Previous Article | Next Article 
Journal of Virology, January 2000, p. 1029-1032, Vol. 74, No. 2
Infectious Diseases Laboratories, Institute
of Medical and Veterinary Science, Adelaide, South Australia 5000, Australia,1 and Max Planck Institute for
Immunobiology, D-79108 Freiburg, Germany2
Received 11 August 1999/Accepted 7 October 1999
Control of ganglionic herpes simplex virus (HSV) infection depends
on CD8+ cells but not on the death of infected neurons.
Primarily, perforin and granzyme B mediate CD8+ cell
cytotoxicity, whereas the in vivo functions of granzyme A, a third
granule protein, are unknown. Here, it is shown that granzyme A
restricts the interneuronal spread of HSV and significantly influences
ganglionic virus load.
CD8+ T cells and natural
killer (NK) cells are major components of the host response to
intracellular pathogens (1, 8, 26), including herpes simplex
virus (HSV) (19). After experimental inoculation of mouse
skin, HSV replicates in epidermal cells and concurrently invades the
peripheral nervous system (PNS), where primary sensory neurons are the
virus's main target (16, 27). Productive infection of
sensory neurons generates the potential for lethal spread of virus
throughout the nervous system, but, in immunocompetent hosts, viral
replication is usually rapidly terminated by timely development of an
adaptive immune response (19). After recovery from primary
infection, virus is not eradicated from the PNS; rather, viral DNA
persists in a latent form in a proportion of neuronal nuclei, creating
a reservoir of virus from which productive infection periodically
reactivates (3). It is becoming increasingly apparent that
the virus load in the PNS during the primary productive infection
directly influences the number of neurons that become latently infected
and the number of viral genomes that persist in each latently infected
cell (14). Factors that limit the spread of HSV in the PNS
are therefore likely to have a profound influence on the ability of the
virus to persist in the host and to reactivate. We showed previously that termination of HSV infection in the PNSs of experimentally infected mice depends on CD8+ cells but, paradoxically, not
on the death of infected neurons (20). CD8+
cells are generally assumed to kill their target cells by membrane disruption and DNA fragmentation, caused, respectively, by perforin and
granzyme B (Gzm B) proteins that are contained within the cytoplasmic
granules of cytotoxic lymphocytes (CTLs) (5, 7, 9, 10, 25).
A third protein, granzyme A (Gzm A), also found exclusively in the
granules of CTLs, is not directly cytolytic (4), and its
biological functions in vivo are not well defined. We reasoned that Gzm
A might contribute to noncytolytic termination of HSV infection in
spinal nerve ganglia, and this hypothesis was addressed by comparing
spinal ganglia of C57BL/6 Gzm A knockout mice (4) and
congenic immunocompetent animals with respect to virus clearance and
virus spread.
To determine the effect of Gzm A deficiency on virus load in the PNS,
several groups of 10 Gzm A knockout mice and congenic C57BL/6 controls
were inoculated with HSV-1 strain SC16; various days after inoculation,
virus in homogenized dorsal root ganglia was quantified by a standard
plaque assay (13). Congenic mice were bred at the Institute
of Medical and Veterinary Science from a breeding pair supplied by A. Müllbacher (Australian National University), and their genetic
authenticity was determined by PCR, as described previously
(4), with tail DNA. The zosteriform model used in these
experiments has been described in detail elsewhere (16, 18).
Briefly, a small patch of depilated skin on the left flank, innervated
by the 8th through the 10th dorsal root ganglia (T8-T10), was
scarified with a 27-gauge needle through a 10-µl drop of virus
suspension containing 5 × 105 PFU. At 4 and 5 days
after inoculation, virus levels were comparable in Gzm
A
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Granzyme A, a Noncytolytic Component of
CD8+ Cell Granules, Restricts the Spread of Herpes Simplex
Virus in the Peripheral Nervous Systems of Experimentally
Infected Mice
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
/ and congenic control mice. However, on days 6 and 7, approximately 10 times more virus was recovered from Gzm A-deficient
mice than from congenic controls (Fig.
1). Both groups of mice survived and
eventually cleared the infection. Similar outcomes were obtained in
three replicate experiments. The major implication of this finding is
that Gzm A either facilitates virus clearance or plays a crucial role
in restricting virus spread but is not critical for eventual clearance
of virus or survival. It is known that development of zosteriform
lesions reflects the spread of virus in the PNS (14);
significantly, in concomitantly infected groups of 10 mice, zosteriform
spread was accelerated in the absence of Gzm A (80% versus 40% of
animals on day 6). These data suggest that the major effect is on the
spread of virus.
View larger version (18K):
[in a new window]
FIG. 1.
Increased virus load in spinal ganglia of Gzm A knockout
mice compared with congenic control animals (B6) at 6 and 7 days after
flank inoculation with HSV-1 (P < 0.01). Vertical bars
indicate ranges of geometric mean titers.
To determine whether the increased virus load in Gzm A/
mice at 7 days after inoculation was associated with an elevated number of infected neurons, virus antigen-positive neuronal profiles were
immunohistochemically quantified (22) in ganglionic sections from Gzm A/ and congenic control mice. Ganglia
(T6-T13) ipsilateral to the inoculation site were fixed in
periodate-lysine-paraformaldehyde and processed as described previously
(22, 24). In concordance with previously reported
observations (24), virus antigen-positive neurons were
sparse by day 7 in immunocompetent mice. However, disappearance of
antigen-positive neurons was severely impaired by Gzm A deficiency (14 of 18,560 neuronal profiles were antigen positive in control mice
versus 959 of 25,856 in Gzm A/ mice [P < 0.0001] [Fig. 2], implying that
Gzm A plays a significant role in reducing interneuronal spread of HSV.
Alternative possibilities are that Gzm A contributes to the death of
infected neurons or to clearance of virus from the skin. However, the
former possibility is not supported by prior data showing that only a
minority of viral antigen-positive neurons are killed during
termination of ganglionic infection (20), and to date we
have found no evidence supporting the latter (data not shown).
|
Histological examination showed that the magnitude and distribution of
the inflammatory response to HSV infection were profoundly altered by
Gzm A deficiency. Seven days after infection, ganglia (T8-T13) were
pooled from C57BL/6 and Gzm A/ mice (five per group)
and paraffin embedded, and sections (5 µm thick) were stained with
hematoxylin and eosin. Tissue from control mice showed moderate
inflammatory infiltration and little edema; i.e., neurons remained
closely apposed to each other, as they would appear in uninfected
tissue (Fig. 3A). In contrast, Gzm A
deficiency was associated with profound edema (demonstrated by
increased distance between neuronal somas) and striking mononuclear cell infiltration, particularly under the ganglionic capsule (Fig. 3B,
arrow), probably indicative of increased viral load and/or tissue
damage. To our knowledge, mononuclear cell infiltration of this
magnitude in HSV-infected spinal ganglia has not been previously
reported. Despite the increased infiltration and edema in Gzm
A/ mice noted at the microscopic level, there was no
overt discernable macroscopic difference in the ganglia of either
group, owing to the inherent ganglionic swelling always associated with
HSV infection. To determine the phenotypes of the infiltrating
inflammatory cells, both in immunocompetent and Gzm A/
mice, ganglia were stained with an antibody to the cytoplasmic component of the CD3 epsilon chain (Dakopatts, Glostrup, Denmark), as
described previously (12). This experiment showed that, in both groups of mice, many of the infiltrating cells were
CD3+ and were therefore either T lymphocytes or NK T cells
(e.g., Gzm A/ mice [Fig.
4]).
|
|
The mechanism by which Gzm A might restrict the spread of HSV in the PNS is unknown. In this respect, we note that Gzm A is known to activate prourokinase (2), initiating a proteolytic cascade leading to the production of plasmin, a protease known to directly inactivate enveloped ectromelia virus (11). Preliminary data (not illustrated) suggest that plasmin might also inactivate extracellular HSV. Generation of plasmin in response to release of Gzm A from CD8+ cells might therefore represent a general noncytolytic mechanism for restricting extracellular virus spread, and further experiments with a wider range of concentrations of plasmin will be required to test this hypothesis.
Several other potential mechanisms of action of Gzm A merit discussion because, in vitro, purified Gzm A has a diverse range of properties. The possibility that Gzm A might directly inactivate HSV has not been formally excluded, though it is noteworthy that Gzm A has no direct antiviral activity against enveloped ectromelia virus (11). Importantly, T cells from Gzm A knockout mice are not impaired in the ability to lyse ectromelia virus-infected targets; nevertheless, control of ectromelia by these mice is impaired (11). Recent data suggest that Gzm A can play an auxiliary role in the induction of apoptosis (15), raising the possibility that the death of infected neurons also contributes to clearance of infectious virus from the PNS. However, prior data do not support this conclusion (20).
Gzm A has also been shown to process interleukin 1
precursor
molecules into active cytokines (6), which might in turn qualitatively enhance other antiviral host defenses. Other established properties of Gzm A include cleavage of extracellular matrix proteins (22) and sensitization of B cells to proliferate
(21), which could be envisioned to enhance local antibody
production. Of potential relevance to this point, we showed previously
that B-cell suppression increases virus load and virus spread in
ganglia, not skin, of HSV-infected mice (17). Finally, in
vivo Gzm A is believed to potentiate the activity of Gzm B
(23).
In conclusion, it is shown here, for the first time, that Gzm A deficiency is associated with impaired ability to restrict the spread of HSV between neurons, resulting in accelerated development of zosteriform lesions, increased inflammation and edema in sensory nerve ganglia, and increased virus load in the PNS. These data indicate that Gzm A, a noncytolytic serine protease of T-cell granules, plays a crucial role in limiting the spread of HSV in the nervous system, perhaps by limiting the number of infected neurons and hence virus load, at the peak of infection.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grants 96-0535 and 98-1300 from the National Health and Medical Research Council of Australia.
We thank Arno Müllbacher (JCSMR, Canberra, Australia) for helpful discussion and for providing breeding pairs of Gzm A knockout mice, and we thank Lidia Mathews for technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. Phone: 61 8 82223434. Fax: 61 8 82223543. E-mail: Rosemary.Pereira{at}imvs.sa.gov.au.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Blanden, R. V. 1974. T cell response to viral and bacterial infection. Transplant. Rev. 19:56-88[Medline]. |
| 2. | Brunner, G., M. Simon, and M. D. Kramer. 1990. Activation of pro-urokinase by the human T cell-associated serine proteinase HuTSP-1 FEBS Lett. 260:141-144[CrossRef][Medline]. |
| 3. |
Cook, M. L.,
V. B. Bastone, and J. G. Stevens.
1974.
Evidence that neurons harbor latent herpes simplex virus.
Infect. Immun.
9:946-951 |
| 4. | Ebnet, K., M. Hausmann, F. Lehmann-Grube, A. Müllbacher, M. Kopf, M. Lamers, and M. Simon. 1995. Granzyme A-deficient mice retain potent cell-mediated cytotoxicity. EMBO J. 14:4230-4239[Medline]. |
| 5. | Heusel, J. W., R. L. Wesselschmidt, S. Shresta, J. H. Russell, and T. J. Ley. 1994. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977-987[CrossRef][Medline]. |
| 6. |
Irmler, M.,
S. Hertig,
H. R. MacDonald,
R. Sadoul,
J. D. Becherer,
A. Proudfoot,
R. Solari, and J. Tschopp.
1995.
Granzyme A is an Interleukin 1-converting enzyme.
J. Exp. Med.
181:1917-1922 |
| 7. | Kagi, D., B. Ledermann, K. Bürki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, and H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin deficient mice. Nature (London) 369:31-37[CrossRef][Medline]. |
| 8. | Karupiah, G., B. E. H. Coupar, M. E. Andrew, D. B. Boyle, S. M. Phillips, A. Mullbacher, R. V. Blanden, and I. A. Ramshaw. 1990. Elevated natural killer cell responses in mice infected with recombinant vaccinia virus encoding murine IL-2. J. Immunol. 144:290-298[Abstract]. |
| 9. | Kojima, H., N. Shimohara, S. Hanaoka, Y. Someya-Shirota, Y. Takagaki, H. Ohnoa, T. Saito, T. Katayama, H. Yagita, K. Okomura, Y. Shiukai, F. W. Alt, A. Matsuzawa, S. Yonehara, and H. Takayama. 1994. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity 1:357-364[CrossRef][Medline]. |
| 10. | Lowin, B., M. Hahne, C. Mattman, and J. Tschopp. 1994. T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature (London) 370:650-652[CrossRef][Medline]. |
| 11. |
Müllbacher, A.,
K. Ebnet,
R. V. Blanden,
R. Tha Hla,
T. Stehle,
C. Museteanu, and M. Simon.
1996.
Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.
Proc. Natl. Acad. Sci. USA
93:5783-5787 |
| 12. |
Pereira, R. A., and A. Simmons.
1999.
Cell surface expression of H2 antigens on primary sensory neurons in response to acute but not latent herpes simplex virus infection in vivo.
J. Virol.
73:6484-6489 |
| 13. | Russell, W. C. 1962. A sensitive and precise assay for herpesvirus. Nature (London) 195:1028-1029[CrossRef][Medline]. |
| 14. | Sawtell, N. M. 1997. Comprehensive quantification of herpes simplex virus latency at the single-cell level. J. Virol. 71:5423-5431[Abstract]. |
| 15. | Shresta, S., T. A. Graubert, D. A. Thomas, S. Z. Raptis, and T. J. Ley. 1999. Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity 10:595-605[CrossRef][Medline]. |
| 16. |
Simmons, A., and A. A. Nash.
1984.
Zosteriform spread of herpes simplex virus as a model of recrudescence and its use to investigate the role of immune cells in prevention of recurrent disease.
J. Virol.
52:816-821 |
| 17. | Simmons, A., and A. A. Nash. 1987. Effect of B cell suppression on primary infection and reinfection of mice with herpes simplex virus. J. Infect. Dis. 155:649-654[Medline]. |
| 18. | Simmons, A., and A. B. La Vista. 1989. Neural infection in mice after cutaneous inoculation with HSV-1 is under complex host genetic control. Virus Res. 13:263-270[CrossRef][Medline]. |
| 19. | Simmons, A., D. C. Tscharke, and P. G. Speck. 1991. Role of immune mechanisms in control of HSV infection in the peripheral nervous system. Curr. Top. Microbiol. Immunol. 179:31-56. |
| 20. |
Simmons, A., and D. C. Tscharke.
1992.
Anti-CD8 impairs clearance of herpes simplex virus from the peripheral nervous system: implications for the fate of virally infected neurons.
J. Exp. Med.
175:1337-1344 |
| 21. | Simon, M. M., H. Hoschützky, U. Fruth, H. G. Simon, and M. D. Kramer. 1986. Purification and characterisation of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes. EMBO J. 5:3267-3274[Medline]. |
| 22. | Simon, M. M., H. G. Simon, U. Fruth, J. Epplen, H. K. Müller-Hermelink, and M. D. Kramer. 1987. Cloned cytolytic effector cells and their malignant variants produce an extracellular matrix degrading trypsin-like serine proteinase. Immunology 60:219-230[Medline]. |
| 23. |
Simon, M. M.,
M. Hausmann,
T. Tran,
K. Ebnet,
J. Tschopp,
R. Tha Hla, and A. Müllbacher.
1997.
In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A × B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells.
J. Exp. Med.
186:1781-1786 |
| 24. |
Speck, P. G., and A. Simmons.
1992.
Synchronous appearance of antigen positive and latently infected neurons in spinal ganglia of mice infected with a virulent strain of herpes simplex virus.
J. Gen. Virol.
73:1281-1285 |
| 25. |
Walsh, C. M.,
M. Matloubian,
C.-C. Liu,
R. Ueda,
C. G. Kurahara,
J. L. Christensen,
M. T. F. Huang,
J. D.-E. Young,
R. Ahmed, and W. R. Clark.
1994.
Immune function in mice lacking the perforin gene.
Proc. Natl. Acad. Sci. USA
91:10854-10858 |
| 26. | Welsh, R. M. 1981. Natural cell-mediated immunity during viral infections. Curr. Top. Microbiol. Immunol. 92:83-97[Medline]. |
| 27. | Wildy, P., H. J. Field, and A. A. Nash. 1992. Classical herpes latency revisited. Symposium 33, p. 133-167. SGM, Cambridge University Press, Cambridge, England. |
Journal of Virology, January 2000, p. 1029-1032, Vol. 74, No. 2
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Regner, M., Pavlinovic, L., Koskinen, A., Young, N., Trapani, J. A., Mullbacher, A.
(2009). Cutting Edge: Rapid and Efficient In Vivo Cytotoxicity by Cytotoxic T Cells Is Independent of Granzymes A and B. J. Immunol.
183: 37-40
[Abstract] [Full Text] -
Walch, M., Rampini, S. K., Stoeckli, I., Latinovic-Golic, S., Dumrese, C., Sundstrom, H., Vogetseder, A., Marino, J., Glauser, D. L., van den Broek, M., Sander, P., Groscurth, P., Ziegler, U.
(2009). Involvement of CD252 (CD134L) and IL-2 in the Expression of Cytotoxic Proteins in Bacterial- or Viral-Activated Human T Cells. J. Immunol.
182: 7569-7579
[Abstract] [Full Text] -
Kuhla, A., Eipel, C., Abshagen, K., Siebert, N., Menger, M. D., Vollmar, B.
(2009). Role of the perforin/granzyme cell death pathway in D-Gal/LPS-induced inflammatory liver injury. Am. J. Physiol. Gastrointest. Liver Physiol.
296: G1069-G1076
[Abstract] [Full Text] -
Knickelbein, J. E., Khanna, K. M., Yee, M. B., Baty, C. J., Kinchington, P. R., Hendricks, R. L.
(2008). Noncytotoxic Lytic Granule-Mediated CD8+ T Cell Inhibition of HSV-1 Reactivation from Neuronal Latency. Science
322: 268-271
[Abstract] [Full Text] -
Mintern, J. D., Guillonneau, C., Carbone, F. R., Doherty, P. C., Turner, S. J.
(2007). Cutting Edge: Tissue-Resident Memory CTL Down-Regulate Cytolytic Molecule Expression following Virus Clearance. J. Immunol.
179: 7220-7224
[Abstract] [Full Text] -
Pao, L. I., Sumaria, N., Kelly, J. M., Dommelen, S. v., Cretney, E., Wallace, M. E., Anthony, D. A., Uldrich, A. P., Godfrey, D. I., Papadimitriou, J. M., Mullbacher, A., Degli-Esposti, M. A., Smyth, M. J.
(2005). Functional Analysis of Granzyme M and Its Role in Immunity to Infection. J. Immunol.
175: 3235-3243
[Abstract] [Full Text] -
Lang, A., Nikolich-Zugich, J.
(2005). Development and Migration of Protective CD8+ T Cells into the Nervous System following Ocular Herpes Simplex Virus-1 Infection. J. Immunol.
174: 2919-2925
[Abstract] [Full Text] -
Rode, M., Balkow, S., Sobek, V., Brehm, R., Martin, P., Kersten, A., Dumrese, T., Stehle, T., Mullbacher, A., Wallich, R., Simon, M. M.
(2004). Perforin and Fas Act Together in the Induction of Apoptosis, and Both Are Critical in the Clearance of Lymphocytic Choriomeningitis Virus Infection. J. Virol.
78: 12395-12405
[Abstract] [Full Text] -
Loh, J., Thomas, D. A., Revell, P. A., Ley, T. J., Virgin, H. W. IV
(2004). Granzymes and Caspase 3 Play Important Roles in Control of Gammaherpesvirus Latency. J. Virol.
78: 12519-12528
[Abstract] [Full Text] -
Colmenares, M., Kima, P. E., Samoff, E., Soong, L., McMahon-Pratt, D.
(2003). Perforin and Gamma Interferon Are Critical CD8+ T-Cell-Mediated Responses in Vaccine-Induced Immunity against Leishmania amazonensis Infection. Infect. Immun.
71: 3172-3182
[Abstract] [Full Text] -
Koelle, D. M., Corey, L.
(2003). Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research. Clin. Microbiol. Rev.
16: 96-113
[Abstract] [Full Text] -
Jerome, K. R., Chen, Z., Lang, R., Torres, M. R., Hofmeister, J., Smith, S., Fox, R., Froelich, C. J., Corey, L.
(2001). HSV and Glycoprotein J Inhibit Caspase Activation and Apoptosis Induced by Granzyme B or Fas. J. Immunol.
167: 3928-3935
[Abstract] [Full Text] -
Koelle, D. M., Chen, H. B., Gavin, M. A., Wald, A., Kwok, W. W., Corey, L.
(2001). CD8 CTL from Genital Herpes Simplex Lesions: Recognition of Viral Tegument and Immediate Early Proteins and Lysis of Infected Cutaneous Cells. J. Immunol.
166: 4049-4058
[Abstract] [Full Text] -
Edelmann, K. H., Wilson, C. B.
(2001). Role of CD28/CD80-86 and CD40/CD154 Costimulatory Interactions in Host Defense to Primary Herpes Simplex Virus Infection. J. Virol.
75: 612-621
[Abstract] [Full Text] -
Koelle, D. M., Schomogyi, M., McClurkan, C., Reymond, S. N., Chen, H. B.
(2000). CD4 T-Cell Responses to Herpes Simplex Virus Type 2 Major Capsid Protein VP5: Comparison with Responses to Tegument and Envelope Glycoproteins. J. Virol.
74: 11422-11425
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»