Human parainfluenza virus type 3 up-regulates major histocompatibility complex class I and II expression on respiratory epithelial cells: involvement of a STAT1- and CIITA-independent pathway

Human parainfluenza virus type 3 (HPIV3) infection causes severe damage to the lung epithelium, leading to bronchiolitis, pneumonia, and croup in newborns and infants. Cellular immunity that plays a vital role in normal antiviral action appears to be involved, possibly because of inappropriate activation, in the infection-related damage to the lung epithelium. In this study, we investigated the expression of major histocompatibility complex (MHC) class I and II molecules on human lung epithelial (A549) and epithelium-like (HT1080) cells following HPIV3 infection. MHC class I was induced by HPIV3 in these cells at levels similar to those observed with natural inducers such as beta and gamma interferon (IFN-beta and -gamma). MHC class II was also efficiently induced by HPIV3 in these cells. UV-irradiated culture supernatants from infected cells were able to induce MHC class I but not MHC class II, suggesting involvement of released factors for the induction of MHC class I. Quantitation of IFN types I and II in the culture supernatant showed the presence of IFN-beta as the major cytokine, while IFN-gamma was undetectable. Anti-IFN-beta, however, blocked the HPIV3-mediated induction of MHC class I only partially, indicating that viral antigens, besides IFN-beta, are directly involved in the induction process. The induction of MHC class I and class II directed by the viral antigens was confirmed by using cells lacking STAT1, an essential intermediate of the IFN signaling pathways. HPIV3 induced both MHC class I and class II molecules in STAT1-null cells. Furthermore, MHC class II was also induced by HPIV3 in cells defective in class II transactivator, an important intermediate of the IFN-gamma-mediated MHC class II induction pathway. Together, these data indicate that the HPIV3 gene product(s) is directly involved in the induction of MHC class I and II molecules. The induction of MHC class I and II expression by HPIV3 suggests that it plays a role in the infection-related immunity and pathogenesis.     

Differential effects of IFN-beta1b on the proliferation of human vascular smooth muscle and endothelial cells

The effect of human interferon (IFN)-beta1b (Betaseron) on the proliferation of cultured human vascular smooth muscle and endothelial cells was tested in vitro. IFN-beta1b inhibited thymidine incorporation and growth of primary cultures of human aortic and coronary artery smooth muscle in a concentration-dependent manner. The same concentrations of IFN-beta1b did not inhibit thymidine incorporation or growth of primary cultures of human aortic or coronary artery endothelial cells. IFN-beta1b induced the expression of MxA (an antiviral protein induced by type I IFNs) in both smooth muscle and endothelial cells, suggesting that both cell types express receptors for type I IFNs. The growth-inhibitory effect of IFN-beta1b could be mimicked by commercially available human IFN-beta, but not by IFN-alpha2 or IFN-alpha8. The effect of IFN-beta1b was species specific, as it did not inhibit thymidine incorporation in aortic smooth muscle cells derived from pig, rabbit, rat, or mouse. The action of IFN-beta1b on smooth muscle cells persisted for at least 4 days following a 24 h preincubation with IFN-beta1b. Human vascular smooth muscle cells treated with IFN-beta1b did not release lactate dehydrogenase, nor did they show any morphologic change, suggesting that IFN-beta1b was not toxic to the human vascular smooth muscle cells. IFN-beta1b inhibited vascular smooth muscle growth while having no growth-inhibitory effect on endothelial cells obtained from the same blood vessel, making it a potential candidate for treating pathologic conditions where abnormal vascular smooth muscle proliferation is implicated, such as restenosis following balloon angioplasty or smooth muscle proliferation following vascular stenting.     

Interferon-delta: the first member of a novel type I interferon family

We have recently described a novel type I interferon (IFN) co-expressed with IFN-gamma by the trophectoderm of the pig conceptus between day 12 and day 18 of gestation, a development stage that corresponds to implantation in the uterus. This IFN, now officially named IFN-delta, is recognized as the first member of a novel type I IFN family. This paper reviews the main published data on IFN-delta, together with some new data, showing that IFN-delta, while being a true type I IFN, has some very specific structural and biological properties. Sequences related to IFN-delta coding sequence were found in the genome of man and other ungulates but the only other potentially functional gene was found, so far, in the horse. The pig IFN-delta mature protein, with 149 amino acids, is the smallest of all known type I IFNs. It is unusually rich in cysteines (seven residues), and has a very basic isoelectric point. Recombinant IFN-delta expressed in insect cells is glycosylated and has a high antiviral activity on porcine cells, but not on human cells. It has high antiproliferative activity, which is significantly enhanced in the presence of IFN-gamma. This new IFN was shown to bind on pig cells to the same type I receptor as IFN-alpha. IFN-delta and IFN-gamma genes are co-regulated in the pig trophectoderm, whose cells on day 14-16 of development simultaneously secrete both IFN proteins. The biological role of porcine IFN-delta in early pregnancy has been found unrelated to the known antiluteolytic effect of trophoblastic IFN-tau in ruminants.     

Differences in activity between alpha and beta type I interferons explored by mutational analysis

Type I interferon (IFN) subtypes alpha and beta share a common multicomponent, cell surface receptor and elicit a similar range of biological responses, including antiviral, antiproliferative, and immunomodulatory activities. However, alpha and beta IFNs exhibit key differences in several biological properties. For example, IFN-beta, but not IFN-alpha, induces the association of tyrosine-phosphorylated receptor components ifnar1 and ifnar2, and has activity in cells lacking the IFN receptor-associated, Janus kinase tyk2. To define the structural basis for these functional differences we produced human IFN-beta with point mutations and compared them to wild-type IFN-beta in assays that distinguish alpha and beta IFN subtypes. IFN-beta mutants with charged residues (N86K, N86E, or Y92D) introduced at two positions in the C helix lost the ability to induce the association of tyrosine-phosphorylated receptor chains and had reduced activity on tyk2-deficient cells. The combination of negatively charged residues N86E and Y92D (homologous with IFN-alpha8) increased the cross-species activity of the mutant IFN-betas on bovine cells to a level comparable to that of human IFN-alphas. In contrast, point mutations in the AB loop and D helix had no significant effect on these subtype-specific activities. A subset of these latter mutations did, however, reduce activity in a manner analogous to IFN-alpha mutations. The effects of these mutations on IFN-beta activity are discussed in the context of a family of related ligands acting through a common receptor and signaling pathway.     

Synthetic oligonucleotides with certain palindromes stimulate interferon production of human peripheral blood lymphocytes in vitro

We studied the ability of synthetic single-stranded 30-mer oligodeoxyribonucleotides (oligoDNAs) with three different kinds of hexamer palindromic sequence to induce interferon (IFN) production of human peripheral blood lymphocytes (PBL). When PBL was cultured with oligoDNA having a palindrome of AACGTT or GACGTC, IFN activity was detected by bioassay in the culture fluid after 8 h, and the amount of IFN reached the maximum after 18 h. IFN-alpha was predominantly produced, and small amounts of IFN-beta and IFN-gamma were also found. OligoDNA with the palindrome ACCGGT had no effect.     

Interferon beta, a cofactor in the interferon gamma production induced by gram-negative bacteria in mice

The interferon (IFN) gamma production of splenocytes from closely related C57BL/10ScSn (Sn) and C57BL/10ScCr (Cr) mice was compared. Concanavalin A and CD3 monoclonal antibodies induced high levels of IFN-gamma in both Sn and Cr splenocytes. By contrast, treatment with gram-negative bacteria induced IFN-gamma only in Sn splenocytes; in Cr splenocytes, the IFN-gamma response was heavily impaired. The IFN-gamma induction by bacteria requires the cooperation of IFN-gamma-producing cells with macrophages. Depletion of macrophages from Sn splenocytes resulted in the loss of ability to produce IFN-gamma after bacterial stimulation. Reconstitution with new Sn macrophages restored the IFN-gamma responsiveness, whereas reconstitution with Cr macrophages failed to do so. Normal function of IFN-gamma-producing cells and a defective function of macrophages of Cr mice was demonstrated by evidence showing that whole or macrophage-depleted Cr splenocytes, when supplemented with Sn macrophages, acquire the ability to produce IFN-gamma in response to bacteria. A similar effect was achieved by supplementing Cr splenocytes with supernatants of bacteria-stimulated Sn macrophages or with recombinant murine IFN-beta or IFN-alpha. Preincubation of active macrophage supernatants with antibodies to IFN-beta suppressed the helper activity for Cr splenocytes. Moreover, the bacteria-induced production of IFN-gamma by Sn splenocytes could be inhibited by antibodies to murine IFN-beta. The results provide evidence that IFN-beta is an important cofactor of IFN-gamma induction, which is not induced in Cr mice by gram-negative bacteria.     

Reovirus induction of and sensitivity to beta interferon in cardiac myocyte cultures correlate with induction of myocarditis and are determined by viral core proteins

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-alpha/beta antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-beta induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-alpha/beta added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-beta levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-alpha/beta compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-alpha/beta is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-alpha/beta, demonstrating that IFN-alpha/beta is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-beta can be a determinant of viral myocarditis and reovirus disease.     

Counteracting effect of IFN-beta on IFN-gamma-induced proliferation of human astrocytes in culture

Recent clinical trials have shown that interferon beta (IFN-beta) is effective in reducing exacerbations in relapsing-remitting MS, while interferon gamma (IFN-gamma) precipitates the relapses. To investigate mechanisms underlying the beneficial effects of IFN-beta and the detrimental effects of IFN-gamma in MS, cell growth-regulatory effects of IFNs were examined in astrocyte-enriched cultures isolated from fetal brains of 12-20 weeks' gestation. Treatment with IFN-gamma (50 or 500 IU ml-1) stimulated significantly the proliferation of astrocytes in 6 out of 9 culture series examined, while IFN-beta (50 or 500 IU ml-1) inhibited the astrocytic proliferation in 3 out of 9 cultures, and IFN-alpha (50 or 500 IU ml-1) did not affect the proliferation IFN-beta and to a lesser degree IFN-alpha reduced the astrocytic proliferation induced by IFN-gamma-treatment in 8 out of 9 culture series. The counteracting effect of IFN-alpha/IFN-beta against IFN-gamma-induced astrocytic proliferation was verified by the DNA content distribution analysis of propidium iodide-labeled cells. The antagonistic effect of IFN-alpha/IFN-beta on the growth-promoting activity of IFN-gamma in cultured human astrocytes suggests that interferons serve as growth regulators of astrocytes at sites of reactive gliosis lesions of MS.     

Protective effects of interferon-gamma in intraocular herpes simplex type 1 infection do not depend on major histocompatibility complex class I or class II expression

Intraocular infection with herpes simplex virus type I strain F (HSV-1) induces bilateral retinitis, the expression of both MHC class I and II molecules and activation of CD4 and CD8 cells. To investigate the role of MHC upregulation in IFN-gamma mediated antiviral effects in intraocular infection with HSV-1, we infected MHC deficient mice and mice with an additional ectopic site of IFN-gamma production in their retina (rho gamma) intravitreally with HSV-1 into one eye. Protective effects of IFN-gamma in intraocular HSV-1 infection were notable as sparing of the contralateral non-inoculated eye from retinitis, and were not dependent on MHC class I and class II expression, thus limiting the importance of MHC expression for the outcome of viral infection in vivo.     

An important role for major histocompatibility complex class I-restricted T cells, and a limited role for gamma interferon, in protection of mice against lethal herpes simplex virus infection

Herpes simplex virus (HSV) inhibits major histocompatibility complex (MHC) class I expression in infected cells and does so much more efficiently in human cells than in murine cells. Given this difference, if MHC class I-restricted T cells do not play an important role in protection of mice from HSV, an important role for these cells in humans would be unlikely. However, the contribution of MHC class I-restricted T cells to the control of HSV infection in mice remains unclear. Further, the mechanisms by which these cells may act to control infection, particularly in the nervous system, are not well understood, though a role for gamma interferon (IFN-gamma) has been proposed. To address the roles of MHC class I and of IFN-gamma, C57BL/6 mice deficient in MHC class I expression (beta2 microglobulin knockout [beta2KO] mice), in IFN-gamma expression (IFN-gammaKO mice), or in both (IFN-gammaKO/beta2KO mice) were infected with HSV by footpad inoculation. beta2KO mice were markedly compromised in their ability to control infection, as indicated by increased lethality and higher concentrations of virus in the feet and spinal ganglia. In contrast, IFN-gamma appeared to play at most a limited role in viral clearance. The results suggest that MHC class I-restricted T cells play an important role in protection of mice against neuroinvasive HSV infection and do so largely by mechanisms other than the production of IFN-gamma.     

Tyrosine phosphorylation of the alpha and beta subunits of the type I interferon receptor. Interferon-beta selectively induces tyrosine phosphorylation of an alpha subunit-associated protein

We studied the phosphorylation of the alpha and beta subunits of the Type I interferon (IFN) receptor induced by Type I IFNs in the human U-266 and MOLT-4 cell lines. Both IFN-alpha and IFN-beta induced tyrosine phosphorylation of the beta subunit of the receptor. The Type I IFN-induced tyrosine phosphorylation of the beta subunit was rapid and transient, being detectable within 1 min of Type I IFN treatment and gradually diminishing to almost base-line levels by 60 min. All Type I IFNs studied were found to induce tyrosine phosphorylation of the alpha subunit of the Type I IFN receptor, the p135tyk2 and JAK-1 tyrosine kinases, and the ISGF3 alpha components. Interestingly, IFN-beta, but not IFN-alpha or IFN-omega, induced tyrosine phosphorylation of an alpha subunit-associated protein with an apparent molecular mass of approximately 100 kDa (p100). These data suggest the existence of a common signaling pathway(s) for Type I IFNs involving the alpha and beta subunits of the receptor, the tyrosine kinases p135tyk2 and JAK-1, and the ISGF3 alpha components. However, differences between the signaling pathways of different Type I IFNs exist, as suggested by tyrosine phosphorylation of an alpha subunit-associated protein only in response to IFN-beta.     

Interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo

Mechanisms responsible for neuroattenuation of herpes simplex virus (HSV) have been defined previously by studies of mutant viruses in cultured cells. The hypothesis that null mutations in host genes can override the attenuated phenotype of null mutations in certain viral genes was tested. Mutants such as those in infected cell protein (ICP) 0, thymidine kinase, ribonucleotide reductase, virion host shutoff, and ICP34.5 are reduced in their capacity to replicate in nondividing cells in culture and in vivo. The replication of these viruses was examined in eyes and trigeminal ganglia for 1-7 d after corneal inoculation in mice with null mutations (-/-) in interferon receptors (IFNR) for type I IFNs (IFN-alpha/betaR), type II IFN (IFN-gammaR), and both type I and type II IFNs (IFN-alpha/beta/gammaR). Viral titers in eyes and ganglia of IFN-gammaR-/- mice were not significantly different from congenic controls. However, in IFN-alpha/betaR-/- or IFN-alpha/beta/gammaR-/- mice, growth of all mutants, including those with significantly impaired growth in cell culture, was enhanced by up to 1,000-fold in eyes and trigeminal ganglia. Blepharitis and clinical signs of infection were evident in IFN-alpha/betaR-/- and IFN-alpha/beta/gammaR-/- but not control mice for all viruses. Also, IFNs were shown to significantly reduce productive infection of, and spread from intact, but not scarified, corneas. Particularly striking was restoration of near-normal trigeminal ganglion replication and neurovirulence of an ICP34.5 mutant in IFN-alpha/betaR-/- mice. These data show that IFNs play a major role in limiting mutant and wild-type HSV replication in the cornea and in the nervous system. In addition, the in vivo target of ICP34.5 may be host IFN responses. These experiments demonstrate an unsuspected role for host factors in defining the phenotypes of some HSV mutants in vivo. The phenotypes of mutant viruses therefore cannot be interpreted based solely upon studies in cell culture but must be considered carefully in the context of host factors that may define the in vivo phenotype.     

Type I IFNs inhibit human dendritic cell IL-12 production and Th1 cell development

We have investigated the role of type I IFNs (IFN-alpha and -beta) in human T cell differentiation using anti-CD3 mAb and allogeneic, in vitro-derived dendritic cells (DC) as APCs. DC were very efficient activators of naive CD4+ T cells, providing necessary costimulation and soluble factors to support Th1 differentiation and expansion. Addition of IFN-alphabeta to DC/T cell cultures resulted in induction of T cell IL-10 production and inhibition of IFN-gamma, TNF-alpha, and LT secretion. Diminished T cell IFN-gamma production correlated with IFN-alphabeta-mediated inhibition of the p40 chain of the IL-12 heterodimer secreted by DC. Suppression of p40 IL-12 and IFN-gamma was not due to increased levels of IL-10 in these cultures, and production of IFN-gamma could be restored by exogenous IL-12. These data indicate that type I IFNs inhibit DC p40 IL-12 expression, which is required for development of IFN-gamma-producing CD4+ T cells. Furthermore, when T cells were restimulated without IFN-beta, these cells induced less p40 IL-12 from DC, suggesting that the functional properties of T cells may regulate DC function. Thus, IFN-alphabeta inhibits both IL-12-dependent and independent Th1 cytokine production and provides a mechanism for inhibition of IL-12-mediated immunity in viral infections.     

Human trophoblast interferons: production and possible roles in early pregnancy

Human villous and extravillous trophoblast populations were isolated from first- and third-trimester placentae and were stimulated with viral and non-viral inducers to produce interferons (IFNs). Polyriboinosinic/polyribocytidylic acid [poly(I:C)] induced exclusively IFN-beta in trophoblast cultures, whereas viruses induced mixtures of IFN-alpha subtypes and -beta. The level of IFN production was dependent on the trophoblast population, type of inducer and the stage of differentiation of the trophoblast. First-trimester extravillous trophoblast cultures produced greater than five-fold more IFN than third-trimester villous trophoblast on a per cell basis, whereas term syncytiotrophoblast produced twice as much IFN as term mononuclear villous trophoblast when stimulated with the same inducer. Pretreatment of trophoblast cultures with platelet-derived growth factor and granulocyte/macrophage-colony stimulating factor (GM-CSF) increased the trophoblast IFN production. Tandem high-performance affinity chromatography of the virus-induced trophoblast IFNs resulted in the isolation of trophoblast IFN-alpha and -beta with specific antiviral activities of 0.75-2.73 x 10(8) IU/ml protein. The trophoblast-induced IFNs have antiproliferative and immunosuppressive properties, and, furthermore, activated natural killer cell activity. These data may suggest the possible roles of these IFNs during embryonic development with regard to protection of the fetus against viral infection and maternal immunity.