Proc. Natl. Acad. Sci. USAVol. 84, pp. 7251-7255, October 1987Immunology

Interferon f32/B-cell stimulatory factor type 2 shares identity withmonocyte-derived hepatocyte-stimulating factor and regulatesthe major acute phase protein response in liver cells

(inflammation/acute phase reactants/gene regulation)

JACK GAULDIE*t, CARL RICHARDS*, DEL HARNISH*, PETER LANSDORPt, AND HEINZ BAUMANN§*Molecular Virology and Immunology Program, Department of Pathology, McMaster University, Hamilton, ON, Canada, L8N 3Z5; tTerry Fox Laboratory,British Columbia Cancer Research Centre, Vancouver, BC, Canada, V5Z 1L3; and §Department of Molecular and Cellular Biology, Roswell Park MemorialInstitute, Buffalo, NY 14263

Communicated by Igor Tamm, July 13, 1987 (received for review May 2, 1987)

ABSTRACT One of the oldest and most preserved of thehomeostatic responses of the body to injury is the acute phaseprotein response associated with inflammation. The liverresponds to hormone-like mediators by the increased synthesisof a series of plasma proteins called acute phase reactants. Inthese studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes tointerferon (82 (IFN-fi2), which has been cloned. Antibodiesraised against fibroblast-derived IFN-13 having neutralizingactivity against both IFN-P1 and -182 inhibited the majorhepatocyte-stimulating activity derived from monocytes.Fibroblast-derived mediator elicited the identical stimulatedresponse in human HepG2 cells and primary rat hepatocytes asthe monocyte cytokine. Finally, recombinant-derived humanB-cell stimulatory factor type 2 (IFN-fi2) from Escherichia coliinduced the synthesis of all major acute phase proteins studiedin human hepatoma HepG2 and primary rat hepatocytecultures. These data demonstrate that monocyte-derivedhepatocyte-stimulating factor and IFN-J82 share immunologicaland functional identity and that IFN-(32, also known as B-cellstimulatory factor and hybridoma plasmacytoma growth fac-tor, has the hepatocyte as a major physiologic target andthereby is essential in controlling the hepatic acute phaseresponse.

The systemic response to tissue injury caused by infection ortrauma is the well-recognized series of humoral and cellularreactions known collectively as the acute inflammatoryresponse. This response consists of leukocytosis, fever,increased vascular permeability, alterations in plasma metaland steroid concentrations, along with increased levels ofliver-derived plasma proteins (1, 2). The liver responds earlyto trauma with increased uptake ofamino acids as well as ironand zinc. In addition, there is a marked increase in thehepatocyte mRNA for a number of plasma proteins. Theseinclude a1-acid glycoprotein, a1-proteinase inhibitor (a1-antitrypsin), a1-antichymotrypsin, haptoglobin, hemopexin,and fibrinogen in most species, along with C-reactive protein,C3, and factor B complement components and serum amyloidA protein in humans, and a2-macroglobulin and a1-cysteineproteinase inhibitor (major acute phase protein) in the rat.The change in mRNA is followed within a few hours by anincrease in the secretion of the proteins by the liver andincreased plasma levels of these acute phase reactants within24 hr. The acute phase reaction has been the subject of recentreviews (3, 4).The identity of the putative hormone-like messenger,

released at the site of injury and traveling to the liver, as

suggested by Koj (5), has received considerable attention oflate. It now appears certain that most nonhepatic acute phasereactions can be attributed to the release of a family ofpolypeptides, originally described as leukocyte endogenousmediator or endogenous pyrogen (6, 7), now known asinterleukin 1 (IL-1), and a second series of peptides known astumor necrosis factor (TNF). These polypeptide cytokinesinteract with a broad spectrum of target tissues and wouldappear to control major components of the inflammatoryresponse (8, 9).Using in vitro hepatocyte cell cultures of primary rat

hepatocytes and rat and human hepatoma cells, we andothers have shown that IL-1 and TNF induce only a restrictedacute phase response in vitro (10-15). The full hepatic acutephase protein response is controlled by a separate cytokine,originally described as fibrinogen-stimulating factor (16) andmore recently known as hepatocyte-stimulating factor (HSF)(17-19). Human monocyte-derived HSF is a polypeptidereleased by activated monocytes and macrophages, whichelutes from molecular sieve chromatography as a 25- to30-kDa protein and has an isoelectric point of 5 (18, 20, 21).Human keratinocytes release HSFs with similar activities onhepatocytes (22). The cytokines IL-1 and TNF control onlya subset of the acute phase protein genes (including a1-acidglycoprotein, C3, and haptoglobin) in human and rathepatocytes while monocytic as well as keratinocytic HSFcontrols the expression of the remaining acute phase proteins(including the antiproteinases and fibrinogen) and has a lesserbut still significant effect on the first subset of acute phaseproteins (refs. 12 and 23; H.B., C.R., and J.G., unpublisheddata). IL-1 and TNF do not induce the expression of thesecond group of proteins (see Table 2; Results). It is impor-tant here to define a HSF on the basis of its functionalproperties: a hormone that interacts with liver cells to inducethe synthesis of a spectrum of plasma proteins that arecharacteristic of a-hepatic acute phase reaction.We have purified human monocyte-derived HSF and noted

a striking similarity in characteristics with a recently de-scribed hybridoma growth factor (24, 25). A number of celltypes including fibroblasts, T cell lines, monocytes, andendotheEuli-cells release such a factor(s) capable of stimulat-ing the growth of murine B-cell hybridoma and plasmacytomacells. This molecule (24) has been shown to be identical to a

Abbreviations: HSF, hepatocyte-stimulating factor; IL-1, interleu-kin 1; rIL, recombinant IL; TNF, tumor necrosis factor; rTNF,recombinant TNF; IFN-,3, -,/I, and -182, interferon (, 1, and 132;BSF-2, B-cell stimulatory factor type 2; rBSF-2, recombinant BSF-2;PBM, peripheral blood monocyte; LAF, lymphocyte-activatingfactor.tTo whom reprint requests should be addressed at: MolecularVirology and Immunology Program, Department of Pathology,McMaster University, 1200 Main Street West, Hamilton, ON,Canada, L8N 3Z5.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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H-cell stimulating factor (BSF-2), which has been cloned andis identical to a protein (26-kDa protein), both of which havethe same cDNA sequence as the originally described se-quence for interferon P2 (IFN-p2) (34). These relationshipshave been summarized in three recent commentaries (27-29).In view of the collective data and the similarity in charac-teristics, we explored the possibility that HSF and IFN-832shared identity. We report here that antibodies raised againsthuman fibroblast-derived IFN-,3 neutralize monocyte-de-rived HSF activity and that purified Escherichia coli-derivedrecombinant human BSF-2 (rBSF-2) (IFN- 2) has many, ifnot all, of the properties of and is likely identical to humanHSF.

MATERIALS AND METHODS

Cytokine Preparations. Human peripheral blood mono-cyte-conditioned medium (PBM-CM) was prepared as de-scribed (18). In brief, monocytes, separated by buoyantdensity centrifugation and further purified by adherence,were stimulated with lipopolysaccharide (10 gg/ml) (E. coli055:B5, trichloroacetic acid-extracted; Sigma) for 24 hr. Thesupernatant was dialyzed (Spectropore 6-8000) against phos-phate-buffered saline and sterilized by filtration through a0.22-I&m membrane (Millipore). Human fibroblast-condi-tioned medium (FCM) obtained from supernatant of humanlung fibroblasts, cultured for 24 hr at 0.2 x 106 cells per mlin T24 Linbro plates, was obtained from primary human lungfibroblast lines that have been established from normal lungexplants (30). Supernatants from fibroblasts cultured inDulbecco's modified Eagle's medium (DMEM) with 10%ofetal calf serum (GIBCO) or from cells cultured in serum-freemedium containing various concentrations (5-20 units/ml) ofplatelet-derived growth factor (Collaborative Research) andcultured for 24 hr, were dialyzed and filter sterilized asdescribed above.Hepatocyte/Hepatoma Acute Phase Protein Assays. The

regulation of plasma protein production was analyzed usinghuman hepatoma HepG2 cells and primary cultures of rathepatocytes as described (12, 20). HepG2 cells were gener-ously provided by B. Knowles (The Wistar Institute, Phila-delphia). These cells were maintained in DMEM containing10% heat-inactivated fetal calf serum and passaged every10-14 days.Primary cultures of rat hepatocytes were prepared from

adult Sprague-Dawley rats by collagenase perfusion andadherence isolation as described (20). The cells were platedat 0.2 x 106 cells per well in T24 Linbro plates and were usedwithin 24 hr of isolation. HSF activity and the induction ofacute phase protein synthesis were determined as describedfor HepG2 cells (12) and for rat hepatocytes (20). One unit ofHSF activity is defined as the concentration of cytokinerequired to induce one-third of the maximal stimulation ofa1-antichymotrypsin in HepG2 cells (12) and one-half of themaximal stimnulation of a2-macroglobulin in rat hepatocytes(20). The assays have previously been shown to be repro-ducible having a within-assay coefficient of variation (CV) of4% and between-assay CV of10% (20). In each case, the cellswere exposed to cytokine-containing medium for varioustimes by repeated addition in fresh medium. The amounts ofacute phase proteins secreted into the medium during thefinal period ofexposure in culture were determined by rocketimmunoelectrophoresis using monospecific antisera fpr thevarious proteins prepared in the author's laboratory for ratproteins and commercially available antisera for humanproteins (18, 19). Purified preparations of plasma proteinswere used as stahdards for quantitation and results arequoted as ,ug of protein synthesized by 106 cells (hepatocytesor hepatoma cells) per 24-hr period.

IL-1 Assay. The assay for lymphocyte-activating factoractivity (LAF) of IL-1 preparations was carried out using thephytohemagglutinin costimulator assay and C3H/HeJ thy-mocytes as described (18).Antibody Preparations and Neutralizations. Rabbit anti-

human recombinant TNF (rTNF) was obtained through R. S.Warren and L. Old (Memorial Sloan-Kettering Cancer Cen-ter), as a gift from M. Palladino (Genentech, South SanFrancisco). One microliter of antiserum neutralizes theactivity of 50 ng of TNF.

Rabbit anti-human recombinant IL-lp (rIL-1/3) was kindlyprovided by K. Matsushima (Frederick Cancer ResearchFacilities, Frederick, MD).Rabbit anti-human rIL-la was kindly provided by M.

Yamada (Dainippon Pharmaceutical Ltd., Osaka, Japan).Rabbit anti-IFN-,31 was provided by K. Chadha (Roswell

Park Memorial Institute, Buffalo, NY). This antibody wasraised against highly purified human fibroblast-derived IFN-P1. One milliliter of antibody neutralizes 1000 units of IFN-f31activity.Sheep anti-IFN-p8 and control sheep antiserum were ob-

tained from the National Institutes of Health (researchreference reagent, catalogue no. G-028-501-568). This anti-body was raised against poly(I-C)-stimulated fibroblast-de-rived IFN-pS and recognizes both the IFN-f31 and IFN-f32molecules.

Calf anti-IFN-f3 was kindly provided by J. Vilcek (NewYork University School of Medicine). This antibody wasraised against poly(I-C)-stimulated human fibroblast-derivedIFN-p8 and recognizes both the IFN-,81 and IFN-p2 mole-cules.Monoclonal anti-human IFN-, was obtained from Boeh-

ringer Mannheim (catalogue no. 853577).Absorptions with the various antisera were carried out by

incubating a standard pool of PBM-CM with increasingamounts of each antibody and control serum. The superna-tant was incubated 1 hr at 37°C, sterilized by milliporefiltration (0.22 ,Lm), and tested in the hepatocyte assay andLAF assay for residual activity.Recombinant Cytokines. Purified human rTNF (5 x 107

units/mg) was kindly provided by M. Palladino. Purifiedhuman rIL-la (6 x 108 units/mg) and rIL-1p (2 x 108units/mg) were generously provided by D. L. Urdal (Im-munex). Purified E. coli-derived rBSF-2 (2 x 105 units/ml)was generously provided by T. Hirano and T. Kishimoto(Institute of Molecular and Cellular Biology, Osaka, Japan)(31).

RESULTSAntibody Absorption. In previous studies using primary rat

hepatocytes and rat and human hepatoma cells, we haveshown that human rIL-la and rIL-1,B as well as human rTNFdo not elicit a full characteristic hepatic acute phase responsein vitro (11, 12). In addition, antibodies against these variouscytokines could not inhibit the HSF activity present in PBMsupernatants. In our first approach to examine the identity ofHSF, we determined the effect of preincubation with variousantibody preparations on the HSF activity of PBM-CM,which contains a complex mixture of these various activities(Fig. 1). Using a standard stimulus of PBM-CM, preabsorp-tion with anti-IL-la and anti-IL-1p inhibited essentially all ofthe LAF activity (150 units/ml down to 3 units/ml), 80% ofthe a1-acid glycoprotein induction and 20-25% ofthe albuminreduction. No significant effect was seen following treatmentofPBM-CM with anti-TNF. Since the antibodies against IL-1and TNF are polyclonal and raised against recombinantmaterial, lack of reactivity with HSF would rule out aserologic relationship between these cytokines. There was nochange in a2-macroglobulin induction, while there was an

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A

AGP

A

A

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1 2 3 4 5 S 7

C

FIG. 1. Antibody inhibition4 of PBM-derived cytokine activ-

HP ity on HepG2 cells and rat

hepatocytes. Crude PBM super-natant (diluted 1:20) was pre-treated for 1 hr at 37°C with

j4* control fetal calf serum (lane 1),rabbit anti-TNF (lane 2), rabbit

3 4 5 6 7 anti-IFN-/3l (lane 3), rabbit anti-IL-la and rabbit anti-IL-1,l (lane4), sheep anti-IFN-j3 (lane 5),

ALB sheep anti-IFN-,B and rabbit an-ti-IL-la and -,8 (lane 6), and

z cultured with hepatocytes ands i compared to hepatocytes cul-l̂r;S tured in medium only (lane 7).

The samples were then assayedby rocket electrophoresis forstimulation of synthesis of a,-acid glycoprotein (AGP) (A), fi-

-7P brinogen (FBG) (B), and hapto-globin (HP) (C) in HepG2 cells;

iSPI a2-macroglobulin (aM) (lightA i*>st precipitin line) and albumin

A J * (ALB) (dark precipitin line) (D)as well as cysteine proteinaseinhibitor (CPI) (E) in rat hepa-

4 5 6 7 tocyte cultures.

apparent increase in the induction of fibrinogen and cysteineproteinase inhibitor, indicating that inhibition of IL-1 doesnot inhibit HSF activity and that IL-1 may actually inhibit theinduction by HSF of these two molecules, confirming ourprevious data (11). Absorption with either sheep or calfanti-IFN-,8 (raised against IFN-P but recognizing both IFN-P1 and IFN-p2 epitopes) totally inhibited the HSF activity(a2-macroglobulin, fibrinogen, etc.) but did not alter the LAFcontent ofthe supernatant. Absorption with a combination ofanti-IFN-f3 and anti-IL-la,3 removed all acute phase proteininduction activity from PBM-CM. Absorption with specificanti-IFN-f31, either monoclonal (data not shown) or polyclo-nal, did not alter any of the responses (Fig. 1). IFN activityof the PBM-CM was not directly determined. Absorptionswith preimmune and control sera (normal rabbit, sheep, calf)had no significant effect on any of the activities.The quantitative nature of the absorptions is shown for a

second experiment in Table 1.Fibroblast Supernatants. Having shown immunologic iden-

tity between monocyte-derived HSF and an activity recog-nized by anti-fibroblast-derived INF-,B, we determinedwhether primary human lung fibroblast lines synthesized amolecule having HSF activity. Table 2 shows that superna-tants from human lung fibroblasts constitutively synthesizedan activity that induced significant stimulation of the samespectrum of acute phase proteins by human and rat

Table 1. Effect of preabsorption of PBM supernatant withantibodies to IL-la,,8 and IFN-,3 and testing for remaininghepatocyte-stimulating activity

Secretion, jig per 24 hrper 106 cells

Treatment Fibrinogen ACH AGP HP

HepG2 cellsControl 0.1 2.0 0.3 0.3PBM (1:20) 0.6 10.4 6.0 2.1PBM (1:20) + anti-IL-1(a,4) 1.4 11.8 1.4 1.5PBM (1:20) + anti-IFN-p 0.1 5.0 2.6 0.8PBM (1:20) + anti-IL-1(a,,/) 0.1 4.2 0.4 0.4and anti-IFN-,B

a2-macro CPI Albumin

Rat hepatocytesControl 7 24 38PBM (1:20) 52 32 15PBM (1:20) + anti-IL-1(a,48) 51 39 21PBM (1:20) + anti-IFN-,B 12 25 28PBM (1:20) + anti-IL-1(a,4) 10 24 35and anti-IFN-,BValues were calculated from rocket immunoelectrophoresis using

purified protein standards for calibration. a2-macro, a2-macroglob-ulin; CPI, cysteine proteinase inhibitor; ACH, al-antichymotrypsin;AGP, a, acid glycoprotein; HP, haptoglobin; PBM (1:20), PBM-CM.

hepatocytes. The same supernatant did not exhibit any IL-1activity in a LAF assay. Stimulation of the cells withplatelet-derived growth factor (5-20 units/ml) resulted in a 5-to 10-fold increase in HSF activity (data not shown). Theamount of HSF activity in platelet-derived growth factor-stimulated FCM was approximately equal, on a per cell basis,to that found in PBM-CM.

rBSF-2. With the antibody and FCM studies suggestingidentity, we next determined whether purified E. coli-derivedhuman rBSF-2 could duplicate the effects of PBM-derivedHSF. Purified human rBSF-2 showed a dose-dependentstimulation of human HepG2 cells and primary rat hepato-cytes with the identical spectrum of acute phase protein gene

Table 2. Stimulation of acute phase protein synthesis bytreatment with human cytokines

Secretion, jg per 24 hrper 106 cells

Treatment Fibrinogen ACH AGP HP

HepG2 cellsControl 0.1 1.3 0.3 0.4rIL-1,B (250 units/ml) <0.01 3.1 1.8 1.2PBM-CM (1:20) 0.4 11.6 5.8 2.2HSF (20 units/ml) 1.0 8.0 1.1 1.2FCM (1:10) 1.4 8.1 0.9 1.5rBSF-2/IFN-32 (60 units/ml) 1.7 9.9 1.4 1.7

a2-macro CPI Albumin

Rat hepatocytesControl 7 25 40rIL-1/3 (250 units/ml) 8 23 33PBM-CM (1:20) 50 30 13HSF (20 units/ml) 45 35 14FCM (1:10) 42 33 29rIFN-p2/BSF-2 (60 units/ml) 46 38 14

Values represent the mean of duplicate cultures. a2-macro, a2-macroglobulin; CPI, cysteine proteinase inhibitor; ACH, a1-antichy-motrypsin; AGP, a,-acid glycoprotein; HP, haptoglobin; FCM,fibroblast conditioned medium (10%6 FCS). Purified protein stan-dards were used to calibrate the rocket immunoelectrophoresis.

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expression as is shown with PBM-derived HSF and fibroblastHSF (Fig. 2). Not only were the various proteins induced, butalbumin was coincidentally decreased in synthesis by rBSF-2. Maximum stimulation of a2-macroglobulin synthesis by rathepatocytes or fibrinogen synthesis by HepG2 cells wasachieved with 60 units of BSF-2 and with 40 units ofPBM-derived HSF. Absorption of the purified rBSF-2 witheither sheep or calf anti-IFN-p totally inhibited the HSFactivity caused by the recombinant molecule (data notshown).

DISCUSSIONInduction of the acute phase protein response during inflam-mation has become an important model for the study oftissue-specific gene regulation. Knowledge of the signals thatinitiate this hepatocyte response is a necessary prerequisiteto a complete understanding of the cellular mechanisms andmolecular machinery involved. While the cytokines IL-1 andTNF have been implicated in this control, in previous workwe and others have shown that these cytokines can onlyaccount for a limited portion of the rat and human in vitrohepatic acute phase response (10-15). Using either purifiedor rIL-1 and rTNF or absorption studies with specificantibodies to these molecules, we have shown that thesecytokines appear to stimulate C3, haptoglobin, and al acidglycoprotein in rat hepatocytes and human and rat hepatomacells (10-12). There is no evidence for stimulation of a2-macroglobulin, cysteine proteinase inhibitor, or fibrinogen.In fact, rIL-1,8 appears inhibitory to HSF stimulation offibrinogen and cysteine proteinase inhibitor in rat and humanHepG2 cells (Fig. 1, Table 1), similar to that seen in Hep3B2cells (13). In addition to these cytokines, glucocorticoid playsa permissive and potentiating role in the induction of theacute phase protein response in rat and human hepatocytes(ref. 19; H.B., C.R., and J.G., unpublished data).Our attempts to purify the molecular activity responsible

for the major spectrum of acute phase protein inductionshowed that lipopolysaccharide-activated monocytes re-leased a hepatocyte-stimulating factor, which stimulated the

r .s ..

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A.lm&:.x.

At

2 3 4 5 6 7

FIG. 2. Activity of rBSF-2 (IFN-92) on rat hepatocyte acutephase protein production. Various concentrations of purified rBSF-2were added to rat hepatocyte cultures. a2-Macroglobulin (lightprecipitin line) and albumin (dark precipitin line) production inhepatocyte supernatants was analyzed by rocket electrophoresis.Hepatocytes were stimulated with 160 units/ml (BSF-2 activity)(lanes 1 and 2), 40 units/ml (lane 3), 4 units/ml (lane 4), 0.4 unit/ml(lane 5). Lanes 6 and 7 contain control (nonstimulated) supernatant.

production in H35 rat hepatoma and primary rat hepatocytesof the antiproteinases (cysteine proteinase inhibitor, a2-macroglobulin, contrapsin, a1-antitrypsin), hemopexin, andfibrinogen in a major way, while having a lesser but signifi-cant stimulating effect on the synthesis of a1-acid glycopro-tein, haptoglobin, and C3 (12). HSF and IL-183 along withpermissive levels of glucocorticoid are likely to represent thefull content of hepatocyte-specific activities in PBM-CM.The similar molecular characteristics that we initially noted

between monocyte-derived HSF and hybridoma growthfactor was confirmed by antibody absorptions of PBM CM(Fig. 1, Table 1). Antibodies raised against human fibroblast-derived IFN-P, having activity against IFN-f31 and -P2 (32),were able to neutralize the HSF activity, leaving only theIL-1-mediated hepatocyte activity and LAF activity. Furtherabsorption with anti-IL-1, which could not neutralize theHSF activity effectively, removed the remaining IL-1-medi-ated hepatocyte specific activity, indicating a lack ofserologic identity between HSF and IL-1. A rabbit antibodymonospecific for fibroblast derived IFN-f31 had no effect onHSF or LAF activity. Thus, we could account for most if notall of the hepatocyte-stimulating activity in human mono-cytes as being due to the presence of a molecule recognizedby anti-IFN-P2 and to the presence of IL-1p8. In addition, weshowed that both sources of anti-IFN-,B antisera couldimmunoprecipitate 23- and 26-kDa proteins from L-[35S]me-thionine-labeled lipopolysaccharide-stimulated monocyte su-pernatants and that the immunoprecipitation could be inhib-ited by rBSF-2 (data not shown), confirming reports that the1.3-kilobase IFN-,82/BSF-2 mRNA is expressed in humanmonocytes (33).We were able to show that fibroblasts release a molecule

that stimulates hepatocytes in an identical manner to mono-cyte HSF (Table 2) and the activity could be totally absorbedwith the same anti-IFN-/3 that inhibited HSF. Interestingly,this neutralization removed all hepatocyte-stimulating activ-ity because the FCM did not contain IL-1 or TNF (data notshown). Thus, the monocyte HSF is serologically related tofibroblast HSF and in turn to fibroblast IFN-,82. The apparentmolecular mass of the monocyte-derived material and thatfrom fibroblasts calculated from elution behavior from HPLCgel filtration columns differs by some 2-4 kDa, even thoughthey share immunologic and functional identity (Fig. 1, Table2). This difference could be due to different degrees ofglycosylation. Such differences would appear not to besignificant to the HSF activity of the molecule as both PBMand fibroblast-derived material showed identical activity.The proposal that HSF and IFN-182 are identical was

further confirmed by showing that purified rBSF-2 (IFN-132)was active on both human and rat cells in a dose-dependentmanner (Fig. 2) and that BSF-2 (IFN-P2) stimulated anidentical spectrum ofprotein synthesis as did monocyte HSF.A comparison of the nucleotide sequences for the various

activities attributed to IFN-p2 suggests (31, 32, 34, 35) limitedhomology with granulocyte colony-stimulating factor (31) inthe amino-terminal region. It is unclear whether the homol-ogy is of biological significance. We were unable to detectany related growth-promoting activity on rat hepatocytes orHepG2 cells with the recombinant sample (data not shown).Human IFN-f32/BSF-2 is effective across species and stim-ulates rat hepatocytes equivalent to human cells in keepingwith the lack of species specificity of its other growth- andsynthesis-stimulating activities on B cells and hybridomas(25, 31). We were unable to detect any HSF activity in humanPDGF or colony-stimulating factor 1 preparations (data notshown).The identification of IFN-f32/BSF-2 as the major inducer of

acute phase protein synthesis completes the spectrum ofcytokines that control hepatocyte acute phase gene expres-sion. IFN-,62/BSF-2 along with IL-183, IL-la, TNF, and

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glucocorticoid can now fully account for the stimulation ofthe acute phase protein response in rat liver cells and formany changes seen in human hepatoma cells. While IL-1 andTNF may participate directly in the hepatocyte response, thefact that these two cytokines are potent inducers of IFN-f2/HSF from fibroblasts (36-39) suggests that in vivo thesecytokines may interact with the hepatocyte indirectlythrough IFN-P32/HSF, the more potent inducer of the livercells.While it had previously been thought that the monocyte

was the sole source for leukocyte endogenous mediator orHSF, it is now clear that many cells can release this potentacute phase protein inducer (27, 28). Since the level ofexpression of many acute phase protein genes is constitu-tively low, it is likely that most cell types will not be activelysecreting IFN-32/HSF. At tissue sites of inflammation,activated monocytes and activated T cells may release themolecule directly. Fibroblasts upon stimulation by injury orwith growth factors such as platelet-derived growth factor(40) or in response to IL-1 or TNF exposure can also releaseIFN-f32/HSF. This would appear to represent a useful am-plification pathway for the release of a molecule that controlsone of the major homeostatic responses in the body. Thesedata, and others showing that monocytes, activated T cells,fibroblasts, and other epithelial cells are all capable ofreleasing IFN-,32/HSF (27, 28), indicate it will be importantto examine which cells are involved in the various inflam-matory responses to determine the origin of the signalcontrolling the hepatic response. It will also be important torepeat the studies of Woloski et al. (41) using recombinantmaterial to confirm the activity of IFN-,32/HSF in causingrelease of corticotropin from pituitary cells, an observationwhich suggests a role for the molecule in the adrenocorticalresponse in host defense. Understanding HSF and the role itplays in the initiation of the acute phase response is a primarygoal, but, in addition, the identification and availability of themediator in pure form will be important in studying themolecular biology of gene regulation in hepatocytes.While there may be a role for the cytokine in the stimula-

tion of the B cell response, and the importance of the IFNactivity of the molecule is not known (27, 28), the dataoutlined above suggest that the most likely primary physio-logic target of IFN-/2 is the hepatocyte with the mostprominent role reflected in the induction of the acute phaseprotein response. In light of these findings, we suggest thatthe molecule previously known as IFN-02, 26-kDa protein,hybridoma growth factor, and BSF-2 should be more appro-priately called hepatocyte-stimulating factor (HSF) or IFN-f32/HSF, acknowledging historical precedence (26, 42).

The authors wish to acknowledge helpful discussions with Dr. A.Koj and Dr. P. B. Sehgal, the technical help of Mrs. J. A. Schroeder,and the secretarial assistance of Mrs. N. Thompson. This work wassupported by Canadian Medical Research Council Grant MRCMA8616 to J.G. and National Institutes of Health Grant CA26222 toH.B. H.B. is a recipient of an American Heart Association Estab-lished Investigatorship Award.

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