Herpesviruses are ubiquitous viruses generally infecting humans early in life. The majority of humans has had a primary infection with one or more herpesviruses and harbour these viruses in a latent state for the rest of their lives. The initial infection is most often asymptomatic, but can be symptomatic depending on the herpesvirus in question and the age and immune status of the host. The viruses are phylogenetically old and humans and herpesviruses have evolved together 1. This co-evolution has created viruses which are well adapted to the human host and environment. Thus, herpesviruses are capable of coping with the human immune defence in a balanced manner generally without serious threads to the life of the host. Infection with a foreign herpesvirus, normally hosted by another species, does not always hold this balance, and the pathology is unpredictable. This is seen when humans are infected with the simian B virus, which often shows serious clinical outcome 2.

The human herpes simplex viruses were initially identified by Lowenstein, who passed it onto rabbits in 1919, and found it to be sensitive to alcohol and higher temperatures 3. The viruses were classified into two serologically different types by Schneweiss in 1962 4, and these are now known to belong to the subfamily of Alphaherpesvirinae together with varicella-zoster virus. These alphaherpesviruses all show neurotropic latency, and mucosal or skin lesions are frequently seen as a result of viral reactivation from sensory nerves. The two types of herpes simplex virus confer the genera Simplexvirus 1 and -2, which were formally designated by the International Committee on Taxonomy of Viruses as Human herpesvirus (HHV) 1 and 2 5.

Herpes simplex virus (HSV) type 1 and type 2 are very closely related, showing a homology at the DNA level of 83% in protein coding regions and less in noncoding regions 6. The genetic map of the two herpes simplex viruses is colinear 6, and the genomes are of approximately the same size, HSV-1 of 152 kbp 7 and HSV-2 of 155 kbp, and code for corresponding genes 6. The minor sequence variations give different cleavage sites for restriction endonucleases, which has been used intensively as an important epidemiological tool 8910.

Macrophages are ubiquitous cells of the mononuclear phagocyte system found throughout the body. Many attempts have been made to classify this range of cells with phagocytic activity. In 1892 Metchnikoff named them macrophages (large eaters) in contrast to microphages (the polymorphonuclear leukocytes)137, and in 1924 Aschoff defined the reticuloendothelial system by the criteria of uptake of vital dye 138. The macrophages are now more precisely defined as an important member of the mononuclear phagocyte system, defined in 1969 by van Furth and colleagues 139. In the tissues they constitute a dynamic pool of cells with many functional capabilities, among which the capacity of phagocytosis, microbial killing, motility, and adherence to surfaces are classic 139.

The macrophages originate from the bone marrow, where proliferating promonocytes give rise to monocytes which enter the blood stream 140. After a mean circulation time of approximately 11/2 day, the blood monocytes migrate to the tissues 140. In the tissues the monocytes differentiate into macrophages with characteristics determined by the environment of the tissue in question 141. The tissue macrophages in the major organs are represented by Kupffer cells in the liver, alveolar and interstitial lung macrophages, spleenic and sinusoidal lymph node macrophages, microglia in the brain, osteoclasts in bone, and Langerhans cells of the skin. Thus, macrophages are strategically situated all over the body taking care of debris from the organism itself and foreign material, among others invading microorganisms, including viruses 142143. Macrophages in different organs have different characteristics and functional capabilities and can not totally substitute one another in studies on macrophages 141144145146147. Likewise, macrophages from different species can possess differences in their functional capability, e.g. the capacity for nitric oxide (NO) production 148149.

Macrophages in tissues are, as described above, in part originating directly from monocytes, but they are also in part originating from local proliferation. This local proliferation in the tissues is performed by newly recruited monocytes, and in the steady state situation they only constitute a small fraction of the mononuclear phagocytes present 150. Of the monocytes produced in the bone marrow of mice and passing through the blood, approximately half are targeting the liver, 15 % are going to the lungs, 25 % to the spleen and 7 % to the peritoneal cavity 150151152. In the lungs, 70% of tissue macrophages in the steady-state originate from monocyte influx and 30% from local proliferation 153. This proportion might vary between different tissues, as the lifespan of tissue macrophages in different organs also varies from around 6 days in mouse spleen to approximately one month for alveolar macrophages 151152. In the skin, Langerhans cells are a very stable and long-lived population of cells staying there for at least 18 month in the steady-state situation. However, in inflammation the Langerhans cells are within 2 weeks replaced and supplemented by circulating mononuclear cells 154. When an inflammatory process is initiated, the dynamics of monocytes and macrophages are changed. Monocytes and other white blood cells are produced and recruited from the bone marrow, and the white blood cell count in the circulation is increased. The monocytes are mainly passing through the blood to become tissue macrophages, and the number of macrophages in the inflamed tissue can be increased by more than ten times 155. In inflamed tissue the local proliferation of macrophages does not seem to increase, although the number of newly recruited cells is high, indicating that the differentiation of monocytes in the tissues is accelerated 155.

The differentiation of monocytes and activation of macrophages have been a focus of interest for many years because of the observation that macrophage activation is crucial in the defence against many intracellular pathogens 156157158159. It became clear relatively early that lymphocytes and soluble factors secreted by these (lymphokines) are important in activation of macrophages for killing of intracellular bacteria, e.g. Listeria 160. In the killing of bacteria, interferon (IFN)-γ was shown to be an important stimulator of macrophage activation 161. As mechanisms in performance of the killing simple toxic substances of reactive oxygen species (ROS) and nitric oxide were identified and seem to conduct their action in synergy 162163164. The toxic substances are chemically simple, but their production and regulation in macrophages are very complex and still a matter of intense studies 149.

The state of the activated macrophage has changed conceptually from being viewed as one specific condition of the cell towards a more dynamic picture, provoked by the fact that macrophages activated by different means show different phenotypical characteristics 163165. The activated macrophage is now viewed as a cell with floating characteristics of many functional capacities regulated by a multitude of stimulating substances, such as the cytokine environment, hormones, and pathogenic and foreign substances 147166. Among variables, controlling macrophage activity in infected individuals, are the genetic constitutions of the host. The genetic background has been shown to be of importance for the regulation of both basic proliferation and function of macrophages and for the more specific antimicrobial responses 167168.

Soluble mediators of lymphocyte activities were described as early as 1953, but the first lymphokines/cytokines found and characterized were the type I interferons. Soon after, many other soluble mediators of lymphocyte and monocyte/macrophage activities were found 169170171. The term lymphokine was introduced by Dumonde et al. in 1969, to describe lymphocyte derived factors, and the term monokine was used as a description of factors coming from the mononuclear phagocyte system, both acting on many cells, primarily leukocytes 172. Because of a broader view on origin and function of these factors, the term cytokine is now more often used. Each cytokine was originally named according to biological activity in a functional assay, which often gave several different names to one cytokine, and thus confusion at the molecular level. To straighten this out, a numerical nomenclature of interleukins (between leukocytes) was introduced in 1979 173. This numbering system has clarified the field, but since it has no mnemonic functional anchorage it has drawn critique since then 174175176.

The cytokines are generally smaller proteins, some composed of two subunits, utilizing specific receptors on target cells for induction of their functional effects. They are structurally related in three families, with the prototypes being IL-1, IL-2 and IL-17 176. Functionally, cytokines are highly potent regulatory proteins acting in a paracrine or autocrine manner at picomolar concentrations 177. The cytokine receptors are also structurally clustered in families, and functionally utilize a battery of overlapping kinases and nuclear binding proteins in their signalling pathway and thus have overlapping functions 178. The final functional capacity of the effector cell thus reflects the cytokine environment experienced by the cell 177. Thus the cytokines comprise a network of factors inducing or inhibiting each others secretion and function in different cells, giving rise to a constantly floating landscape of a large array of functional capacities 177. In the early hours of a viral infection, the cytokines produced by cells infected or coming into contact with viral products are vital in conduction of the innate immune response to the infection 168179.

An important model used in the study of resistance mechanisms in defence against generalized infection with HSV is a mouse model, where mice infected intra-peritoneally or intra-venously experience a generalized infection with HSV replication in most organs, including the liver, spleen, and eventually the brain 284. The dissemination of infection to the brain and the severity of infection of the peripheral organs depend in part on the age of the mice, as is the case in humans, where neonates have difficulties in controlling a HSV infection 281285286287. The course of infection in mice also depends on the type of HSV in question. Furthermore, in 1975 Lopez described a differential susceptibility of inbred mice to generalized infection with HSV, and this genetic difference in sensitivity has since been used for analysis of resistance factors of importance for the anti-herpetic defence 288. In generalized infections, the genetics of the relative resistance to HSV-2 was shown to segregate with the X-chromosome 289. This pattern of resistance to the generalized infection was for both HSV-1 and -2 attributed to a genetically determined difference in the capacity for IFN-α/β production 179290291, and it was shown that the X-linked pattern of resistance segregated with the HSV-2-induced production of IFN-α/β in macrophages during the first hours of infection 168. Furthermore, macrophages from female mice respond to HSV with higher IFN-α/β production than macrophages from male mice 168. This observation is in line with female mice being more resistant to HSV infection in vivo 291.

Early production of IFN-α/β has been correlated to resistance of HSV infections in several other studies. Treatment of mice with antibodies to IFN-α/β increases and accelerates mortality of a generalized HSV-1 infection and with higher doses of virus, mice are dying already after three to four days, a period where antigen-specific mechanisms are still in the induction and proliferation phase 292. Furthermore, mice treated with mercuric chloride showed higher titres of HSV-2 in the first days of infection, an effect which could be correlated to impaired production of IFN-α/β 293294. In studies on peripheral HSV infections, such as cutaneous or corneal infections, IFN-α/β has been shown to be produced locally and to restrict the local replication of HSV and infection of nervous ganglia cells of the area, an effect which has also been correlated to the genetic constitution of the host 295296297298.

The genetic background for the X-linked trait of HSV resistance and IFN-α/β production of macrophages remains unravelled. Induction of IFN-α/β upon HSV infection seems to be governed by different mechanisms in different cells 299. IFN-α/β can be induced early by both infectious and UV-inactivated HSV in various cells, with the infectious virus being the more potent inducer in mouse peritoneal macrophages, whereas the UV-inactivated virus showed most potency in human peripheral blood mononuclear cells (PBMC) 168299300301302. Production of IFN-α/β was induced by gD of HSV-1 in PBMC, but not in murine macrophages 17303304. In PBMC-derived dendritic cells, however, the cellular mannose receptor was shown to be involved 299305. Furthermore, different Toll-like receptors (TLRs) have been shown to react with HSV 306. TLRs are transmembrane pattern recognition receptors (PRRs) that detect redundant microbial molecular motives and induce antiviral and proinflammatory cytokines in response to alerting signals. In dendritic cells, TLR9-signalling, induced by the GC-rich HSV genome, has been shown to govern the induction of IFN-α/β, but TLR9-KO mice are still capable of controlling HSV infections in vivo 307308309. However, in mouse macrophages the TLRs do not seem to be crucial for IFN-α/β induction upon HSV infection 304. This is in agreement with the observation that the majority of IFN-α/β produced by spleen cells and dendritic cells and the total production from bone marrow macrophages was independent of TLR9 or MyD88, which is necessary for signalling by most TLRs 308. In this study, heat inactivated virus was shown still to induce IFN-α/β in cells utilizing TLR9. As resident peritoneal macrophages do not produce IFN-α/β in response to even high doses of heat inactivated HSV, this gives an additional indication of independency from TLR9 of IFN-α/β production in macrophages 300. Moreover, efficient induction of IFN-α/β by HSV in macrophages required dsRNA-activated protein kinase (PKR) activity and infectivity of the virus 304. This is in agreement with the observation that dsRNA, which is produced by most viruses during replication, induces IFN through PKR, and not through TLR3, which also binds dsRNA 310. Furthermore, another mechanism of IFN induction by dsRNA through a RNA helicase has been proposed 311.

The different induction patterns in different cells types, and the fact that IFN-α/β seems largely to be induced by other mechanisms than TLRs, explain the fact that knocking out TLR-signalling by MyD88 did not influence the in vivo infection with HSV in mice 309. Other TLRs have also been shown to mediate signals in HSV infections. In HSV encephalitis in TLR2-KO mice, viral replication seemed unchanged or slightly increased during the first 4 days of infection, and the production of IL-6 and monocyte chemoattractant protein 1 were impaired, but interestingly pathological changes and mortality were reduced 134.

In relation to the X-linked resistance pattern of HSV infection and IFN production upon HSV infection, it is interesting that some of the TLRs are coded from the X-chromosome 312. These are the TLR7 and TLR8, which are triggered by guanosine- or uridine-rich ssRNA in the endosomal compartment of cells 313314. There are, however, no indications that this pathway is implicated in IFN induction in cells during HSV infection, but the question has still not been directly addressed.

Regulation of the IFN-α/β gene induction is in part governed by activation of the transcription factors IRF-3 and -7, which are induced by IFN-α/β itself, resulting in a positive feed back loop, an effect which has been known for years without knowledge of the signalling mechanisms 315316317. Thus, one possible explanation for the genetic differences in HSV-induced IFN-α/β production could be an elevated physiological level of this IFN self-stimulating system 318319320321. An analysis of the levels of IRF-3 and -7 in normal macrophages from these mice could be of interest. Analysis of the levels of the IFN-induced enzyme 2'-5'-oligoadenylate synthetase (OAS) in uninfected cells showed low but slightly higher levels in cells from relatively resistant mice 322. With LPS, cells from the relatively resistant (C57Bl/6) mice show an early induction pattern of IFN-α/β, peaking within 2 hours, whereas cells from the susceptible BALB/c mice demonstrate a delayed response, peaking 7 hours after induction 323.

Among other transcription factors involved in induction of the various IFN-α/β genes are the heterodimeric NF-κB family, which is activated by TLRs, IL-1R, and TNFR 324. During a HSV infection NF-κB is activated and translocated to the nucleus 325. Many regulatory mechanisms of NF-κB activation exist, one of them exerted through TNF, which is produced by macrophages very early during HSV infection (fig. 2) 293300325326. Thus, the responsible mechanisms might be exerted by other regulatory signals, influencing the magnitude of the HSV-triggered IFN-α/β induction pathway, and perhaps not by this pathway in itself 327.

A number of X-linked immunodeficiencies have been described, one of them being the Wiskott-Aldrich syndrome with defects in a protein expressed in haematopoietic cells, facilitating reorganization of the actin cytoskeleton, and thus influencing the mobility of immune cells and chemotaxis of macrophages. Patients with this X-linked immunodeficiency show aggravated herpetic infections, and cells from some patients seem to produce lower amounts of IFN in response to HSV 328329330. Cells from patients with another X-linked immunodeficiency with mutations in the CD40-ligand, a member of the TNF family, showed decreased IFN-α/β production when infected with HSV-1, but these patients apparently show a normal response to viral infections 331332. This supports the notion above that other regulatory signals might be involved.

The overall effects of the IFN-α/β system, besides the production as described above, are determined by the sensitivity of cells to the secreted IFN-α/β. The effector mechanisms of IFN-α/β on HSV replication are not fully elucidated. Several IFN-α/β-activated systems are involved, including the dsRNA-activated PKR, which phosphorylates, and thereby inhibits, the elongation initiation factor (eIF)-2α, resulting in inhibition of translation 333. Another important mediator of the antiviral activity is the OAS system, which activates 2'-5'oligoadenylate-dependent RNase L with the capacity to degrade single-stranded RNA 333. Lately, the PML bodies have been described as crucial for the anti-HSV effect of IFN-α/β 334.

In mice exhibiting a relatively HSV-resistant phenotype, the direct antiviral effect of IFN-α/β in embryonic cells was found to be approximately three-fold higher than in cells from susceptible mice 322. Data from another study showed comparable results on IFN-α/β sensitivity concerning the replication of encephalomyocarditis virus (EMCV) in cells from the same mouse strains 335. This phenomenon was inherited as a co-dominant autosomal trait without any apparent influence of X-linked genes 322336. Further studies in mouse fibroblasts have revealed that TNF intensify the antiviral effect of IFN-α/β and, thus, the in vivo situation seems more complicated (fig. 2) 300337. In the original publication on genetics of HSV susceptibility in inbred mice, Lopez reported fibroblasts from the different mice to replicate HSV equally, and the same was found in the cells showing differential sensitivity to IFN-α/β 288322. In line with these results, the IFN-activated OAS, an inhibitor of HSV replication, was induced to a higher degree in cells from the resistant mice upon IFN-α/β treatment 322333338. Furthermore, the level of stimulated and unstimulated OAS was generally found to vary between different inbred mouse strains 339. Thus, the genetic difference in antiviral action of type I IFNs seems to affect the replication of several different viruses and to correlate with resistance to HSV.

The viral host protein synthesis shutoff, exerted by the HSV vhs-protein of the tegument, has major effects on the cytokine production of infected cells and reduces the effect of IFN-α/β on HSV replication 340. Furthermore, the tegument proteins have been shown to induce cellular inhibitors of the JAK/STAT pathway, resulting in inhibition of both IFN signalling and production 341342. The IE protein ICP0 inhibits activation of IRF-3 and thereby also restricts IFN-induced pathways 717273, and ICP0, ICP4 and ICP27 induce late shutoff of protein synthesis with decreased mRNA stability and thus reduced cytokine production 81343. As outlined, it thus seems HSV has evolved several mechanisms to evade the consequences of the IFN-α/β system, which underline the importance of these cytokines in the antiviral defence.

During HSV infection macrophages are activated and possess an increased antiviral potential 281344. Classically, the macrophage antiviral activity has been described as intrinsic or extrinsic 345. Resting macrophages possess a high degree of intrinsic activity against HSV, generally being non-permissive to viral replication. The macrophages are thus a blind end for the HSV infection, and they can in that way protect other cells from infection, for example as a barrier lining the liver sinusoids 344. The extrinsic antiviral activity refers to the ability of macrophages to inactivate virus outside the macrophage itself or to inhibit viral replication in other cells 346. The intrinsic antiviral activity depends among other factors on macrophage differentiation and has been correlated to IFN activity, either physiological levels of "spontaneous" pre-infection-synthesized or rapidly acting autocrine IFN-α/β 344. In that respect, macrophages from mice of the resistant phenotype showed higher intrinsic activity by being less permissive to HSV replication 281347.

One potential antiviral mechanism of macrophages may be the production of ROS. These were originally assigned to bacterial killing, but the effect of ROS has also been correlated to antiviral functions, although they might not be of major importance 348. The ROS are mainly produced by NADPH-oxidases (Nox), which are membrane-bound multi-component enzymes primarily situated in the phagolysosome 349. Activation of the NAHPD-oxidase, by phosphorylation and fusion of the enzyme subunits, primarily results in production of superoxide anion (O₂^-), which by superoxide dismutase can be converted to hydrogen peroxide (H₂O₂). The H₂O₂ in turn is then by Fe²⁺ (Fenton reaction) or by Fe³⁺ and O₂^- (Haber-Weiss reaction) converted to hydroxyl radical (·OH), hydroxyl anion (OH^-) and singlet oxygen (¹O₂), or by the myeloperoxidase to hypochlorous acid (HOCl) 349350. Small amounts of ROS are also produced by the mitochondria and may be of importance as signalling molecules from TNF 351352.

During HSV infection in vivo, macrophages are activated and achieve an increased capacity to react with a respiratory burst of ROS when appropriately triggered, i.e. by phorbol esters (fig. 2) 353. This macrophage activation is induced early in response to HSV infection, reaching a plateau within the first 12 hours of i.p. infection 353. In vitro, macrophages were shown to be the cell type responding with an oxidative burst, and this capacity peaked after only 8 hours of infection with HSV 353. This HSV-induced capacity for an increased respiratory burst was shown to be governed by autocrine IFN-α/β as a sine qua non phenomenon 300353. Nevertheless, TNF was also found to influence the macrophage activation. By itself, TNF reduced the macrophage capacity for a respiratory burst, but in combination with IFN-α/β it synergistically enhanced the IFN-induced activation 293300. Interestingly, a secreted portion of the HSV-gG acts as a phagocyte chemoattractant and induces production of ROS by signalling through the receptor activated by the phorbol esters 354.

The HSV-induced activation of macrophages in vivo is influenced by the genetic constitution of the host, with the most pronounced activation of macrophages originating from resistant mice, as expected on the basis of the genetics of IFN-α/β production in response to the infection. Furthermore, the genetics of the efferent part of the IFN-α/β-mediated HSV-induced activation of macrophages, displayed a co-dominant autosomal trait, as was the case with the antiviral effect of IFN-α/β in fibroblasts 336. Thus, the genetically-determined sensitivity to IFN-α/β seems to be expressed in different cell-types. The influence of TNF on the genetics of this phenomenon has not been addressed. In Contrast to these observations, the genetics concerning the antiproliferative effect of IFN-α/β in bone marrow cells seems to be reversed 335355. This might, however, be linked, in that ROS are shown to activate various signalling molecules, mediate apoptosis, and exhibit antiproliferative effects depending on the dose and time of exposure 352.

Little is known on the potential antiviral effect of ROS. By examining peroxidized lipids, which is an oxidative product from ROS in tissues, it has been documented that these are produced during the acute HSV infection in vivo, and speculations on antiviral mechanisms have focused on induction of apoptosis 348356357. HSV triggers apoptosis of infected cells by several pathways, and the importance of this phenomenon is indicated by the fact that the virus has evolved mechanisms to counteract each of these pathways 358. Macrophages generally suppress apoptosis in HSV infections, as seen by increased apoptosis in macrophage-depleted mice 359.

Several studies on the mechanisms involved in the early battle against HSV, performed in in vivo animal models, have pointed to IFN-α/β as a crucial player. In adoptive transfer experiments, the effect of adult mouse spleen cells on the initial phase of a generalized HSV infection in suckling mice was conducted by IFN-α/β 360. Furthermore, administration of a hematopoetic growth factor to neonatal mice increases the number of dendritic cells, B cells and NK cells, and confers resistance in a cutaneous model of HSV infection. The effect in this model could also largely be attributed to the actions of IFN-α/β, with some additional contributions by IFN-γ 361362. In KO-mice IFN-α/β was able to control the initial phase of a generalized HSV infection without contributions from NK, T- or B cells, but these latter players were necessary for survival and long term control of the infection 363.

The importance of an early, local IFN-response in models including in vivo progression and evaluation of final outcome of infection is more unclear, in that many other viral and host factors are of importance in these more complicated models with several stages of infection and involvement of different organs. Such models are, however, more close to the normal human HSV infection, starting at an epithelial surface, but to expect that one resistance factor in such a complicated system will come out clear as the responsible factor for the outcome downstream the sequence of events, is too simplistic. Nevertheless, induced expression of IFN-α/β in the eye by plasmid DNA or an adenovirus vector was shown to inhibit early local replication of HSV and the concomitant spread of virus to the brain and death from encephalitis 333364, and in IFN-α/βR KO-mice HSV replicated to much higher titres than in normal mice 297.

This tells us that the innate and adaptive immune systems exhibit much redundancy, and that IFN-α/β is of vital importance in local inhibition of HSV replication. The multitude of antiviral mechanisms, be it innate or adaptive, have varying effects and importance in the different phases of infection, such as initial local infection, dissemination to other organs, establishment of latency and reactivation, and conclusions can not be drawn from one situation to another.

A few hours after the type I IFN and TNF response, macrophages react upon HSV infection with production of IL-12, which is seen from 8 to 12 hours after infection and on 238365. The same was found with other viruses 12 to 24 hours after infection 366. In these and other studies, the producers of IL-12p40 during viral infection seem to be inflammatory cells, including macrophages, and not the infected stromal cells 365367. The IL-12 induction during HSV infection requires infectious virus, and it was shown to be regulated at the transcriptional level 238, as it is also the case when it is induced by LPS 247367. The dependence on infectivity is, however, in conflict with results from in vivo production of IL-12p40 and IFN-γ in draining lymph nodes from sites injected with UV-inactivated HSV 302. High doses of UV-inactivated virus were used, and some minimal transcription of viral genes could have taken place, although the virus was not replication competent. Transcription of the IL-12p40 gene in macrophages requires de novo protein synthesis during the inducing HSV infection, which could explain the relatively late appearance of IL-12 production 238367. The κB-sequence of the IL-12p40 promoter binds NF-κB in HSV-infected cells, and the production of IL-12p40 was found to be repressed by an inhibitor of NF-κB activation 238. Both these observations indicate that signalling through NF-κB is of significance in HSV-induced IL-12 production.

In human macrophages, TNF has been shown to inhibit IL-12p40 production, but not p35 production, by a mechanism not involving NF-κB 368. Furthermore, IL-12 has in a mouse model been shown to stimulate TNF expression 255, indicating that TNF can participate in a negative feed-back loop in the regulation of the IL-12 system 369. Likewise, IFN-α/β has been shown to inhibit IL-12 production in both humans and mice 370371372. The implication of such inhibition by IFN-α/β and TNF, which are secreted very early in HSV infections, well before the production of IL-12, has so far not been elucidated.

As described earlier, the IL-12p40 induction is influenced by IFN-γ in a positive feed back loop. IFN-γ could activate IL-12 transcription through binding of IRF-1, -2, and -8 to an ISRE site in the promoter-region of IL-12 373374. Upon HSV infection, IFN-γ is produced as part of the non-specific response to the virus. A marked synergism between HSV and IFN-γ in IL-12 induction has been demonstrated 238, indicating that the IL-12 / IFN-γ auto-accelerating system is of importance during HSV infections.

The IFN-γ-inducing activity of the produced IL-12 is pronounced in mouse peritoneal cells after 24 hours of infection with HSV 238. In a study by Kirchner et al. IFN-γ was detected as early as on day 3 of in vivo HSV infection, and the IFN-γ production was correlated to the genetics of HSV resistance 375. During HSV infection, the production of IFN-γ is mainly induced as a concerted action of several factors and not by IL-12 alone. IFN-α/β by itself was shown to be a weak inducer of IFN-γ production by NK cells, but in synergy with IL-12 the production of IFN-γ was markedly enhanced 376. In elicited peritoneal macrophages, HSV induced efficient IFN-γ production through cooperation of IL-12, IFN-α/β and IL-18 377. In such a proinflammatory environment even other cells than NK and T cells, e.g. macrophages, might produce lower levels of IFN-γ 200202245. IL-12 signals through STAT4, but STAT4 translocation to the nucleus of NK cells has also been seen after IFN-α/β stimulation 206378. Likewise, IFN-α/β induces STAT4 phosphorylation in T cells 379, indicating that IL-12 and IFN-α/β at this point act through a shared signalling pathway. Furthermore, the synergistic action of IL-12 and IL-18 in IFN-γ production by macrophages was shown to be dependent on STAT4 197. In addition to these factors, TNF and IL-1 have also been shown to act in synergy with IL-12 in IFN-γ induction 206380381 and vice versa, IFN-γ has been shown to synergize with HSV in induction of TNF production 325. This further emphasizes the concept of positive feed-back mechanisms in the regulation of early IFN-γ production.

The important direct effect of IFN-α/β on HSV replication was found to be enhanced synergistically by IFN-γ in both cell culture and in vivo in mice 363382383384. This is, however, in conflict with an early study, which could not reveal any synergism between IFN-α/β and IFN-γ on the replication of HSV in human blood mononuclear cells 385. Synergistic action of the two types of IFN is further supported by the observation of synergism between IFN-γ and TNF on HSV replication in corneal cells, and the fact that this was exerted through production of IFN-β 386387388. The effect was, however, greatly dependent on the cell type examined, which could explain the above-mentioned inconsistency. Synergism between TNF and IFN-γ in inhibition of HSV replication has now been shown to be mediated by activation of a tryptophan-depleting enzyme 389. Thus, relatively small amounts of early IFN-γ produced by NK cells in response to IL-12, IFN-α/β, TNF, and IL-18 could in collaboration with the already present IFN-α/β and TNF have important local effect on HSV replication in permissive cells (fig. 3). This conclusion is further supported by observations in KO mice, indicating that collaborated action of IFN-α/β and IFN-γ is of importance in control of subcutaneous HSV infections 362.

In vivo studies on HSV infections in immunodeficient, KO, and antibody-treated mice have shown that the IL-12, -23 / IFN-γ system is able to control the infection, affecting both the survival rate and the HSV titres early in infection 390391. The effect of IL-12 in HSV infections seems to be conducted in synergy with IL-18 390, as it has also been shown for vaccinia virus 392. In HSV corneal infections in KO mice, IL-12 was shown to participate in the immune pathogenesis 393, but in another study utilizing IL-12 encoding plasmid DNA, corneal expression of IL-12 reduced the angiogenesis, and thus the pathology of the infection 394. However, both studies agreed that IL-12 does not affect the local titres of HSV in the eye. After a thermal injury, wide-spread HSV infections are an important risk, and treatment of injured mice with IL-12 combined with soluble IL-4R results in augmentation of the IFN-γ production and decreased viral replication and mortality 395.

In mice infected with murine cytomegalovirus (MCMV) production of IL-12-induced IFN-γ by NK cells has been demonstrated in vivo, and the system was further shown to lower the viral titres 396397. The IL-12 / IFN-γ system seems, however, not to be of importance in all viral infections, in that the latter study could not detect any production of early IL-12 or IFN-γ in a model of infection with the arenavirus lymphocytic choriomeningitis virus. Analyses of the IL-12, -23 / IFN-γ system in humans with genetic defects and in KO-mice reveal more redundancy in man than in mouse and indicate that the system is of more importance in DNA- than in RNA-virus infections 219.

The producers of early IFN-γ, the NK and NKT cells, and the cytokine IL-15 and the transcription factor T-bet, which are both crucial for the differentiation and function of these cells, have all been shown to be decisive for the early control of HSV infection in vivo 268398399400. Although NK cells but not IFN-γ was shown to be decisive for survival from ocular infections 401, such an effect of IFN-γ has been seen by others 402. Furthermore, a review of genetic functional NK cell defects found NK cells and their innate IFN-γ production to be of central importance in herpesvirus infections 403.

Overall, it can be concluded that the IL-12 / IFN-γ system is active in HSV infections and possesses an important antiviral potential, capable of controlling viral replication during the early phases of infection.

In macrophages exposed to IFN-γ, the enzyme inducible nitric oxide synthase is induced, which eventually results in production of NO from molecular oxygen and a guanidino nitrogen by conversion of L-arginine to L-citrulline 404. Upon HSV infection, the iNOS gene is induced, as shown by detection of iNOS-mRNA in infected mouse peritoneal cells and corneal neutrophils 405406. The production of NO in HSV-infected cultures of resting mouse peritoneal cells, which comprise a mixed population of macrophages, lymphocytes, NK cells etc., is dependent on the virus being infectious 405. This is in line with the requirement of infectious HSV for IL-12 production and thus for production of IFN-γ as described previously 238. NO could itself be involved in a positive feed-back, in that signalling of IL-12 utilizing Tyk2 requires the activity of NO 407. When exogenous IFN-γ is added to virus-infected cells, a marked synergism is seen. This synergistic effect of HSV on the IFN-γ-induced NO production in macrophages was shown to be mediated by autocrine secretion of TNF 325405. In line with this, mice with a targeted disruption of the TNF gene showed impaired resistance to HSV and increased viral replication within the first days of infection 408, and antibodies to TNF and an inhibitor of NO production impaired early control of HSV infection in peripheral nervous tissue 409.

The induction of iNOS and the following production of NO in response to IFN-γ and HSV is a relatively slow reaction, coming up after about 18 hours of infection 405. In in vivo vaginal HSV infections iNOS mRNA could be detected after 24 hours of infection 410. Thus, the production of this relatively toxic substance is part of the second wave of innate defence mechanisms. The retarded production of NO and the requirement for two or more signals for induction of iNOS are logic considering the toxicity of NO and the potentially harmful consequences for the host.

In HSV-infected macrophages exposed to IFN-γ, iNOS is induced synergistically though TNF-induced NF-κB activation and translocation to the nucleus, as shown by binding of a heterodimeric complex of p55/p65 and a homodimer of p55 to the κB-site of the iNOS promoter during infection 325. The crucial position of NF-κB in the induction of iNOS and production of NO is also indicated by experiments showing that antibodies to TNF inhibit activation of NF-κB and production of NO in HSV-infected cells and abolish the synergism between the virus and IFN-γ, an observation which was also seen with inhibitors of NF-κB activation 325. Further analysis of the signalling mechanism has revealed that the synergism upon HSV infection is influenced by physical interaction of IRF-1 and the NF-κB subunit p65 and controlled by the ISRE-site and the distal κB-site of the iNOS promoter (fig. 5) 411. A further support for this notion comes from the observation that the DNA-binding capacity of NF-κB and the nuclear translocation of IRF-1 have similar kinetics upon HSV infection 411 and the fact that IRF-1 is essential for iNOS induction 412. Induction of other genes such as IFN-β and vascular cell adhesion molecule 1 also involve physical interaction of IRF-1 and NF-κB 413, and both IRF-1 and IRF-2 have in other cells types been shown to form complexes with NF-κB 414415. Another potential mechanism in the synergistic induction of iNOS could involve complex formation of IFN consensus sequence-binding protein (ICSBP or IRF-8) and IRF-1, which is also important for high-output NO production but has still not been studied in HSV infections 416.

Thus, high-output NO production from activated macrophages is controlled by a "double-lock" signalling mechanism restricting the production of this antiviral toxic substance to sites of active viral replication, and sparing uninfected tissue from the detrimental effects (fig. 4).

The antiviral effects of NO have been documented in several viral infections, although there clearly exist viruses and conditions where NO does not exhibit major antiviral properties. NO is thus not a magic bullet against virus infections 417. In HSV infections, NO has been shown to confer a substantial part of the antiviral activity induced by IFN-γ in a macrophage cell line and to participate in the extrinsic anti-HSV effect of macrophages 418419420421. An exogenously added donor of NO has in several cell lines been shown to reduce the replication of HSV 422. In vivo, analysis of mice treated with an inhibitor of NO production showed higher titres of HSV in the lungs but increased survival rates due to reduced inflammation 135. Recently, a study using another inhibitor of NO production has confirmed the anti-herpetic effect of NO during a HSV respiratory infection, but in this study mice with inhibited NO production showed increased inflammatory responses, symptoms of infection, and mortality 423. Replication of HSV during vaginal infection was increased in the presence of an inhibitor of NO production, and this enhanced viral replication was most prominent during the first 24 hours of infection 410. In iNOS-KO mice, the herpes virus MCMV replicates to higher titres in various organs and in macrophages, and this results in impaired survival of the animals 424. Weanling mice with a targeted disruption of the iNOS gene showed increased HSV replication, but apparently without differences in HSV titres during the first days of infection 425, and in adult KO mice, we could not detect any significant effect of NO during the early days of a generalised HSV infection (Ellermann-Eriksen, unpublished results). Probably, these in vivo results are due to redundancy of the antiviral system. 426.

The final effects of NO on HSV infections therefore appear to be balanced between antiviral versus toxic effects, and the final outcome seems to depend on the timing, infectious dose, and tissues involved. Thus NO production in the early phases of HSV infection is one of the effector mechanisms of the innate immune response inhibiting HSV replication, but when overproduced, NO might itself result in pathology, as discussed in the following section.

As outlined above, positive feed-back mechanisms exist at the afferent side of the early cytokine response, involving especially the production of IFN-γ, IL-12, IFN-α/β and TNF, and synergisms at the efferent side, resulting in high-output NO production. As a result of coordinated induction of the iNOS gene by several transcription factors, activated by especially IFN-γ and TNF, a potent early antiviral system is activated. However, NO causes damage to DNA, proteins and lipids in cells and tissues and could thus be deleterious for the host 427428429430. A study in KO-mice indicates that NO can be responsible for inflammation and life-threatening symptoms to HSV infection of the lungs 135. This effect of NO on pulmonary symptoms is also observed in influenza virus infections 431, although NO inhibits replication of both influenza virus and severe acute respiratory syndrome coronavirus 432433. Consequently, when this system is activated, it has to be controlled and eventually closed down, as it would otherwise induce unnecessary harm to the host. Such negative regulations of the iNOS gene induction in IFN-γ activated macrophages is conducted by IL-4 and IL-13 272273434. Furthermore, TGF-β can exhibit down-regulation of NO production through several post-transcriptional regulatory mechanisms, but the contribution of these pathways have not been analysed in HSV infections 435436. IL-4 production during HSV infection has in vaginal and CNS infections been demonstrated on day 2 of infection and to increase for the next days 437438. In peritoneal cells from mice infected i.p. production of IL-4 could be detected at day 5 of infection 439.

At low IFN-γ concentrations, IL-4 has been shown to inhibit iNOS induction through STAT6 competition with STAT1 binding to the GAS element of the IRF-1 promoter region. This results in reduced expression of the transcription factor IRF-1, which is crucial for induction of iNOS 440. Generally, STAT6 was shown to be a key factor in IL-4- and IL-13-induced inhibition of iNOS gene transcription induced by IFN-γ (fig. 5) 441.

At higher IFN-γ concentrations, activated STAT6 is no longer able to compete with the high amounts of activated STAT1 dimer 440. However, in this situation IL-4 is still able to inhibit the production of NO from IFN-γ-stimulated macrophages 272273434. In the presence of high levels of IFN-γ, IL-4 is not able to alter the induction of IRF-1, but the production of IRF-2 is increased 434. The human promoter region of IRF-2 contains a SBE, and the induction of IRF-2 could thus potentially be mediated by STAT6 binding to this element 442. This is in agreement with the fact that IRF-2 is known to compete with the binding of IRF-1 to ISRE sites and to antagonize the trans-activating activity of IRF-1 in the regulation of other IFN-induced genes 443444445. Inhibition of iNOS expression by high concentrations of IRF-2 relative to IRF-1 has thus been proposed as a controlling mechanism in situations with high levels of IFN-γ (fig. 5) 434. Furthermore, another mechanism could evolve from the observation that IL-4 signalling can result in disruption of the complex formation of ICSBP and IRF-1 and thereby inhibit iNOS induction 416. Other mediators of IL-4-induced repression of iNOS induction might exist, in that another DNA-binding transcriptional repressor competing with IRF-1 has been described 446.

In IFN-γ activated macrophages the IL-4- and IL-13-induced inhibition of iNOS induction can thus be overruled by HSV infection, leading to a sustained NO production (fig. 4) 439447. This effect of HSV infection is mediated through TNF production and NF-κB activation 439447. However, pre-treatment with IL-4 has in a Theiler's murine encephalomyelitis virus model showed inhibition of NF-κB activation 448. In thioglycollate-induced peritoneal cells, LPS and TNF could only overcome the inhibiting effect of IL-4 in situations, where IL-4 was added simultaneously or after the stimulators 272, a sequence of events which, however, is in agreement with the sequence of cytokine production in HSV infections. When activated, the NF-κB p65 physically interacts with IRF-1 and trans-activate iNOS transcription in HSV-infected and TNF-treated cells 411449. It is thus tempting to speculate that the NF-κB-IRF-1 complex has higher affinity for the combined DNA-binding site and thus is able to obstruct the binding of IRF-2 to the ISRE site of the iNOS promoter and in that way turn the competition towards transcriptional activity (fig. 5) 411. This will block the inhibiting effect of IL-4 in foci of HSV replication and open up for NO production at sites where the antiviral effect is of more importance than the potential toxicity.