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Regulation of the acute phase and immune responses in viral disease. Enhanced expression of the beta 2-interferon/hepatocyte-stimulating factor/interleukin 6 gene in virus-infected human fibroblasts.

We have defined the expression of the mRNA for, and secretion of, IFN- beta 2/hepatocyte-stimulating factor/IL-6 (IFN-beta 2/IL-6) in human diploid fibroblasts (FS-4 strain) infected with different RNA- and DNA- containing viruses. RNA blot-hybridization analyses carried out 6-8 h after the beginning of infection showed that the RNA-containing Sendai virus (paramyxoviridae) enhanced IFN-beta 2/IL-6 mRNA levels 10-fold, followed, in decreasing order, by encephalomyocarditis (EMC, picornaviridae), vesicular stomatitis (VSV, rhabdoviridae), Newcastle disease virus (NDV, paramyxoviridae), and influenza A (Flu, myxoviridae) viruses. The DNA-containing pseudorabies virus (PR, herpesviridae) enhanced IFN-beta 2/IL-6 mRNA levels sixfold, while the effect of adenovirus type 5 (Ad5, adenoviridae) was considerably less and comparable with that of NDV or Flu. A rabbit antiserum raised against E. coli-derived human IFN-beta 2/IL-6 was used in immunoprecipitation experiments to monitor the secretion of 35S- methionine-pulse-labeled IFN-beta 2/IL-6 proteins by fibroblasts up to 7 h after the beginning of infection. Enhanced levels of secretion of IFN-beta 2/IL-6 (2-14-fold) were observed in every instance evaluated (Sendai, EMC, VSV, Flu, PR, Ad5 viruses). A biological consequence of enhanced secretion of IFN-beta 2/IL-6 was the ability of media from infected FS-4 cell cultures to enhance by 8-15-fold the synthesis and secretion of a typical acute phase plasma protein (alpha 1- antichymotrypsin) by human hepatoma Hep3B2 cells. These observations make it likely that IFN-beta 2/IL-6 mediates, in part, the host response to acute virus infections.


Materials and Methods
The human diploid foreskin fibroblast strain FS-4 was used throughout ; procedures for its growth in cell culture, for IFN-02/IL-6 mRNA induction, for RNA blot-hybridization analyses, and for "S-methionine-labeling of secreted IFN-02/IL-6 proteins have been described earlier (4,12,13) . The human hepatoma cell line Hep3B clone 2 was obtained from the American Type Culture Collection, Rockville, MD (No . HB8064) . Hep3B2 cells were grown in T-75 flasks in Eagle's MEM supplemented with 10% (vol/vol) heat-inactivated FCS (Gibco, Grand Island, NY), 0 .1 mM pyruvate (Gibco), and "MEM nonessential amino acids" (Gibco) . The procedure for assaying hepatocyte-stimulating factor activity (increased synthesis and secretion of a t -antichymotrypsin) has been described elsewhere (4) . Medium from virus-infected FS-4 cell cultures was centrifuged at 40,000 rpm for 90 min in an SW50 .1 rotor (Beckman Instruments, Inc ., Palo Alto, CA) and then y-irradiated (90,000 rad) before addition to hepatocyte cultures .
Virus Stocks and Procedurefor Infection of Fibroblasts. Seed stocks of Sendai and Newcastle disease viruses were passaged in embryonated chicken eggs . Egg-grown Sendai and Newcastle disease virus preparations had titers of 8,000 and 4,000 hemagglutination units (HAU) per ml, respectively (1 HAU/ml corresponds to -105 plaque forming units (PFU) per ml) . Vesicular stomatitis virus (Indiana strain) was grown in chicken embryo fibroblast cultures (titer in the range of 10'-to 10' PFU/ml) . Encephalomyocarditis virus was grown in murine L cell cultures (titer 6 x 10' PFU/ml) . Influenza virus type A strains WSN and Udorn (titers in the range of 1,000-4,000 HAU/ml) were grown in embryonated chicken eggs . Pseudorabies virus was grown in HeLa cells (titer 4 .8 x 10' PFU/ml). Adenovirus type 5 grown in HeLa cells was purified through a cesium chloride gradient (titer 6 .4 x 10' 2 virus particles/ml). Hyperimmune horse anti-Sendai virus antiserum was used in some of the neutralization experiments .
Confluent cultures of FS-4 cells in T175 flasks 5-8 d after the previous medium change were rinsed twice with PBS and then incubated with 2 ml/flask of Eagle's MEM containing the virus at an appropriate concentration . The virus was allowed to adsorb for 1-1 .5 h at 37°C, the cell sheets were then washed 10 times with PBS, and the flasks were replenished with either Eagle's MEM supplemented with 5% FCS or with the appropriate medium for "'IS-methionine labeling .
Measurement ofBacterial LPS(Endotoxin) Levels. Endotoxin levels in the different virus preparations at the highest concentrations used in Fig . 1 were measured using the QCL-1000 Quantitative Chromogenic Limulus Amebecyte Lysate assay kit (Whittaker M . A . Bioproducts, Walkersville, MD) calibrated using an Escherichia coli strain 0111 :134 endotoxin standard . In this assay, which was verified to be linear in the range of 0 .1-1 .0 endotoxin U/ml (10-100 pg/ml), only the pseudorabies virus preparation contained a measurable level of endotoxin (-50 pg/ml) . This concentration has already been shown to be insufficient to affect IFN-02/IL-6 gene expression in human diploid fibroblasts (13) .

Results and Discussion
Enhancement of IFN-/IZ/IL-6 mRNA Levels in Virus-infected Fibroblasts. into the culture medium (2-15-fold increase) . In these gels, the 28-30-kD species are clearly resolved from the 23-25-kD proteins . The relative levels of IFN-02/IL-6 secretion observed approximately correlate with the RNA blot-hybridization data in Fig . 1 . It is remarkable that even as the EMC-infected cells are undergoing pathological changes (note the additional high molecular mass protein bands that contaminate the immunoprecipitate, particularly at the higher multiplicity of infection), the cultures continue to secrete high levels of IFN-02/IL-6 ( Fig . 2) . Thus, although the RNA data in Fig. 1 did not reveal a clear enhancement in IFN-02/IL-6 mRNA levels after Ad5 or Flu infection (this experiment represents a comparison of mRNA levels at one particular time after virus infection), monitoring of the cumulative synthesis and secretion of IFN-R2/IL-6 over a 6-h period (Fig . 3) reveals increased production of the cytokine (also see Fig . 4) .
In additional experiments, we have observed that stimulation of FS-4 cells with different concentrations of Sendai or PR viruses combined with bacterial LPS in the concentration range of 100-10 ng/ml did not display any synergistic or even an additive enhancement of IFN-02/IL-6 protein secretion (not shown). The induction of IFN-02/IL-6 proteins by the viral stimuli was the dominant determinant in these experiments .
Hepatocyte-stimulating Activity of Medium from Virus-infected FS-4 Cell Cultures . The outstanding biological effect of IFN-02/IL-6 is its ability to elicit the major acute phase alterations in plasma protein secretion by hepatocytes . We have evaluated medium from virus-infected FS-4 cell cultures for its ability to enhance atantichymotrypsin secretion by human hepatoma Hep3B2 cells ; secretion of atantichymotrypsin by hepatocytes is enhanced by IFN-R2/IL-6 but not by other cytokines such as TNF (6) . Fig . 4 illustrates an experiment showing the enhanced synthesis and secretion of the positive acute phase protein al-antichymotrypsin by Hep3B2 cells exposed to culture media from FS-4 cells infected with Sendai, NDV  . Culture media from virus infected human fibroblasts enhance acute phase plasma protein secretion by hepatocytes . Human Hep3B2 cells in 24-well multiwell plates were exposed to a 1:5 dilution of ultracentrifuged and irradiated samples of media from FS-4 cell cultures infected with Sendai, NDV, VSV, Ad5, and Flu as described in legend to Fig. 1. The synthesis and secretion of al'-antichymotrypsin was evaluated by labeling the Hep3B2 cells with "S-methionine followed by immunoprecipitation of the labeled protein from the culture medium ; the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. By densitometric scanning, there was an 8-15-fold increase in al-antichymotrypsin secretion in this assay when Hep3B2 cells were exposed to media from virusinfected fibroblasts. Hep3B2 cells treated with media from EMC-or PR-infected FS-4 cells rapidly lifted off the substratum . VSV, Ad5, or Flu viruses (8-15-fold enhancement) . Culture media from EMC and PR-infected fibroblasts had cytocidal effects on Hep3B2 cells ; thus these assays are not shown .
In this article, we report that virus infection strongly enhances the expression and secretion of IFN-R2/IL-6 proteins by the ubiquitous stromal fibroblast . The secreted IFN-02/IL-6 may mediate aspects of the host response (acute phase and immune) to viral infections and may itself contribute to the symptoms of viral disease such as pyrexia (recombinant E. toll'-derived human IFN-R2/IL-6 has been reported to be highly pyrogenic ; see reference 9) . The development of assays that detect IFN-02/IL-6 in body fluids (9), the cloning and expression of the murine IFN-02/IL-6 gene, and the availability of serologic reagents that neutralize IFN-02/IL-6 (4) now permit an evaluation of the biological importance of IFN-02/IL-6 in human and murine virus infections .

Summary
We have defined the expression of the mRNA for, and secretion of, IFN-R2/hepatocyte-stimulating factor/IL-6 (IFN-R2/IL-6) in human diploid fibroblasts (FS-4 strain) infected with different RNA-and DNA-containing viruses . RNA blothybridization analyses carried out 6-8 h after the beginning of infection showed that the RNA-containing Sendai virus (paramyxoviridae) enhanced IFN-R2/IL-6 mRNA levels 10-fold, followed, in decreasing order, by encephalomyocarditis (EMC, picornaviridae), vesicular stomatitis (VSV, rhabdoviridae), Newcastle disease virus (NDV, paramyxoviridae), and influenza A (Flu, myxoviridae) viruses . The DNA-containing pseudorabies virus (PR, herpesviridae) enhanced IFN-02/IL-6 mRNA levels sixfold, while the effect of adenovirus type 5 (Ad5, adenoviridae) was considerably less and comparable with that of NDV or Flu . A rabbit antiserum raised against E. coli-derived human IFN-02/IL-6 was used in immunoprecipitation experiments to monitor the secretion of "S-methionine-pulse-labeled IFN-02/IL-6 proteins by fibroblasts up to 7 h after the beginning of infection . Enhanced levels of secretion of IFN-02/IL -6 (2-14-fold) were observed in every instance evaluated (Sendai, EMC, VSV, Flu, PR, Ad5 viruses) . A biological consequence of enhanced secretion of IFN-(32/IL-6 was the ability of media from infected FS-4 cell cultures to enhance by 8-15fold the synthesis and secretion of a typical acute phase plasma protein (a,-