Role of interferon in lymphocyte recruitment into the skin

Large numbers of lymphocytes are recruited from the blood into sites of cutaneous DTH reactions. Our goal was to investigate the factors controlling this recruitment. 111In-labeled peritoneal exudate lymphocytes were injected iv and the accumulation of these cells in skin sites injected with a variety of stimuli, was used to measure lymphocyte recruitment in rats. Large numbers of lymphocytes migrated into vaccinia- and KLH-injected sites in sensitized animals, but only into the viral and not the KLH lesions in non-immune animals. Lymphocytes also migrated efficiently into sites injected with the alpha-interferon (IFN) inducers, uv-inactivated vaccinia virus and poly I:C, as well as into sites injected with IFN. In each case there was a dose-response relationship. Analysis of the kinetics of lymphocyte recruitment demonstrated that the peak rate of migration occurred most rapidly after the injection of IFN, later after poly I:C, and was slowest to be reached after vaccinia virus. Rabbit anti-IFN blocked the recruitment of lymphocytes by uv-inactivated vaccinia and by IFN. Histologically, all of these sites demonstrated a dense mononuclear cell infiltrate in the dermis. It is suggested that IFN may be an important mediator in the recruitment of lymphocytes into inflammatory reactions.     

Specific and nonspecific T-cell recruitment in viral meningitis: Possible implications for autoimmunity

Specific and nonspecific T-cell invasion into cerebrospinal fluid has been investigated in the nonfatal viral meningoencephalitis induced by intracerebral inoculation of mice with vaccinia virus. At the peak of the inflammatory process on Day 7 approximately 5 to 10% of the Lyt 2⁺ T cells present are apparently specific for vaccinia virus. Concurrently, in mice primed previously with influenza virus, 0.5 to 1.0% of the appropriate T-cell set located in cerebrospinal fluid is reactive to influenza-infected target cells. This vaccinia virus-induced inflammatory exudate may thus contain as many as 500 influenza-immune memory T cells. These findings are discussed from the aspect that such nonspecific T-cell invasion into the central nervous system during aseptic viral meningitis could result in exposure of potentially brain-reactive T cells to central nervous system components.     

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.     

Polyinosinic-polycytidylic acid treatment of Friend retrovirus-infected mice improves functional properties of virus-specific T cells and prevents virus-induced disease

The induction of type I IFN is the most immediate host response to viral infections. Type I IFN has a direct antiviral activity mediated by antiviral enzymes, but it also modulates the function of cells of the adaptive immune system. Many viruses can suppress type I IFN production, and in retroviral infections, the initial type I IFN is weak. Thus, one strategy of immunotherapy in viral infection is the exogenous induction of type I IFN during acute viral infection by TLR ligands. Along these lines, the TLR3/MDA5 ligand polyinosinic-polycytidylic acid [poly(I:C)] has already been used to treat viral infections. However, the immunological mechanisms underlying this successful therapy have not been defined until now. In this study, the Friend retrovirus (FV) mouse model was used to investigate the mode of action of poly(I:C) in antiretroviral immunotherapy. Postexposure, poly(I:C) treatment of FV-infected mice resulted in a significant reduction in viral loads and protection from virus-induced leukemia. This effect was IFN dependent because type I IFN receptor-deficient mice could not be protected by poly(I:C). The poly(I:C)-induced IFN response resulted in the expression of antiviral enzymes, which suppressed FV replication. Also, the virus-specific T cell response was augmented. Interestingly, it did not enhance the number of virus-specific CD4(+) and CD8(+) T cells, but rather the functional properties of these cells, such as cytokine production and cytotoxic activity. The results demonstrate a direct antiviral and immunomodulatory effect of poly(I:C) and, therefore, suggests its potential for clinical treatment of retroviral infections.     

Immunobiology of infection with recombinant vaccinia virus encoding murine IL-2. Mechanisms of rapid viral clearance in immunocompetent mice.

CD8+ cytotoxic T (Tc) lymphocytes mediate recovery from vaccinia virus (VV) infection. In mice, anti-VV Tc cells are detectable on or after day 3 after infection, and cytolytic activity peaks between days 5 and 6. A rVV encoding murine IL-2, VV-hemagglutinin (HA)-IL-2, was cleared more rapidly, compared with a control rVV, VV-HA-thymidine kinase (TK), from tissues of infected euthymic normal mice. The mechanism of VV-HA-IL-2 clearance was operative early in infection and correlated with an elevated NK cell response, before the induction of anti-VV Tc cell response. We have investigated the roles of NK cells, T cells, and IFN-gamma in the rapid clearance of VV-HA-IL-2, by using specific mAb. Depletion of NK cells with mAb significantly enhanced VV-HA-IL-2 but not VV-HA-TK titers 3 days after infection. NK cells alone could not account for rapid viral clearance, because VV-HA-IL-2 titers in NK cell-depleted mice were not comparable to VV-HA-TK titers. Treatment with a mAb to IFN-gamma completely abrogated the IL-2-induced mechanism(s) of VV-HA-IL-2 clearance, and titers of the IL-2-encoding virus were comparable to control virus titers. In addition, the elimination of CD4+ but not CD8+ T cells resulted in significant increases in VV-HA-IL-2 titers.     

Virus-induced transient immune suppression and the inhibition of T cell proliferation by type I interferon

Vaccine-induced memory is necessary for protective immunity to pathogens, but many viruses induce a state of transient immune suppression that might contribute to the inability of a vaccine to elicit immunity. We evaluated here the fate of bystander T cells activated by third party cognate antigens during acute viral infections in vivo, using distinct models to track and specifically activate HY and P14 transgenic bystander CD8 T cells in vivo during acute arenavirus infections of mice. Viral infections acted as stimulatory adjuvants when bystander T cells were exposed to an inflammatory milieu and cognate antigens at the beginning of infections, but bystander CD8 T cell proliferation in response to cognate antigen was inhibited 3 to 9 days after virus infection. Reduced proliferation was not dependent on Fas-FasL- or tumor necrosis factor (TNF)-induced activation-induced cell death or on deficiencies of antigen presentation. Instead, reduced proliferation was associated with a delayed onset of division that was an intrinsic defect of T cells. Inhibition of proliferation could be simulated by exposure of T cells to the Toll-like receptor agonist and type I interferon (IFN) inducer poly(I · C). T cells lacking IFN-α/β receptors resisted both the suppressive effects of preexposure to poly(I · C) and the stimulatory effects of type I IFN, indicating that the timing of exposure to IFN can have negative or positive effects on T cell proliferation. Inhibition of T cell receptor-stimulated bystander CD8 T cell proliferation during acute viral infections may reflect the reduced ability of vaccines to elicit protective immunity when administered during an acute illness.     

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.     

Stimulation of lymphokines in Jurkat cells persistently infected with vaccinia virus.

The response of the human CD4+ T-cell line Jurkat to infection with vaccinia virus was investigated. Virus titers peaked approximately 3 to 4 days after infection, while cell growth paralleled that of uninfected cells, indicating that growth rates were not appreciably affected by viral infection. Results from plaque assays and fluorescence-activated cell sorter (FACS) analyses of virus antigens demonstrated that a persistent infection in which the percentage of infected cells and the virus titers fluctuated from passage to passage was established. Further characterization of the persistent infection revealed that the virus influences cellular functions. Induction of interleukin-2 (IL-2) and IL-2 receptor alpha (IL-2R alpha) in Jvac cells was shown by enzyme-linked immunosorbent assay and FACS analysis, respectively. Hybridization of cellular RNA with cloned probes confirmed the increased IL-2 expression and demonstrated that Jvac cells also expressed more IL-6 but not gamma interferon (IFN-gamma) or IL-1 beta. Dual-antibody staining and FACS analysis for vaccinia virus antigens and IL-2R alpha indicated that IL-2R alpha expression was restricted to the infected cells. Jvac cells were also resistant to superinfection, an additional proof that persistent infection elicited phenotypic changes in the cell population.     

CD127+CCR5+CD38+++ CD4+ Th1 effector cells are an early component of the primary immune response to vaccinia virus and precede development of interleukin-2+ memory CD4+ T cells

The stages of development of human antigen-specific CD4+ T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5+CD38+++ CD4+ effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-gamma)-producing vaccinia virus-specific CD4+ T cells. The majority of these initial vaccinia virus-specific CD4+ T cells were CD127+ and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-gamma and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5+CD38+++ vaccinia virus-specific CD4+ T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4+ T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2+ vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4+ T cells in healthy adults and their dysfunction in HIV-1 infection.     

MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways inflammation and hyperresponsiveness

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.     

Interferon induction in marrow-derived macrophages: regulation by L cell conditioned medium

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti-CSF serum to LCM medium also reduced IFN titers in response to polyI:C but had no effect on NDV induction. The inhibitory effect of anit-CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with polyI:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with polyI:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.     

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.     

Sendai virus infection induces efficient adaptive immunity independently of type I interferons

Adaptive immunity in response to virus infection involves the generation of Th1 cells, cytotoxic T cells, and antibodies. This type of immune response is crucial for the clearance of virus infection and for long-term protection against reinfection. Type I interferons (IFNs), the primary innate cytokines that control virus growth and spreading, can influence various aspects of adaptive immunity. The development of antiviral immunity depends on many viral and cellular factors, and the extent to which type I IFNs contribute to the generation of adaptive immunity in response to a viral infection is controversial. Using two strains (Cantell and 52) of the murine respiratory Sendai virus (SeV) with differential abilities to induce type I IFN production from infected cells, together with type I IFN receptor-deficient mice, we examined the role of type I IFNs in the generation of adaptive immunity. Our results show that type I IFNs facilitate virus clearance and enhance the migration and maturation of dendritic cells after SeV infection in vivo; however, soon after infection, mice clear the virus from their lungs and efficiently generate cytotoxic T cells independently of type I IFN signaling. Furthermore, animals that are unresponsive to type I IFN develop long-term anti-SeV immunity, including CD8+ T cells and antibodies. Significantly, this memory response is able to protect mice against challenge with a lethal dose of virus. In conclusion, our results show that primary and secondary anti-SeV adaptive immunities are developed normally in the absence of type I IFN responsiveness.     

Type I IFN modulates innate and specific antiviral immunity

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.     

Natural killer cell depletion enhances virus synthesis and virus-induced hepatitis in vivo

The role of natural killer (NK) cells in the natural resistance of mice to infections by several viruses was examined. Mice were specifically depleted of NK cells by i.v. injection of rabbit antiserum to asialo GM1, a neutral glycosphingolipid present at high concentrations on the surface of NK cells. Control mice were left untreated or were injected with normal rabbit serum. Four to 6 hr later, these mice were infected with lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), murine cytomegalovirus (MCMV), or vaccinia virus. The mice were sacrificed 3 days post-infection and assayed for virus in liver and spleen, spleen NK cell activity, and plasma interferon (IFN). All mice treated with anti-asialo GM1 antibody had drastically reduced NK cell-mediated lysis. Correlating with NK cell depletion, these mice had significantly higher (up to 500-fold) titers of MCMV, MHV, or vaccinia virus in their livers and spleens as compared to control mice. NK cell-depleted MCMV and MHV-infected mice had higher levels of plasma IFN than controls, correlating with the higher virus titers. These NK cell-depleted, virus-infected mice had more extensive hepatitis, assayed by the number of inflammatory foci in their livers, as compared to control virus-infected mice; these foci were also larger and contained more degenerating liver cells than those in control mice. In contrast to the results obtained with MHV, MCMV, and vaccinia virus, NK cell depletion had no effect on virus titers in the early stages of acute LCMV infection or during persistent LCMV infection. Mice depleted of NK cells had similar amounts of LCMV in their spleens and similar plasma IFN levels. Because this antibody to asialo GM1 does not impair other detectable immunologic mechanisms, these data support the hypothesis that NK cells act as a natural resistance mechanism to a number of virus infections, but suggest that their relative importance may vary from virus to virus.     

Anti-influenza virus cytotoxic T lymphocytes recognize the three viral polymerases and a nonstructural protein: responsiveness to individual viral antigens is major histocompatibility complex controlled

It has recently been shown that antiviral major histocompatibility complex class I-restricted cytotoxic T lymphocytes can recognize proteins that serve as internal viral structural components (influenza A virus nucleoprotein, vesicular stomatitis virus nucleocapsid protein). To further examine the role of internal viral proteins in cytotoxic T-lymphocyte recognition, we constructed recombinant vaccinia viruses containing individual influenza A virus genes encoding three viral polymerases (PB1, PB2, PA) and a protein not incorporated into virions (NS1). We found that cells infected with each of these recombinant vaccinia viruses could be lysed by anti-influenza cytotoxic T lymphocytes. Cytotoxic T-lymphocyte responsiveness to the individual viral antigens varied greatly between mouse strains. By using congenic mouse strains, responsiveness to PB1 and PB2 was found to cosegregate with major histocompatibility complex haplotype. These findings provide further evidence that internal antigens play a critical role in cytotoxic T-lymphocyte recognition of virus-infected cells. Additionally, they suggest that the cytotoxic T-lymphocyte response to viral antigens may often be restricted to only a fraction of the major histocompatibility complex class I repertoire.     

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in?vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.     

Factor determining the antigenic type of interferons produced in human lymphoblastoid cell lines

The factor that determines the antigenic type of IFN produced in human lymphoblastoid cell lines was examined using live Sendai virus, ultraviolet (UV)-irradiated virus, HANA spikes exposed on L cells persistently infected with Sendai virus (L-HVJ) and poly-inosinic acid poly-cytidylic acid (poly I: C). When Sendai virus was irradiated with UV-light for 300 sec, its abilities to infect chicken eggs and induce IFN were diminished, but its HA activity was unaffected. HANA spikes exposed on L-HVJ could not induce IFN in human lymphoblastoid cell lines, although they induced IFN in mouse spleen cells. These results suggest that the induction of IFN in human lymphoblastoid cells is closely related to viral nucleic acid. Poly I: C also induced IFN in some human lymphoblastoid cell lines in which IFN production is induced by Sendai virus. The antigenic types of IFN induced by poly I: C were the same as those induced by Sendai virus. These results suggest that the antigenic type of IFN produced depends on the nature of the IFN producer cells rather than on the kind of IFN inducer.