Tumor necrosis factor increases the number of epidermal growth factor receptors on human fibroblasts

Tumor necrosis factor (TNF) was shown previously to stimulate the growth of human FS-4 fibroblasts. Here we show that human recombinant TNF can increase the binding of epidermal growth factor (EGF) to these cells. Incubation with TNF resulted in a 40-80% increase in the number of EGF-binding sites with no apparent change in receptor binding affinity. The increase in EGF binding was apparent 8-12 h after the addition of TNF. TNF also increased the amount of EGF receptor protein immunoprecipitated from cells labeled with [35S]methionine. Stimulation of EGF receptor protein synthesis was demonstrable 2-4 h following TNF treatment. TNF increased EGF binding with a dose-response relationship similar to that reported earlier for the mitogenic action. Increased expression of EGF receptors, due to enhanced synthesis of the EGF receptor protein, may be functionally related to the mitogenic action of TNF in human fibroblasts.     

Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts

The effect of cell density on low density lipoprotein (LDL) binding by cultured human skin fibroblasts was investigated. Bound LDL was visualized by indirect immunofluorescence. Cellular lipid and cholesterol were monitored by fluorescence in cells stained with phosphine 3R and filipin, respectively. LDL binding and lipid accumulation were compared in cells in stationary and exponentially growing cultures, in sparsely and densely plated cultures, in wounded and non-wounded areas of stationary cultures, and in stationary cultures with and without the addition of lipoprotein-deficient serum. We conclude that LDL binding and cholesterol accumulation induced by LDL are influenced by cell density. It appears that, compared to rapidly growing cells, quiescent (noncycling) human fibroblasts exhibit fewer functional LDL receptors.     

Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.     

Stimulation of receptor-dependent and receptor-independent pathways of low-density lipoprotein degradation in arterial smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF), a powerful mitogen released by platelets, promoted the degradation of low-density lipoprotein (LDL) by cultured primate arterial smooth muscle cells and human skin fibroblasts by stimulating both receptor-mediated and LDL-receptor-independent uptake of LDL. Stimulation of LDL-receptor-independent LDL uptake and degradation by PDGF was demonstrated in three ways. First, the small amount of LDL that was degraded by LDL-receptor-negative skin fibroblasts was stimulated by PDGF. Second, PDGF led to increased degradation of LDL that had been reductively methylated to prevent its binding to LDL receptors. Third, 125I-labeled LDL degradation was stimulated by PDGF in the presence of high concentrations of unlabeled LDL, i.e., conditions under which the contribution of the LDL receptor to cellular uptake and degradation is reduced. These observations suggest that mitogens, as typified by PDGF, can facilitate the cellular delivery of LDL cholesterol by both LDL-receptor-mediated and non-LDL-receptor-mediated mechanisms to provide exogenous cholesterol for use during cell replication.     

Regulation of low-density lipoprotein metabolism by cell density and proliferative state

Stimulation of the proliferation of human skin fibroblasts by platelet-derived growth factor increased the binding and degradation of low-density lipoproteins at cell densities of 2000-30,000 cells/cm2. Binding and degradation of low-density lipoprotein was an inverse function of cell density in both proliferating and quiescent cells, indicating that the effect of cell density on the LDL receptor has proliferation-dependent and proliferation-independent components. The effect of medium conditioned by confluent fibroblasts on LDL metabolism was tested to determine if the effects of cell density on LDL metabolism might be mediated by cellular secretion products. Fibroblast-conditioned medium increased LDL metabolism, suggesting secretion products do not mediate these effects of cell density. These data indicate that regulation of the low-density lipoprotein receptor is not a simple on/off response to growth stimulation, but is responsive to extracellular cues such as cell density.     

Effects of tumor necrosis factor on cell growth and expression of transferrin receptors in human fibroblasts

Human recombinant tumor necrosis factor (TNF) stimulated the growth of confluent human fibroblasts (FS-4) in the presence of fetal calf serum. Epidermal growth factor (EGF) similarly stimulated cellular growth; however other mitogenic factors such as insulin, fibroblast growth factor, 12-O-tetradecanoyl-phorbol-12-acetate and Ca2+ ionophore A23187 did not. The growth-stimulating action of TNF was not synergistic with the activity of EGF in the presence of serum. TNF induced a rapid increase in the binding of transferrin to the cell surface, followed by a return to the basal level within 5 min. A similar increase in transferrin binding was observed in FS-4 cells exposed to EGF. In contrast, insulin caused a prolonged stimulation of transferrin binding. These results suggest that TNF and EGF generate similar or identical intracellular signals for cellular growth and the regulation of transferrin receptor expression.     

Lipoprotein(a) mediates high affinity low density lipoprotein association to receptor negative fibroblasts.

Lipoprotein(a) (Lp(a)) is an acute phase protein with unknown function. Lp(a) binds to low density lipoprotein (LDL) receptors, as well as to plasminogen (Plg) receptors. Preincubation of normal human skin fibroblasts with Lp(a) or with apo(a) cause a severalfold increase of LDL binding. Plg and kringle-4 of Plg have no effect. LDL receptor-negative fibroblasts respond upon preincubation with apo(a) with high affinity binding of LDL with Kd values that are almost identical with those of LDL binding to the LDL receptor. Incubation of apo(a)-pretreated fibroblasts with anti-apo(a) completely abolishes the increment of LDL binding. The high affinity LDL binding to LDL receptor-negative fibroblasts could be dissociated by approximately 80 and 54% with 5 mg/ml proline and 30 mg/ml NaCl, respectively, but not with dextran sulfate. The Lp(a)- and apo(a)-triggered LDL binding to fibroblasts have no effect on LDL internalization. These findings may reflect a key function in the role as an acute phase protein and may be relevant to the high atherogeneicity of Lp(a).     

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation

A synthetic heparin-mimicking polyaromatic anionic compound RG-13577 (polymer of 4-hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr approximately 5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of (14)C-RG-13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor-related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG-13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild-type and heparan sulfate-deficient Chinese hamster ovary (CHO) cells, as well as normal- and LDL receptor negative- human skin fibroblasts bind RG-13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor-related protein (LRP) expressed a markedly reduced binding of RG-13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG-13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG-13577, suggesting that this inhibition is mediated by RG-13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and thereby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.     

Lipid metabolism in cultured cells. XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic and tumor virus-transformed human fibroblasts.

Serum lipoproteins control cell cholesterol content by regulating its uptake, biosynthesis, and excretion. Monolayers of cultured fibroblasts were used to study interactions with human high density (HDL) and low density (LDL) lipoproteins doubly labeled with [(3)H]cholesterol and (125)I in the apoprotein moiety. In the binding assay for LDL, the absence of specific LDL receptors in type II hypercholesterolemic fibroblasts was confirmed, whereas monolayers of virus-transformed human lung fibroblasts (VA-4) exhibited LDL binding characteristics essentially the same as normal lung fibroblasts. In the studies of HDL binding, specific HDL binding sites were demonstrated in normal and virus-transformed fibroblasts. In addition, type II hypercholesterolemic cells, despite the loss of LDL receptors, retained normal HDL binding sites. No significant competition was displayed between the two lipoprotein classes for their respective binding sites over a 5-fold concentration range. In VA-4 cells, the amount of lipoprotein required to saturate half the receptor sites was 3.5 micro g/ml (9 x 10(-9) M) for LDL and 9.1 micro g/ml (9 x 10(-8) M) for HDL. Pronase treatment reduced LDL binding by more than half but had no effect on HDL binding. Chloroquine, a lysomal enzyme inhibitor, stimulated net LDL uptake 3.5-fold by increasing internalized LDL but had essentially no effect on HDL uptake. Further experiments were conducted using doubly labeled lipoproteins to characterize the interaction of LDL and HDL with cells. While the cholesterol and protein moieties of LDL were incorporated into cells at similar rates, the uptake of the cholesterol moiety of HDL was 5 to 10 times more rapid than that of the protein component. Furthermore, the apoprotein component of LDL is extensively degraded following exposure, whereas the apoprotein moiety of HDL retains its macromolecular chromatographic characteristics. These results indicate that HDL and LDL bind to cultured cells at separate sites and that further processing of the two lipoprotein classes appears to take place by fundamentally different mechanisms.-Wu, J-D., J. Butler, and J. M. Bailey. Lipid metabolism in cultured cells XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic, and tumor virus-transformed human fibroblasts.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.     

Purification and characterization of a calf thymus protein active on lipid metabolism

1. This work concerns the purification of a calf thymus protein that increases the binding of human 125I-labeled low density lipoprotein (LDL) on both human skin fibroblasts and a special line of rat liver cells, BRL 3A. 2. It was found that the thymus gland affects cholesterol metabolism via an activation of the LDL receptor pathway. 3. Moreover, the thymus protein active on the LDL receptor pathway has a different amino acid composition and molecular weight from other well-characterized thymic peptides.     

Calmodulin antagonists stimulate LDL receptor synthesis in human skin fibroblasts

The LDL receptor synthesis of human skin fibroblasts in the presence of the specific calmodulin antagonists trifluoperazine, condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) (W-7) was studied. Labelling of cells with [35S]methionine followed by immunoprecipitation of radioactive LDL receptor protein with monospecific antibodies revealed that calmodulin antagonists caused a 3-fold increase in the radioactivity of the LDL receptor protein as compared with values found in control cells. A corresponding increase of high-affinity binding and internalization of 125I-labelled LDL was observed. The drugs did not influence the overall protein synthesis or the half-life of the LDL receptor. A concomitant suppression of cholesterol synthesis from [14C]mevalonolactone was found to be an independent effect. The calmodulin antagonist-produced stimulation of LDL receptor synthesis could not be simulated by preincubation of cells with cyclic nucleotide analogues, cholera toxin or 3-isobutyl-1-methylxanthine, known as specific effectors of adenylate cyclase and cyclic nucleotide phosphodiesterase, respectively. Modulation of calcium concentration in the incubation medium had no reproducible effect on the rate of LDL receptor synthesis. The results implicate calmodulin as an intracellular suppressor of LDL receptor synthesis in human skin fibroblasts.     

Sphingolipid-induced enhancement of receptor-mediated uptake of low density lipoproteins in normal and receptor-deficient human skin fibroblasts

(1) The receptor mediated endocytosis of homologous LDL by human skin fibroblasts can be significantly enhanced by prior incubation of the cells with sphingolipids. Gangliosides G_M1 or G_D1a, their desialylated derivatives and sphingosine stimulate binding and uptake to LDL by up to 40% of normal values. The effect is observed in normal fibroblasts, LDL receptor deficient fibroblasts or in tunicamycin-treated cells with a reduced number of functional receptors but is dependent on the time of preincubation of the cells and the concentration of the sphingolipid in the medium. (2) Detailed studies on the ganglioside effect revealed, that cell bound gangliosides intensify the LDL-induced supression of [¹⁴C]acetate incorporation into cholesterol. (3) The receptor dependence and relative receptor specificity of the sphingolipid effect is evident from the fact that (a) after complete suppression of receptor synthesis gangliosides fail to stimulate uptake of LDL, that (b) fatty acids or lipids not containing sphingosine are without effect and that (c) the receptor specific internalisation of α₂-macroglobulin or epidermal growth factor is not influenced by exogenous sphingolipids.     

Role of net charge of low density lipoproteins in high affinity binding and uptake by cultured cells.

Selective modification of arginine residues of LDL by cyclohexanedione or acetylation of lysine residues of LDL deminishes their high affinity binding and internalisation by human skin fibroblast up to 50% as compared with native LDL. The enhanced negative charge of the modified LDL particles results in an accelerated electrophoretic mobility towards the anode. Neuraminidase treatment of cyclohexanedione-modified LDL and acetyllysine-LDL normalizes not only their electrophoretic mobility, but also restores more than 80% of the original binding and uptake capacity, the specificity of this effect being indicated by using fibroblasts deficient in LDL receptor and by competitive binding and internalization experiments.     

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL (d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were prepared by centrifugation from the plasma of apo E-3/3 or 4/3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50-150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 micrograms protein/10 micrograms IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B,E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with Kd values of 2.50, 0.93 micrograms protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2-3 molecules/particle) and enrichment of IDL with apo E-3 (to 4-5 molecules/particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.     

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.     

Induction of beta 2-interferon by tumor necrosis factor: a homeostatic mechanism in the control of cell proliferation

Earlier studies showed that tumor necrosis factor (TNF) exerts a mitogenic effect in human diploid fibroblasts. Here we demonstrate that purified E. coli-derived recombinant human TNF inhibits encephalomyocarditis virus replication in "aged" human fibroblasts. Addition of neutralizing antibodies to human beta interferon (IFN-beta) blocked the antiviral action of TNF, indicating that this action is mediated by the generation of IFN-beta. We also show that antiserum to IFN-beta enhanced the mitogenic effect of TNF in confluent, serum-starved human fibroblasts, suggesting that induction of IFN-beta by TNF represents a physiological negative feedback mechanism regulating cell proliferation. Blot hybridization analysis of cytoplasmic polyadenylated RNA showed that TNF induced IFN-beta 2 mRNA, whereas no induction of IFN-beta 1 mRNA could be demonstrated. The results suggest that IFN-beta 2 has biological functions distinct from the other interferons.