Interferon-beta. A potential autocrine regulator of human vascular smooth muscle cell growth.

Positive and negative signals regulate the proliferation in vitro of vascular smooth muscle cells (SMC), a principle cell type in the blood vessel wall. Immune interferon (IFN-gamma, a type II IFN) retards the growth of human SMC, but the effect of type I IFN (IFN-alpha or beta) is unknown. Furthermore, the capacity of SMC to produce IFN is uncharacterized. If type I IFN alters SMC growth and is produced by this cell type, an autocrine inhibitory loop could operate in vascular growth control. To test this possibility, we compared the effects of IFN-alpha, beta, and gamma on the growth of SMC stimulated by platelet-derived growth factor, interleukin-1 or tumor necrosis factor alpha. IFN-beta and IFN-gamma, but not IFN-alpha, consistently retarded growth of SMC cultures (measured by net DNA accumulation and cell number). We investigated whether SMC could produce IFN-beta, a mediator characteristically produced by fibroblasts. Vascular SMC treated with poly(I):poly(C) or tumor necrosis factor-alpha expressed IFN-beta mRNA. SMC treated with poly(I):poly(C) or Newcastle Disease virus elaborated biologically active IFN-beta as well. Our results establish that IFN-beta inhibits human vascular SMC growth and that these cells can express the IFN-beta gene. These findings show that human vascular SMC have the capacity of producing a potential autocrine growth regulator.     

Opposite effects of murine interferons on erythroid differentiation of Friend cells

Interferons (IFNs), in addition to inducing an antiviral state in uninfected cells, are able to affect cell physiology, including cell differentiation. In this respect hematopoiesis is certainly the area in which most data have accumulated. In general IFN-alpha or -beta inhibit cell growth of normal progenitors of hematopoietic lineages. In leukemia cell cultures IFNs may either stimulate or inhibit cell growth and differentiation. We report here different biological effects of murine (mu) IFN-alpha 1, -beta, and -gamma species on the erythroid differentiation of dimethyl sulfoxide (DMSO)-induced Friend leukemia cells. Treatment with mu recombinant IFN-beta enhances DMSO-induced FLC differentiation, whereas treatment with IFN-alpha 1 species as well as with natural and recombinant mu IFN-gamma preparations only inhibits it. All these observed effects are neutralized by monoclonal antibodies against IFN-alpha, -beta, and -gamma species. When mu fibroblast IFN (a mixture of alpha and beta species) was used, the inhibitory effect attributable to IFN-alpha was partly overshadowed by the simultaneous presence of a majority of IFN-beta molecules exerting the opposite effect. This is in agreement with data obtained neutralizing fibroblast IFN preparations with excess amounts of monoclonal antibodies against IFN-beta (G.B. Rossi et al., 1988, "The Status of Differentiation Therapy of Cancer," Raven Press, New York) and with our previous reports indicating that mu fibroblast IFN can either enhance or inhibit DMSO-induced differentiation when administered at low (less than 500 U/ml) or high (greater than 5000 U/ml) doses, respectively. The inhibitory effect of IFN-alpha 1 on cell differentiation is not linked to any inhibitory effect on cell growth. Results obtained analyzing the effect of IFN-alpha 1 and -beta on various IFN-resistant FLC clones indicate that different mechanisms underlie the stimulatory effect of IFN-beta and the inhibitory effect of IFN-alpha 1. These results shed light on possibly distinct physiological roles of the various species of IFNs.     

HIV-1 gp41 and type I interferon

HIV-1 gp41-like human type I interferon (IFN) could inhibit lymphocyte proliferation and up-modulate MHC class I and II and ICAM-1 molecule expression. Sequence comparison indicates that a similar epitope RILAV-YLKD exists between N-domain of gp41 and two regions in IFN-alpha(aa29-35 and 113-129), IFN-beta (aa31-37 and 125-138) and IFN-omega (aa29-35 and 123-136), which was shown to form IFN-alpha/beta-receptor binding site. Weak sequence similarity was also found to exist in both regions on gp41 and type I IFN of murine and bovine. Experimental studies indicated that a common immunological epitope exists between gp41 and IFN-alpha and -beta. Antibodies against human IFN-alpha and -beta recognized the common immunological epitope and inhibited gp41-binding to the potential cellular receptor protein p45. Moreover, the polyclonal antibody to IFN-beta completely inhibited gp41-binding to human T, B cells and monocytic cells, while IFN-alpha could only inhibit this binding incompletely. It was interestingly observed that human IFN-beta after preincubating with cells could incompletely inhibit the binding of gp41 to human B cells and monocytic cells, and very weakly inhibit the binding to human T cells, indicating that the receptor for IFN-beta-binding may be involved in gp41 binding. This potential relationship may be based on the amino acid sequence homology in the receptor binding region between gp41 and IFN-beta. It was observed that the increased levels of antibodies against human IFN-alpha and -beta exist in HIV-1-infected individuals and are associated with the common epitope on gp41. Besides, several studies provided experimental evidence that the common immunological epitope could induce protective activity against HIV-1. The IFN-alpha-based vaccine has showed a significant reduction of disease progression in IFN-alpha-vaccine-treated HIV-infected patients. Recent experimental evidence indicates that gp41 and IFN-beta were involved in downregulation of CCR5 expression and induction of cell activation or signal transduction. Whether it may be performed by a similar mechanism is still to be investigated.     

Human interferon-beta inhibits binding of HIV-1 gp41 to lymphocyte and monocyte cells and binds the potential receptor protein P50 for HIV-1 gp41

Previous findings have indicated that HIV-1 gp41 like human type I interferon (IFN) could inhibit lymphocyte proliferation and up-modulate MHC class I, II and ICAM-1 molecule expression, and a common epitope exists between gp41 and type I interferon (IFN-alpha and -beta) in the receptor binding regions. To clarify the relationship between human type I interferon and HIV-1 gp41, we tried to inhibit recombinant soluble gp41-binding to human T, B and monocyte cell lines by human IFN-alpha, -beta and -gamma. It was interestingly observed that IFN-beta after preincubating with cells could inhibit the binding of rsgp41 to H9, Raji and U937 cells (T, B and monocyte cell lines), while this binding could not be inhibited by another type I interferon (IFN-alpha) and a type II interferon (IFN-gamma). It was further examined whether human IFN-alpha and -beta bind to the gp41 binding protein P50. In ELISA-assay, the human IFN-beta, but not IFN-alpha, could bind to P50 which was identified as a potential cellular receptor protein for gp41-binding. By the affinity capillary electrophoresis (ACE) analysis, formation of stable IFN-beta-P50 complex was observed. These results indicate that IFN-beta binds the potential receptor protein P50. Based on these experimental evidences and previous studies, it was presumed that the potential cellular receptor protein P50 may be the 51 kDa subunit of human IFN-alpha/beta receptor, which needs to be verified in the future.     

The immediate-early protein, ICP0, is essential for the resistance of herpes simplex virus to interferon-alpha/beta

Herpes simplex virus type 1 (HSV-1) is resistant to the antiviral effects of interferon (IFN)-alpha, -beta, or -gamma. The fact that ICP0(-) mutants replicate like wild-type virus in IFN-alpha/beta receptor knockout mice (Leib et al., 1999, J. Exp. Med. 189, 663) suggested that ICP0 may serve a direct role in the resistance of HSV-1 to IFN. To test this hypothesis, the effects of IFN-alpha, -beta, and -gamma were compared against wild-type HSV-1 and an ICP0(-) mutant virus, 7134. In Vero cells, 7134 was more sensitive to inhibition by low doses of type I IFN (-alpha/beta) or type II IFN (-gamma) than vesicular stomatitis virus, a well-studied IFN-sensitive virus. At a concentration of 100 U/ml, IFN-alpha, -beta, or -gamma reduced the efficiency of 7134 plaque formation by 120-, 560-, and 45-fold, respectively. In contrast, none of the IFNs reduced wild-type HSV-1 plaque formation by more than 3-fold. Even when Vero cells were infected with 10 pfu per cell, IFN-alpha and -beta inhibited 7134 replication by over 100-fold, but inhibition by IFN-gamma decreased to less than 10-fold. While IFN-beta efficiently inhibited 7134 replication in primary mouse kidney and SK-N-SH cells, IFN-gamma did not inhibit 7134 to a comparable extent in these cells. ICP0 provided in trans from an adenovirus vector allowed 7134 to replicate efficiently in Vero cells in the presence of IFN-alpha, -beta, or -gamma. While IFN-beta or -gamma efficiently repressed the ICP0 promoter-lacZ reporter gene in 7134 (i.e., approximately 60-fold reduction in beta-galactosidase activity), ICP0 provided in trans almost completely reversed IFN-mediated repression of the lacZ gene in 7134. The results suggest that the rate of ICP0 expression in infected cells in vivo may be critical in determining whether host IFNs repress the HSV-1 genome. This concept is discussed in light of its potential relevance to the establishment of latent HSV-1 infections.     

Induction of interferon-alpha by interferon-beta, but not of interferon-beta by interferon-alpha, in the mouse

The antigenicity of the interferon (IFN) produced in transgenic mice carrying an extra mouse IFN-beta gene under the control of mouse metallothionein-I enhancer-promoter was examined after induction with Cd2+. Unexpectedly, IFN-alpha in addition to IFN-beta was detected in the serum. Induction of IFN-alpha was also observed when recombinant mouse IFN-beta was injected into normal mice. IFN-alpha was first detected in the circulation 6-10 hr after the administration of IFN-beta, and after 12 hr, IFN-alpha became the major component of serum IFN. On the other hand, when IFN-alpha was injected, no production of IFN-beta was observed. Messenger RNAs specific for IFN-alpha and endogenous IFN-beta were detected in the spleen, though the amount of IFN-beta mRNA was much less than that of IFN-alpha mRNA. These mRNAs were not detected in other organs including the liver where exogenous IFN-beta gene was markedly expressed. These observations showed that the expression of IFN-alpha is inducible by IFN-beta in the mouse, and the spleen was suggested to be the main site of production. Possible mechanisms of the induction are discussed.     

Spontaneous production of gamma interferon and induced production of beta interferon by human T-lymphoblastoid cell lines.

Two human T-lymphoblastoid cell lines, CCRF/CEM and Molt 4, produced beta interferon (IFN-beta) upon infection with Sendai virus. Molt 4, but not CCRF/CEM, spontaneously produced up to 300 U of IFN-gamma per ml, apparently not contaminated with IFN-alpha or -beta. Phytohemagglutinin, a T-cell mitogen, did not stimulate IFN production in these lines. A third T-lymphoblastoid line, CCRF/HSB2, produced no IFN either spontaneously or after infection with Sendai virus or treatment with phytohemagglutinin. The Molt 4 cells contained an mRNA which could be translated by oocytes to give IFN-gamma. Molt 4 cells therefore provide a convenient source of human IFN-gamma and its mRNA for experimental purposes.     

Type I interferons (IFN-alpha and -beta) suppress cytotoxin (tumor necrosis factor-alpha and lymphotoxin) production by mitogen-stimulated human peripheral blood mononuclear cell.

We studied the effect of the different types of interferons on the production of cytotoxin by human peripheral blood mononuclear cells (PBMCs) stimulated with the mitogen phytohemagglutinin (PHA). Maximum secreted levels of cytotoxin were observed at day 3 in culture and consisted of both tumor necrosis factor alpha (TNF-alpha) and lymphotoxin as determined by specific antibodies. Type I interferons (IFN-alpha and IFN-beta) consistently suppressed cytotoxin production. Both TNF-alpha and lymphotoxin were significantly suppressed. Mean suppression by IFN-alpha and IFN-beta (1000 U/ml) was 56 and 66%, respectively, in PBMCs from 18 different donors. The suppressive effects of IFN-alpha and IFN-beta on cytotoxin production were dose responsive over a range of 10 to 1000 U/ml. Type II interferon (IFN-gamma) did not have consistent significant effects. Pretreatment with IFN-alpha or IFN-beta for 24 or 48 h prior to PHA stimulation also resulted in significant suppression. Supplementation with interleukin-2 (10 U/ml) or IFN-gamma (1000 U/ml) did not overcome cytotoxin suppression by IFN-alpha or IFN-beta. Cytotoxin suppression by IFN-alpha and IFN-beta together appeared to be noninteractive. Suppression appeared not to be due to blockade of the cytotoxin release, since both cell-associated cytotoxin and secreted cytotoxin were suppressed to the same level. These results demonstrated that cytotoxin and lymphotoxin production by PHA-stimulated PBMCs could be down-regulated by type I interferons and that there is a substantial difference between the action of type I interferons and type II interferons (IFN-gamma) in modulating the biosynthesis of cytotoxins.     

Activity of hybrid type I interferons in cells lacking Tyk2: a common region of IFN-alpha 8 induces a response, but IFN-alpha2/8 hybrids can behave like IFN-beta.

Type I interferons (IFNs) are a family of pleiotropic cytokines with antiviral, antiproliferative, and immunomodulatory properties. The type I IFN family consists of 12 IFN-alpha subtypes, IFN-beta, and IFN-omega. Cells lacking the receptor-associated protein kinase Tyk2 (U1A) are responsive only to IFN-beta and partially to IFN-alpha8. We constructed a series of IFN-alpha2/alpha8 hybrids and mutants and identified the region within IFN-alpha8 responsible for its activity in Tyk2-deficient cells. The same domain mediates the interactions between IFN and IFN-alpha receptor (IFNAR) in Tyk2-complemented and Tyk2-deficient cells (U1A). The presence or absence of Tyk2 altered the inhibitory effects of anti-IFNAR antibodies, suggesting that the IFN-alpha binding domain on IFNAR is altered by the presence of Tyk2. The activity of IFN-beta was not significantly affected by the deletion of Tyk2, and, surprisingly, one of our IFN-alpha2/alpha8 hybrids (IFN-alpha288) behaved like IFN-beta in a number of assays that distinguish IFN-alphas from IFN-beta. This suggests that this hybrid mimics the interactions of IFN-beta with the receptor and also suggests the existence of a distinct binding site(s) on IFNAR for IFN-beta and some hybrid IFN-alphas.     

Production of Interferon-β by Murine T-Cell Lines Induced by 10-Carboxymethyl-9-Acridanone

Besides the established T-cell property of producing gamma interferon (IFN-gamma), murine T cells additionally possess the ability to produce IFN-alpha and IFN-beta when appropriate inducers such as 10-carboxymethyl-9-acridanone (CMA) or Newcastle disease virus (NDV) are used. Interleukin 2 (IL-2)-dependent murine T-cell lines, but not purified resting splenic T cells, responded to CMA and NDV with production of IFN-alpha, beta. The IFN production by these T cells was not restricted to a special subset, since T cells expressing the Lyt 1+2- and the Lyt 1-2+ phenotype responded to these inducers with IFN production. After prolonged passaging of the T-cell lines in IL-2-containing medium, the ability to respond to CMA with production of antiviral activity was sustained longer than the ability for concanavalin A-induced IFN-gamma production. Whereas the NDV-induced T-cell supernates contained both IFN-alpha and IFN-beta, the induction with CMA resulted exclusively in the synthesis of IFN-beta by the T-cell lines.     

Interferon-induced inhibition of Chlamydia trachomatis: dissociation from antiviral and antiproliferative effects.

The yield of infectious Chlamydia trachomatis was analyzed in human (HeLa) and mouse (McCoy) cell lines treated with the human interferon (IFN) subtypes IFN-alpha A and IFN-alpha D, with their hybrids [IFN-alpha AD (BglII), IFN-alpha AD (PvuII), and IFN-alpha DA (BglII)] constructed in vitro from their expression plasmids, or with IFN-beta 1 or buffy coat IFN. In HeLa cells, a significant inhibition of Chlamydia infectivity was obtained with IFN-alpha D, IFN-alpha DA (BglII), and buffy coat IFN. In McCoy cells, IFN-alpha AD (BglII) and IFN-alpha AD (PvuII) induced a strong degree of inhibition of Chlamydia infectivity. In McCoy cells, there was a correlation among the antichlamydial, antiviral, and antiproliferative activities of the different IFNs tested. In HeLa cells, however, the ability of a particular IFN subtype to inhibit Chlamydia infectivity did not always correlate with its inhibitory effects on encephalomyocarditis virus replication or with its antiproliferative activity.