J Gen Virol Try IJSEM Online
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Gen Virol 84 (2003), 2497-2500; DOI 10.1099/vir.0.19251-0

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Malmgaard, L.
Right arrow Articles by Paludan, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Malmgaard, L.
Right arrow Articles by Paludan, S. R.
Agricola
Right arrow Articles by Malmgaard, L.
Right arrow Articles by Paludan, S. R.
© 2003 Society for General Microbiology

Short Communication

Interferon (IFN)-{alpha}/{beta}, interleukin (IL)-12 and IL-18 coordinately induce production of IFN-{gamma} during infection with herpes simplex virus type 2

Lene Malmgaard and Søren R. Paludan

Department of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark

Correspondence
Søren Paludan
srp{at}microbiology.au.dk


   ABSTRACT
TOP
 ABSTRACT
MAIN TEXT
 REFERENCES
 
Interferons (IFNs) are important mediators of antiviral activity. In this study we have investigated how production of IFN-{gamma} is induced during herpes simplex virus type 2 (HSV-2) infection in murine peritoneal cells (PCs). We found that HSV-2 infection of thioglycolate-activated PCs from BALB/c mice rapidly led to expression of type 1 IFNs (IFN-{alpha}/{beta}) and interleukin (IL)-12, which was followed by production of IFN-{gamma}. IL-12 alone induced the most expression of IFN-{gamma}, which was augmented by cotreatment with IFN-{alpha} or IL-18, or combinations of IFN-{alpha} and IL-18. Moreover, neutralization of any of these cytokines in vitro strongly reduced the production of IFN-{gamma}, and neutralization of all three cytokines totally prevented HSV-2-induced IFN-{gamma} expression. Our data suggest that IFN-{gamma} production is induced during HSV-2 infection through the coordinated action of IFN-{alpha}/{beta}, IL-12 and IL-18.


   MAIN TEXT
TOP
 ABSTRACT
 MAIN TEXT
REFERENCES
 
Herpes simplex virus (HSV) is a human pathogenic virus that infects through mucocutaneous membranes and is able to replicate in most cell types (Whitley, 2001Down). After a primary HSV infection, the virus establishes latent infections in neurons, from where it can be reactivated in response to various forms of stress. HSV infections can give rise to gingivistomatitis, cold sores and encephalitis (HSV-1), as well as genital and neonatal herpes (HSV-2).

The host reaction to infection is a highly regulated response aimed at eliminating the infectious agent from the organism. Cytokines are soluble secreted factors that play important roles in regulation of the immune response. With respect to virus infections, the interferons (IFNs), a special subset of cytokines, have been ascribed particularly important roles (Muller et al., 1994Down; Mikloska & Cunningham, 2001Down; Salazar-Mather et al., 2002Down). IFN-{alpha}/{beta}, or type I IFNs, are produced rapidly after infection (Ellermann-Eriksen, 1993Down; Malmgaard et al., 2002Down) and form part of the very first line of defence (Stark et al., 1998Down). IFN-{gamma} is produced both in the early stages of infection by natural killer cells and at later stages by activated T cells (Boehm et al., 1997Down). IFN-{gamma} is a strong activator of macrophages (Boehm et al., 1997Down; Paludan et al., 1998Down; Malmgaard et al., 2000Down) and is also involved in driving the development of CD4+ T cells towards a Th1 response (Boehm et al., 1997Down). Not surprisingly, IFN-{gamma} is crucial for elimination of many virus infections, including HSV infections (Kodukula et al., 1999Down; Harandi et al., 2001Down; Mikloska & Cunningham, 2001Down). In this work, we have investigated how expression of IFN-{gamma} is regulated during the early stages of HSV infection in a peritoneal cell culture system containing a mixture of leukocytes.

In order to examine how IFN-{gamma} production is regulated during HSV-2 infection, we first examined the expression of IFN-{alpha}/{beta}, interleukin (IL)-12 and IL-18, all cytokines reported to play a role in the regulation of IFN-{gamma} production (Orange & Biron, 1996Down; Nakamura et al., 1989Down; Pien et al., 2000Down; Nguyen et al., 2002Down). Thioglycolate-elicited peritoneal cells (PCs) were harvested from the peritoneal cavity of 8-week-old female BALB/c mice and cultured in RPMI 1640 medium supplemented with 5 % foetal calf serum (Bio-Whittaker). The following day, the cells were infected with the MS strain of HSV-2 (3x106 p.f.u. ml-1) and incubated for different intervals up to 24 h at 37 °C. Supernatants were harvested and cytokine levels measured by ELISA (IL-12, IL-18 and IFN-{gamma}) or bioassay (IFN-{alpha}/{beta}). As seen in Fig. 1Down(a), IFN-{alpha}/{beta} production was induced within 4 h of infection. IL-12p40 was also induced by HSV-2 infection with a significant increase observed after 8 h of infection (Fig. 1bDown). For both cytokines, the levels increased continuously throughout the 24 h of the experiment. IL-18 was produced constitutively in amounts well above the detection limit (Fig. 1cDown) and HSV-2 infection did not affect the production of IL-18 in PCs. This finding, however, does not exclude enhanced IL-18 signalling following HSV-2 infection, since we have previously reported that expression of both the IL-18 receptor and the pro-IL-18-cleaving enzyme caspase-1 are induced by HSV (Paludan et al., 2002Down). In order to correlate the induction of IFN-{gamma} with expression of the cytokines shown in Fig. 1(a–c), we also examined the kinetics of expression of IFN-{gamma}. Interestingly, accumulation of IFN-{gamma} was detectable after 12 h of HSV-2 infection (Fig. 1dDown) and hence occurred with delayed kinetics compared with IFN-{alpha}/{beta} and IL-12. These findings therefore indicate a possible role for these cytokines in HSV-2-induced IFN-{gamma} expression.



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1. Expression of cytokines during HSV-2 infection in vitro. Thioglycolate-elicited PCs were harvested from BALB/c mice and left overnight in culture to settle. The cells were infected with HSV-2 (3x106 p.f.u. ml-1; m.o.i. 1·7) (closed circles) or left untreated (open triangles). Supernatants were harvested after the indicated periods of infection for measurement of IFN-{alpha}/{beta} (a), IL-12p40 (b), IL-18 (c) and IFN-{gamma} (d). The results are shown as the mean of measurements from triplicate cultures±SEM. Similar results were seen in three independent experiments.

 
In the next set of experiments, we wanted to examine whether type I IFNs, IL-12 and IL-18 did in fact stimulate production of IFN-{gamma} in the cell population used. Elicited PCs were harvested, cultured in RPMI and treated with recombinant cytokines. As seen in Fig. 2Down, none of the cytokines alone was a potent inducer of IFN-{gamma} expression, with only IL-12 stimulating detectable IFN-{gamma} production. The ability of IL-12 to promote IFN-{gamma} production was strongly augmented by IL-18 and modestly by IFN-{alpha}. Stimulating the cells concomitantly with IFN-{alpha} and IL-18 only marginally affected IFN-{gamma} expression. When the cells received all three stimuli, we observed high levels of IFN-{gamma}, although these were lower than in response to a combination of IL-12 and IL-18 alone. These findings thus show that IFN-{alpha}, IL-12 and IL-18 do induce expression of IFN-{gamma} in thioglycolate-activated PCs from BALB/c mice, and also confirm previous reports in the literature that IFN-{alpha}/{beta} can both promote and inhibit production of IFN-{gamma}, depending on the context in which it acts (Cousens et al., 1997Down; Nguyen et al., 2002Down).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2. Induction of IFN-{gamma} production by recombinant cytokines. PCs harvested from BALB/c mice were treated in vitro for 24 h with recombinant cytokines at the following concentrations: 1 ng IL-12p70 ml-1, 1 ng IL-18 ml-1 and 2x103 U IFN-{gamma} ml-1. Supernatants were harvested and IFN-{gamma} levels were measured by ELISA. The results are shown as the mean of measurements from triplicate cultures±SEM. Similar results were obtained in three independent experiments.

 
In order to investigate whether IFN-{alpha}/{beta}, IL-12 and IL-18 play a role in the induction of IFN-{gamma} production during HSV-2 infection, we infected PCs with virus and cotreated with either neutralizing antibodies against IL-12p40, IL-18 or IFN-{alpha}/{beta}, or with control antibodies. Depletion of the culture medium for functional IL-12p40 or IFN-{alpha}/{beta} reduced the production of IFN-{gamma} by 85–90 % (Fig. 3Down). Elimination of IL-18 also reduced HSV-2-induced IFN-{gamma} production, although not to the same extent. By using combinations of neutralizing antibodies, we found that the absence of IL-12 and any or both of the two other cytokines totally inhibited virus-induced expression of IFN-{gamma}. When IL-18 and IFN-{alpha}/{beta} were neutralized, IFN-{gamma} production was inhibited by between 85 and 95 %. Taken together, these results show that IL-12, IL-18 and IFN-{alpha}/{beta} all play a role in HSV-induced IFN-{gamma} production and also suggest that these three cytokines define the major IFN-{gamma}-inducing cytokines during the early phase of infection, since no residual IFN-{gamma} production was detected when all three were neutralized. It should be noted that we cannot formally rule out an involvement of IL-23, since the p40 subunit of IL-12 is also part of IL-23, which is known to promote IFN-{gamma} expression (Lankford & Frucht, 2003Down). However, this cytokine primarily targets memory T cells and hence is unlikely to play any major role in the early IFN-{gamma} response to virus infections.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3. Effects of cytokine-neutralizing antibodies on IFN-{gamma} production in HSV-2-infected BALB/c PCs. Cells were infected with HSV-2 (3x106 p.f.u. ml-1; m.o.i. 1·7) and treated with neutralizing antibodies (left panel) or isotype controls (right panel) in the indicated combinations. The antibodies were used at the following concentrations: rat monoclonal anti-IL-12p40 (BD-Pharmingen, clone C17.8) at 1 µg ml-1; rat monoclonal anti-IL-18 (MBL, clone 93-10C) at 1 µg ml-1; sheep polyclonal anti-IFN-{alpha}/{beta} (PBL) at 0·2 µg ml-1; rat control IgG2a (BD-Pharmingen, clone R35-95) at 1 µg ml-1; rat control IgG1 (BD-Pharmingen, clone R3.34) at 1 µg ml-1; normal sheep serum at 0·2 µg ml-1. Supernatants were harvested after 24 h and IFN-{gamma} levels were measured by ELISA. The results are shown as the mean of measurements from triplicate cultures±SEM. Similar results were seen in three independent experiments.

 
In this study we have examined how the production of IFN-{gamma} is regulated during the early stages of HSV-2 infection. We found that a complex cytokine network is involved, with all of the cytokines IFN-{alpha}/{beta}, IL-12 and IL-18 playing non-redundant roles. IL-12 and IL-18 have long been considered to be the prime inducers of IFN-{gamma} (Okamura et al., 1998Down). However, more recent work has unveiled a more complex picture, with many other cytokines also being able to stimulate IFN-{gamma} production (Carson et al., 1995Down; Oppmann et al., 2000Down; Nguyen et al., 2002Down; Pflanz et al., 2002Down). The present findings by us and previous reports by others (Sareneva et al., 1998Down; Nguyen et al., 2002Down; Pien et al., 2002Down) suggest that IFN-{alpha}/{beta} is a central regulator of IFN-{gamma} production in the context of virus infections. The finding that IFN-{alpha}/{beta} is involved in regulation of IFN-{gamma} production sheds new light on the function of type I IFNs, which appear to be important not only for the initial antiviral response, but also for shaping of the subsequent immune response.


   ACKNOWLEDGEMENTS
 
The authors are thankful to Birthe Søby and Elin Jacobsen for their skilful technical assistance. L. M. was supported by a fellowship from The Faculty of Health Sciences, University of Aarhus. This work was supported by research grants from The Danish Medical Research Council (grant no. 22-01-0379), Leo Pharma Research Foundation and Fonden til Lægevidenskabens Fremme.


   REFERENCES
TOP
 ABSTRACT
 MAIN TEXT
 REFERENCES
 
Boehm, U., Klamp, T., Groot, M. & Howard, J. C. (1997). Cellular responses to interferon-{gamma}. Annu Rev Immunol 15, 749–795.[CrossRef][Medline]

Carson, W. E., Ross, M. E., Baiocchi, R. A., Marien, M. J., Boiani, N., Grabstein, K. & Caligiuri, M. A. (1995). Endogenous production of interleukin 15 by activated human monocytes is critical for optimal production of interferon-gamma by natural killer cells in vitro. J Clin Invest 96, 2578–2582.

Cousens, L. P., Orange, J. S., Su, H. C. & Biron, C. A. (1997). Interferon-{alpha}/{beta} inhibition of interleukin 12 and interferon-{gamma} production in vitro and endogenously during viral infection. Proc Natl Acad Sci U S A 94, 634–639.[Abstract/Free Full Text]

Ellermann-Eriksen, S. (1993). Autocrine secretion of interferon-alpha/beta and tumour necrosis factor-alpha synergistically activates mouse macrophages after infection with herpes simplex virus type 2. J Gen Virol 74, 2191–2199.[Abstract/Free Full Text]

Harandi, A. M., Svennerholm, B., Holmgren, J. & Eriksson, K. (2001). Interleukin-12 (IL-12) and IL-18 are important in innate defense against genital herpes simplex virus type 2 infection in mice but are not required for the development of acquired gamma interferon-mediated protective immunity. J Virol 75, 6705–6709.[Abstract/Free Full Text]

Kodukula, P., Liu, T., van Rooijen, N., Jager, M. J. & Hendricks, R. L. (1999). Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system. J Immunol 162, 2895–2905.[Abstract/Free Full Text]

Lankford, C. S. R. & Frught, D. M. (2003). A unique role for IL-23 in promoting cellular immunity. J Leukoc Biol 73, 49–56.[Abstract/Free Full Text]

Malmgaard, L., Paludan, S. R., Mogensen, S. C. & Ellermann-Eriksen, S. (2000). Herpes simplex virus type 2 induces interleukin-12 in macrophages through a mechanism involving NF-{kappa}B. J Gen Virol 81, 3011–3020.[Abstract/Free Full Text]

Malmgaard, L., Salazar-Mather, T. P., Lewis, C. A. & Biron, C. A. (2002). Promotion of alpha/beta interferon induction during in vivo viral infection through alpha/beta interferon receptor/STAT1 system-dependent and -independent pathways. J Virol 76, 4520–4525.[Abstract/Free Full Text]

Mikloska, Z. & Cunningham, A. L. (2001). Alpha and gamma interferons inhibit herpes simplex virus type 1 infection and spread in epidermal cells after axonal transmission. J Virol 75, 11821–11826.[Abstract/Free Full Text]

Muller, U., Steinhoff, U., Reis, L. F., Hemmi, S., Pavlovic, J., Zinkernagel, R. M. & Aguet, M. (1994). Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921.[Abstract/Free Full Text]

Nakamura, K., Okamura, H., Wada, M., Nagata, K. & Tamura, T. (1989). Endotoxin-induced serum factor that stimulates gamma interferon production. Infect Immun 57, 590–595.[Abstract/Free Full Text]

Nguyen, K. B., Watford, W. T., Salomon, R., Hofmann, S. R., Pien, G. C., Morinobu, A., Gadina, M., O'Shea, J. J. & Biron, C. A. (2002). Critical role for STAT4 activation by type 1 interferons in the interferon-{gamma} response to viral infection. Science 297, 2063–2066.[Abstract/Free Full Text]

Okamura, H., Kashiwamura, S., Tsutsui, H., Yoshimoto, T. & Nakanishi, K. (1998). Regulation of interferon-gamma production by IL-12 and IL-18. Curr Opin Immunol 10, 259–264.[CrossRef][Medline]

Oppmann, B., Lesley, R., Blom, B. & 22 other authors (2000). Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715–725.[CrossRef][Medline]

Orange, J. S. & Biron, C. A. (1996). Characterization of early IL-12, IFN-alpha/beta and TNF effects on antiviral state and NK cell responses during murine cytomegalovirus infection. J Immunol 156, 4746–4756.[Abstract]

Paludan, S. R., Ellermann-Eriksen, S. & Mogensen, S. C. (1998). NF-kappaB activation is responsible for the synergistic effect of herpes simplex virus type 2 infection on interferon-gamma-induced nitric oxide production. J Gen Virol 79, 2785–2793.[Abstract]

Paludan, S. R., Melchjorsen, J., Malmgaard, L. & Mogensen, S. C. (2002). Expression of genes for cytokines and cytokine-related functions in leukocytes infected with herpes simplex virus: comparison between resistant and susceptible mouse strains. Eur Cytokine Netw 13, 306–316.[Medline]

Pflanz, S., Timans, J. C., Cheung, J. & 18 other authors (2002). IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+ T cells. Immunity 16, 779–790.[CrossRef][Medline]

Pien, G. C., Satoskar, A. R., Takeda, K., Akira, S. & Biron, C. A. (2000). Selective IL-18 requirements for induction of compartmental IFN-{gamma} responses during viral infection. J Immunol 165, 4787–4791.[Abstract/Free Full Text]

Pien, G. C., Nguyen, K. B., Malmgaard, L., Satoskar, A. R. & Biron, C. A. (2002). A unique mechanism for innate cytokine promotion of T cell responses to viral infections. J Immunol 169, 5827–5837.[Abstract/Free Full Text]

Salazar-Mather, T. P., Lewis, C. A. & Biron, C. A. (2002). Type I interferons regulate inflammatory cell trafficking and macrophage inflammatory protein 1{alpha} delivery to the liver. J Clin Invest 110, 321–330.[CrossRef][Medline]

Sareneva, T., Matikainen, S., Kurimoto, M. & Julkunen, I. (1998). Influenza A virus-induced IFN-{alpha}/{beta} and IL-18 synergistically enhance IFN-{gamma} gene expression in human T cells. J Immunol 160, 6032–6038.[Abstract/Free Full Text]

Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. & Schreiber, R. D. (1998). How cells respond to interferons. Annu Rev Biochem 67, 227–264.[CrossRef][Medline]

Whitley, R. J. (2001). Herpes simplex virus. In Fields Virology, pp. 2461–2509. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.

Received 24 March 2003; accepted 13 May 2003.



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Malmgaard, L.
Right arrow Articles by Paludan, S. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Malmgaard, L.
Right arrow Articles by Paludan, S. R.
Agricola
Right arrow Articles by Malmgaard, L.
Right arrow Articles by Paludan, S. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS