivo of P2X7 receptors in control of virulent Mycobacterium tuberculosis was examined in a low-dose aerosol infection mouse model. P2X7–/– mice controlled infection in lungs as well as wild-type mice, suggesting that the P2X7 receptor is not required for control of pulmonary M. tuberculosis infection.
TEXT
Signaling through the purinergic P2X7 receptor by mM concentrations of extracellular ATP has been shown to induce apoptosis and lead to killing of mycobacteria in infected human and murine macrophages (4, 6, 10). The mechanism of this Mycobacterium tuberculosis growth reduction is due to increased phagosome-lysosome fusion after signaling of the P2X7 receptor (3, 5, 15). Previously, we created an in vitro human system where lysed cells provided bystander ATP in the local microenvironment of mycobacterium-infected monocytes. No evidence of mycobacterial killing was observed in spite of achieving significant local concentrations of ATP (2). It is not clear whether sufficient extracellular ATP concentrations can be achieved in vivo or if there are modulating factors in vivo that cannot be recreated with in vitro models. As a result, we wanted to determine if ATP signaling of the P2X7 receptor plays a role in control of mycobacteria in vivo.
Control of M. tuberculosis after aerosol infection in P2X7–/– mice. P2X7–/– mice backcrossed six generations to C57BL/6 (Pfizer) mice and wild-type C57BL/6 mice (Charles River) received low-dose (50 CFU) aerosol infection with virulent M. tuberculosis Erdman as previously described (14). At various time points postinfection, mice were sacrificed to determine bacillary burdens in the lungs and draining mediastinal lymph node (LN) (13). In two independent experiments, there were no differences in total lung CFU from 14 to 100 days postinfection between P2X7–/– and control wild-type mice (Fig. 1A). A third experiment examined very early time points, and no difference in CFU in the lungs at day 5 or 10 was detected (data not shown). Bacillary burdens were also determined in the mediastinal LN in three experiments. There was no significant difference in numbers of M. tuberculosis CFU between the P2X7–/– mice and wild-type controls (Fig. 1B). This suggests that the P2X7 receptor is not required for the control of pulmonary M. tuberculosis infection.
Mononuclear cells in naive and infected mice. Initial experiments were performed to determine whether the baseline number and composition of mononuclear cells were similar in P2X7–/– and control mice. There were no significant differences in absolute numbers of cells or CD4+-, CD8+-, CD11c+-, or CD11b+-expressing cells in lungs or spleen between naive P2X7–/– and control mice (data not shown).
After aerosol M. tuberculosis infection, there were significantly higher total numbers of CD4+ T cells at weeks 4 (P = 0.05) and 11 (P = 0.005) in the lungs of P2X7–/– mice (Fig. 2A). CD8+ T-cell numbers in the lungs were similar in both groups after infection (Fig. 2B). There were no statistically significant differences in the percentages of CD11c+ and CD11b+ cells in lungs and mediastinal LN after infection in two experiments (data not shown).
Several experiments were performed on infected P2X7–/– and wild-type mice, using Annexin V and 7-aminoactinomycin D (7-AAD) staining, that did not show consistently different levels of apoptosis in either the T cells, dendritic cells (DC), or macrophages (data not shown).
Antigen-specific responses in lung and lymph node after infection. After infection, T-cell responses were assessed using two methods. First, T-cell responses from lungs and mediastinal LN were analyzed by intracytoplasmic gamma interferon (IFN-) staining after anti-CD3 and anti-CD28 stimulation. We have shown previously that this correlates well with T-cell responses of previously activated CD44high T cells (13). Total numbers of CD4+ T cells producing IFN- in the lungs of P2X7–/– mice were significantly higher at weeks 4 (P = 0.02) and 11 (P = 0.02) after infection, with a trend also at weeks 6 and 14 (Fig. 2C). Similarly, total numbers of CD8+ T cells producing IFN- in the P2X7–/– lungs were higher at weeks 4 (P = 0.02) and 11 (P = 0.007) (Fig. 2D).
To confirm that these responses were M. tuberculosis specific, IFN- enzyme-linked immunospot analysis was performed on the lungs and LN of infected animals in two separate experiments, using DC with and without M. tuberculosis infection to restimulate the T cells as previously described (7). In the lungs, there was no significant difference in M. tuberculosis-specific IFN--secreting T cells between the groups (Fig. 2E). There were significantly higher numbers of M. tuberculosis-specific IFN--secreting T cells in the LN of P2X7–/– mice at 2 and 11 weeks postinfection (P < 0.05). Otherwise, there was a trend at all time points tested for higher M. tuberculosis-specific IFN--secreting-T-cell frequencies in P2X7–/– mice.
In vitro priming and antigen presentation. We sought to determine a mechanism for the increased numbers of antigen-specific T cells in P2X7–/– mice. Antigen presentation and priming functions were examined. Bone marrow-derived macrophages (BMM) were activated with IFN- and infected in vitro with M. tuberculosis. Antigen processing and presentation by BMM was determined using a M. tuberculosis antigen 85B-specific T-cell hybridoma (11). T-cell responses were identical after stimulation with either P2X7–/– or wild-type BMM (Fig. 3A), demonstrating no difference in the intrinsic ability of P2X7–/– macrophages to present M. tuberculosis antigens.
The ability of P2X7–/– CD11c+ antigen-presenting cells (APC) to prime naive T cells was then determined. CD11c+ APC, primarily DC, were purified by immunomagnetic beads (Miltenyi) from spleens of naive P2X7–/– and wild-type mice. T cells from naive ovalbumin (OVA)-specific T-cell transgenic mice (1) were incubated with OVA antigen and CD11c+ APC. An [3H]thymidine (ICN, Costa Mesa, CA) incorporation lymphoproliferation assay was performed. P2X7–/– CD11c+ APC were similar to wild-type APC in their ability to prime naive T cells (Fig. 3B).
Overall, our studies demonstrate that the P2X7 receptor is not required in mice to contain M. tuberculosis infection. There was a modest but statistically significant increase in the number of IFN--secreting CD4+ T cells in lungs of M. tuberculosis-infected P2X7–/– mice. The etiology of this increase is not clear from our studies. It is not related to increased efficiency of P2X7–/– APC in presenting antigen or in priming naive T cells. In addition, we were unable to detect consistent differences in apoptosis between the groups to account for the difference in T cells.
Polymorphisms in the human P2X7 gene have not been associated with increased risk of tuberculosis (8, 9, 12). Our previous in vitro work with human macrophages exposed to ATP from degranulating T cells or lysed bystander cells did not induce P2X7-mediated control of mycobacterial growth (2). These human data, along with the data presented in this in vivo P2X7–/– mouse study, bring into question the importance of the P2X7 receptor in the control of mycobacterial disease. Perhaps the observation that high-dose extracellular ATP induces killing of mycobacteria through increases in phagolysomal fusion is just an interesting in vitro phenomenon that does not have a role in mycobacterial disease.
REFERENCES
1. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76:34-40.
2. Canaday, D. H., R. Beigi, R. F. Silver, C. V. Harding, W. H. Boom, and G. R. Dubyak. 2002. ATP and control of intracellular growth of mycobacteria by T cells. Infect. Immun. 70:6456-6459.
3. Fairbairn, I. P., C. B. Stober, D. S. Kumararatne, and D. A. Lammas. 2001. Atp-mediated killing of intracellular mycobacteria by macrophages is a p2x(7)-dependent process inducing bacterial death by phagosome-lysosome fusion. J. Immunol. 167:3300-3307.
4. Kusner, D. J., and J. Adams. 2000. ATP-induced killing of virulent Mycobacterium tuberculosis within human macrophages requires phospholipase D. J. Immunol. 164:379-388.
5. Kusner, D. J., and J. A. Barton. 2001. ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion. J. Immunol. 167:3308-3315.
6. Lammas, D. A., C. Stober, C. J. Harvey, N. Kendrick, S. Panchalingam, and D. S. Kumararatne. 1997. ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors. Immunity 7:433-444.
7. Lazarevic, V., A. J. Myers, C. A. Scanga, and J. L. Flynn. 2003. CD40, but not CD40L, is required for the optimal priming of T cells and control of aerosol M. tuberculosis infection. Immunity 19:823-835.
8. Li, C. M., S. J. Campbell, D. S. Kumararatne, R. Bellamy, C. Ruwende, K. P. McAdam, A. V. Hill, and D. A. Lammas. 2002. Association of a polymorphism in the P2X7 gene with tuberculosis in a Gambian population. J. Infect. Dis. 186:1458-1462.
9. Li, C. M., S. J. Campbell, D. S. Kumararatne, A. V. Hill, and D. A. Lammas. 2002. Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter. FEBS Lett. 531:127-131.
10. Molloy, A., P. Laochumroonvorapong, and G. Kaplan. 1994. Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guerin. J. Exp. Med. 180:1499-1509.
11. Noss, E. H., C. V. Harding, and W. H. Boom. 2000. Mycobacterium tuberculosis inhibits MHC class II antigen processing in murine bone marrow macrophages. Cell Immunol. 201:63-74.
12. Saunders, B. M., S. L. Fernando, R. Sluyter, W. J. Britton, and J. S. Wiley. 2003. A loss-of-function polymorphism in the human P2X7 receptor abolishes ATP-mediated killing of mycobacteria. J. Immunol. 171:5442-5446.
13. Serbina, N. V., and J. L. Flynn. 1999. Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect. Immun. 67:3980-3988.
14. Serbina, N. V., C. C. Liu, C. A. Scanga, and J. L. Flynn. 2000. CD8+ CTL from lungs of Mycobacterium tuberculosis-infected mice express perforin in vivo and lyse infected macrophages. J. Immunol. 165:353-363.
15. Stober, C. B., D. A. Lammas, C. M. Li, D. S. Kumararatne, S. L. Lightman, and C. A. McArdle. 2001. ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guerin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes. J. Immunol. 166:6276-6286.
16. Zhi-Jun, Y., N. Sriranganathan, T. Vaught, S. K. Arastu, and S. A. Ahmed. 1997. A dye-based lymphocyte proliferation assay that permits multiple immunological analyses: mRNA, cytogenetic, apoptosis, and immunophenotyping studies. J. Immunol. Methods 210:25-39.(Amy J. Myers, Brandon Eil)