Schistosomiasis is one of the most important helminthic diseases, estimated to afflict 200 million people in the tropics (Walsh et al., 1979). Two of the schistosome species that infect humans (Schistosoma mansoni and S . japonicum) can cause serious morbidity (including portal hypertension and gastrointestinal hemorrhage) as a result of a form of hepatic fibrosis (Cheever et al., 1967). However, only a relatively small subpopulation (3-6%) of infected individuals develop this scarring; the others remain generally healthy (Chen et al., 1988).

Traditional forms of antihelminthic therapy have a number of undesirable side effects, and treatment with the relatively new drug, praziquantel, is very expensive, thus making antihelmithic treatment of all infected individuals impractical. In addition, while early antihelminthic therapy may aid in preventing liver scarring, it has not been established that this is an invariable outcome of treatment (Homeida et al., 1988). Alternatively, early aggressive treatment of infected individuals with anti-inflammatory or cytotoxic drugs including cytotoxin and various corticosteriods may aid to prevent scarring. However, given that these drugs are known to produce a number of relatively severe side effects, treatment of all infected individuals, 94-97% of which would never develop the progressive fibrotic form of the disease, is both undesirable and impractical. Several other chronic inflammatory diseases, including pulmonary fibrosis, progressive systemic sclerosis, sarcoidosis, sceroderma, sclerosing

colangitis, and rheumatoid arthritis are also known to be involved in the organ dysfunction associated with fibrosis. As in Schisotsomiasis, only a subpopulation of individuals with these diseases develop severe fibrotic scarring. Thus, methods are needed that would make it possible to predict which patients will develop the progressive fibrotic forms of these inflammatory diseases so that more aggressive, antifibrotic courses of treatment can be limited to only those individuals which would benefit from these treatments.

Summary of the Invention In general, the invention features a method for identifying individuals with a propensity for fibrotic scarring. The method comprises providing a sample from an individual with a chronic inflammatory disease, contacting the sample with an antibody specific for Fsf-1 under conditions which permit immunocomplex formation, and detecting an increase in the relative level of said immunocomplex as an indication of a propensity to form fibrotic scarring. By "relative level" is meant the relative amount of immunocomplex detected when compared to the level in a sample from a normal individual.

The sample may be any biological sample which contains lymphocytes. Preferably, the sample is a blood sample, but may also be a urine or tissue sample. Also preferably, the sample is obtained from a mammal, and even more preferably, the mammal is a human.

In one preferred embodiment, the fibrotic scarring results from hepatic fibrosis. In another related embodiment, hepatic fibrosis is the result of the disease Schistosomiasis. In other embodiments, fibrotic scarring is a result of various chronic inflammatory diseases including sarcoidosis, scleroderma, sclerosing cholangitis, rheumatoid arthritis, and pulmonary fibrosis.

The invention also features a substantially pure fibroblast stimulating factor-1 (Fsf-1) polypeptide. Preferably the polypeptide includes the contiguous amino acid sequence: Leu-Gln-Thr-Glu-Ala-Tyr Lys-Gly. (SEQ. ID NO: 1)

By "Fsf-1 polypeptide" is meant all or part of a novel lymphokine which is also a heparin-binding growth factor which stimulates fibroblast proliferation, and which is distinct from other previously characterized heparin-binding growth factors. Preferably, Fsf-1 is produced in CD4 ⁺ lymphocytes, and the intact polypeptide is characterized as being of molecular weight of about 60kD as measured by SDS-PAGE, and being of isoelectric point of about 6.2. By "polypeptide" is meant any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation) .

A further feature of the invention is a substantially pure antibody which specifically binds Fsf- 1. By "specifically binds" is meant an antibody which binds to Fsf-1 and which does not substantially recognize and bind to other antigenically-unrelated molecules. Antibodies according to the invention may be prepared by a variety of methods. For example, cells expressing Fsf- 1 protein or antigenic fragments thereof can be administered to an animal in order to induce the production of polyclonal antibodies. Alternatively, antibodies according to the invention may be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol . 6:511, 1976; Kohler et al., Eur J. Immunol . 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981) .

As used herein, the term "substantially pure" describes a compound, e.g., a protein, polypeptide, or

antibody, that is substantially free from the components that naturally accompany it. Typically, a compound is substantially pure when at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99%, of the total material (by weight) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The Fsf-1 polypeptide, according to the invention, may be used as the active ingredient of therapeutic compositions. In such therapeutic compositions, the active ingredient may be formulated with a physiologically-acceptable carrier or anchored in the membrane of a cell. Such therapeutic compositions are . used to stimulate fibroblast proliferation, e.g. , to promote wound healing. The method involves applying the therapeutic composition, preferably topically, to a wound of a mammal in a dosage effective to stimulate fibroblast proliferation.

In addition, a kit for detecting the presence of FSF-1 may encompass any or all of the embodiments described herein.

Pathological fibrosis in schisotosomiasis is preceded by a chronic granulomatous inflammatory reaction to helminth eggs deposited in the liver. We have purified a potent fibroblast mitogen, Fsf--1, and have demonstrated that this polypeptide is overproduced by the CD4 ⁺ cells contained within these egg granulomas. Thus, we have concluded that Fsf-l plays an important role in the progression of fibrotic pathogenesis in liver, and is also likely to contribute to fibrotic scarring in other chronic inflammatory disease.

In addition, we have also found that Fsf-1 is overproduced in spleen cells of these infected animals,

and it is likely that relatively high amounts (i.e., compared to animals which do not develop fibrosis) of Fsf-1 may be found in lymphocytes from other sources including blood and urine. Thus, detection of Fsf-1 in the lymphocytes from patients with chronic inflammatory diseases using the method of the present invention provides a relatively simple and rapid means to detect those individuals with a propensity to develop the progressive fibrotic forms of these diseases. This provides the advantage of allowing clinicians to limit treatment to only the subpopulation of infected individuals which require antifibrotic and/or antihelminthic therapy.

Detailed Description The drawings will first briefly be described.

Fig. 1 is a gel filtration chromatography (Biogel P-30) of unconcentrated egg granuloma culture supernatant. Samples of each fraction were assayed at a final concentration of 1/20 fro their ability to stimulate fibroblast proliferation (uptake of [ ³ H]- thymidine) . Each point is a mean of three determinations (SEM < 10% of the mean) . Elution positions of relevant m.w. standards are indicated. Shown is a representative experiment of more than six experiments. Fig. 2 is a heparin-sepharose affinity chromatography of a pool of biologically-active fractions obtained by gel filtration chromatography (Biogel P30) of crude granuloma culture supernatant. Thirty 1-ml fractions were collected. Each fraction was tested for its ability to stimulate fibroblast proliferation. The mean cpm of triplicate determinations is indicated (SEM < 10%) . Shown are the results of a representative experiment of two performed.

Fig. 3 is a FPLC anion exchange chromatography of purified FSF-1. The eluted material from heparin-

Sepharose was applied to a Mono Q column and eluted at a rate of 1 ml/min with a gradient of NaCl (0 to 2.2 M NaCl) . Arrow marks the elution position of commercial heparin. Each point represent the mean of three determination: SEM < 10%. Shown is a representative experiment of two performed.

Fig. 4 is a SDS-PAGE (10% acrylamide) of FsF-1 (lane 2) prepared from granuloma culture supernatants by our published methods and used for immunizing rabbits to prepare anti FsF-1 IgG described in this report. For comparison, the electropherogram of proteins present in unfractionated granuloma supernatant is shown in lane 1. Note that the migration position of FsF-1 (lane 2) corresponds to that of a major protein present in the staring material (lane 1) . 10% acrylamide; silver stain. Fig. 5 is a western blot of cell-free supernatants from egg granuloma cultures (granuloma supernatant) probed with anti FsF-1 antibody. Granuloma supernatant was subjected to SDS-PAGE, electrophoretically transferred to Immobilon-P Transfer Membranes (Millipore) and then either stained with Coomassie Blue (lane a) ; or treated with anti FsF-1 IgG (lane b) , or pre-immune IgG (lane c) , followed by alkaline phosphatase-conjugated goat anti rabbit IgG (Promega Corp. , Madiaon, WI) and developed with substrate as described. The migration position of standard molecular weight markers is shown. Fig. 6 is a dot blot ELISA of FSF-1 (10 ng) , FN. (20 ng) , FGF (100 ng, and PDGF (40 ng) applied to nitrocellulose paper in a volume of 1 μl to 5 μl and then probed with antibodies.

Fig. 7 is a heparin-sepharose eluate (FSF-1:10 ng/ml) or FGF (5 ng/ml) was incubated with either normal rabbit IgG (open bar) or with anti-FSF-1 (blosed bar) at a final concentration of 2.5 mg/ml. Remaining fibroblast-mitogenic activity is represented as percent

cpm obtained with untreated mitogenε. B, PDGF (10 ng/ml) , biologically active peak from P-30 chromatography, or heparin-Sepharose eluate (FSF-1:~ 10 ng/ml) was treated with either normal rabbit IgG (open bar) or anti-PDGF IgG (closed bar; 50 mg/ml) . The supernatants were then tested for fibroblast mitogenic activity. Shown is a representative of three performed. Fig. 8 shows growth of bovine aortic endothelial cells (open symbols) and human fibroblasts (solid symbols) in response to FGF (A) or to heparin-Sepharose purified FSF-1 (B) . Growth response was determined by counting numbers of cells per culture. Baseline counts (medium alone) were: fibroblasts, 6.5 ± 0.1 x 10 ⁴ ; endothelial cells, 6.1 ± 0.1 10 ⁴ . The first two points in B represent responses to FSF-1 at concentrations of

0.01 and 0.1 vol %. Shown is a representative experiment of two performed. Each point represents a mean of four determinations (SEM < 10%) .

Fig. 9 is a curvilinear representation of flow cytometry analysis of dissociated granuloma cells stained with NRS plus FITC-conjugated goat anti rabbit IgG (a) or antiFsF-l IgG followed by FITC-conjugated goat anti rabbit IgG (b) .

Fig. 10 is a contour plot of flow cytometry analysis of monodispersed cells obtained from isolated hepatic egg granulomas. Cells enzymatically dissociated from intact granulomas were stained in the following manner and then analyzed with a FACScan: (a) unstained cells; (B) phycoerythrin-conjugated rat anti mouse CD4 antibody; (C) anti FsF-1 IgG followed by FITC- conjugated goat anti rabbit IgG; (D) anti FsF-1 IgG, followed by FITC-conjugated anti rabbit IgG, followed by rat anti CD4.

Fig. 11 is an autoradiograph of metabolically- labeled proteins produced by granuloma CD4 ⁺ lymphocytes

following SDS-PAGE. CD4 ⁺ lymphocytes were purified by FACS from suspensions of enzymatically-dissociated granuloma cells and incubated for 24 h in the presence of ³⁵ S-methionine- ³⁵ S-cysteine. Cell-free supernatants were then either subjected to 10% SDS-PAGE directly (lane 1) or were precleared by incubation with Sepharose- conjugated normal rabbit IgG and then treated with anti • FsF-1 IgG using two different antibody concentration (5 μg, lane 2;15 μg, lane 3) prior to electrophoresis. The migration position of standard molecular weight markers is indicated.

Animals and Schistosomiasis infection C57BL/6NcrLBr female mice (18 to 20 g; Taconic Farms, Inc., Germantown, NY) were infected by the intraperitoneal injection of 35-50 cercariae of S. mansoni (Puerto Rican strain) suspended in 0.5 ml sterile saline. The mice were euthanized eight weeks later by inducing C0 ₂ narcosis and their livers were placed in cold Hanks'' buffered salt solution (HBSS) . Isolation of granulomas and preparation of crranuloma supernatant. Isolation of granulomas was performed as described previously (Wyler et al. , Science. 202:438 (1978), Pellegrino et al. , J. Parasito1, 42:564 (1956)) . Briefly, livers were homogenized in cold HBSS using a Waring Blendor (New Hartford, CT) . Granulomas were isolated from hepatic parenchymal debris by three to five cycles of serial sedimentation at 1 x g in HBSS. Granulomas were suspended at 10% (v/v) in serum-free culture medium RPMI 1640 supplemented with antibiotics and L-glutamine and cultured for 20 to 24h at 37°C in an atmosphere containing 5% C0 ₂ -95% air. Cell-free supernatant from the granuloma cultures was retrieved by centrifugation (1000 x g 20 min at 4° C) and filter sterilized (0.22 μm diameter; Millipore Corporation,

Bedford, MA) . The supernatants were stored in aliquots at -20 or -70°C.

In some experiments cell-free supernatants were retrieved by centrifugation (200 g x 10 min) and used to purify FsF-l (5) for immunization of rabbits, or to conduct Western blot analysis. In other experiments, granuloma cells were dissociated by collagenase treatment using methods as described in (Wyler et al. , J. Immunol. 132:3142 (1984)). The granuloma cell suspension was first washed with HBSS and then with a solution of phosphate-buffered 0.15M NaCl containing NaN ₃ (0.015 M) and goat serum (1% v/v; Sigma Chemical Co., St. Louis, MO) . The washed cells were pelleted and incubated at 4°C for 0.5 h each in the presence of 10-15 μg/ml of normal rabbit IgG or of rabbit anti-FsF-1 IgG, washed, and then treated with 10 μg/ml goat anti rabbit IgG (H and L chain specific) antibody conjugated with fluorescein isothiocyanate (Fisher Scientific, Pittsburg, PA) . In some experiments, unstained or the FITC-stained cells were treated with phycoerythrin-conjugated rat anti mouse CD4 antibody (5μ/ml; Becton-Dickinson, Mountain View, CA) .

Antibody-treated cells were sorted FACStar Plus flow cytometry and analyzed by a FACScan (Becton- Dickinson) or a Coulter Epics 541 (Coulter Electronics Inc. , Hialeah, FL) flow cytometer. The fields were gaited to exclude autofluorescent and dead cells. The CD4+ sorted cells were washed and suspended (0.6-1.0 x 10 ⁶ /ml) in serum-free medium RPMI 1640 supplemented with 0.3 g/ml bovine serum albumin (BSA; Sigma, St. Louis, MO) and incubated for 24h at 30°C in 5% C0 ₂ - 95% air humid atmosphere. Cell-free supernatants were retrieved by centrifugation (200 g x 10 min; 4° C) and analyzed for fibroblast mitogenic activity.

Culture of cells and cell proliferation assays. Human diploid fibroblast cultures were established from newborn foreskin as described previously (Wyler et al., J. Immunol. 130:1371 (1983)). Primary cultures of BAEC were prepared by and were a kind gift of Dr. Michael Gimbrone (Harvard Medical School, Boston, MA) (Gimbrone et al. , J. Cell. Biology 60:673 (1974)). All cells were grown to confluency in 75 cm ² polystyrene tissue culture flasks (Nuncalon, Rochester, NY) in supplemented medium RPMI 1640 containing 10% inactivated FCS (GIBCO

Laboratories, Grand Island, NY) . When the cultures reached confluency (approximately every 4d) , cells were passaged by treatment with 0.2% trypsin-0.1% sodium EDTA (trypsin-EDTA) . For the proliferation assay, cells were suspended in serum-containing supplemented medium at a density of 5 to 6 x 10 ⁴ .ml. One ml of the cell suspension was seeded in each well of a 24-well polystyrene tissue culture plate (Nuncalon) and incubated overnight. Cells were then washed twice with warm (37°C) HBSS and replenished with serum-free medium. On the next day, 100 μl test samples were added to each well. Twenty hours later, 1 μCi[ ³ H]-thymidine (specific activity 6.7 Ci/mM, Dupont- NEN Research Products, Boston, MA) was added to each well for 4 h. Cells were then trypsinized and harvested onto glass fiber filters with a cell harvester (Titertek, Flow Laboratories, Rockville, MD) . The magnitude of incorporation of [ ³ H]-thymidine was estimated by scintillation spectrometry. In selected experiments, cell growth was also assessed by direct quantitation. The cells were cultured as described above. Test samples were added for 96 h, after which the cultures were washed and detached from the monolayer by treatment with trypsin-EDTA.

Monodispersed cells were counted in a hemocytometer chamber (Cambridge Instruments, Inc., Buffalo, NY).

Gel filtration chromotography and heparin affinity chromatography. A 1 x 40 cm column of Bio-Gel P-30 (Bio- Rad Laboratories, Rockville Center, NY) was equilibrated with PBS (0.15 M) (pH 7.4) and calibrated with the following .w. markers: blue dextran, OVA,chymotrypsin A., and myoglobin (gel filtration markers; Sigma Chemical Co., St. Louis, MO). Approximately 1 ml of unconcentrated crude granuloma culture supernatant was loaded onto the column, which was then run at 4°C with PBS at a rate of 5 to 6 ml/h. One ml fractions were collected, 0.3 mg/ml BSA (Sigma) was added as a carrier, and the fractions were then dialyzed (Nomincal exclusion, 6 to 8 kDa) , first against HBSS, followed by RPMI 1640. The dialyzed material was filter-sterilized before testing in the proliferation assay.

The two or three fractions eluting from the P-30 column with peak fibroblast-sti ulating activity (Fig. 1) were pooled and mixed with an equal volume of washed- heparin-Sepharose CL-6B (Pharmacia LKB, Uppsala, Sweden) . The mixture was rocked gently over-night at 37°C in polyprolylene tubes (Corning, Glassworks, Corning, NY) that had been pretreated by incubation with BSA (1 mg/ml) followed by washing with PBS. The slurry was poured into a column (1 x 15 cm. Bio-Rad) . Material that was not adsorbed (fall-through fraction) was reapplied to the column. The column was then washed extensively with PBS (0.15 M NaCl) and elution was carried out over a 2- to 3- h period with a 30 ml continuous salt gradient (0 to 2.5 M NaCl) and 1-ml fractions were collected. The conductance (ohms ^-1 ) of each fraction was determined (model CDM, Radiometer, Copenhagen, Denmark) . Fractions were dialyzed against medium RPMI 1640 and filter sterilized before testing in the biologic assay.

After determining the concentration of NaCl with which the fibroblast proliferative activity eluted from heparin-Sepharose, a "batch elution" procedure was used for preparing biologically active material from heparin- Sepharose beads. The biologically active fractions prepared by initial gel filtration chromatography were adsorbed ro heparin-Sepharose as described above. The tubes were centrifugated (1000 x g for 10 min) and the supernatant was recovered and filter-sterilized- (0.22-μm diameter pore) . The beads were then washed three times with PBS. After the last wash the beads, suspended in an equal volume of 3.0 M NaCl, were gently mixed at 37°C for 1 h. The supernatant was removed by centrifugation and dialyzed before testing in the biologic assays. The heparin-Sepharose purified material was designated FSF-1. FPLC anion exchange chromatography. The fractions eluting from heparin Sepharose with 1.5 M NaCl were dialyzed (6 to 8 kDa cutoff) at 4°C against two to three changes of PBS, followed by two changes of 20mM Tris-HCl, pH 8.0, and applied to a Mono Q column (HR 5/5,

Pharmacia Uppsala, Sweden) that had been equilibrated in the starting buffer (20 mM Tris-HCl, pH 8.0). The fast- performance liquid chromatography apparatus (Pharmacia) was operated at 4°C. Elution was achieved with a gradient of 0 2.2 M NaCl in 20 mM Tris-HCl, pH 8.0, at a flow rate of 1 ml/min. Forty 1 ml fractions were collected. Absorbance (280 nm) and the conductance of each fraction was monitored and 0.3 mg/ml of BSA was added as a carrier protein before each fraction was dialyzed against medium and tested in the proliferation assays.

SDS-PAGE. Samples were combined with an equal volume of buffer consisting of 10 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 2% dithiothreitol, and 0.01% bromophenol blue. Samples were analyzed by SDS-PAGE as

described by Laem li (Laemmli et al. , Nature 227:680 (1970)). Separating gels of 10% acrylamide and stacking gels of 7% were used. Gels were stained with silver nitrate (Morrissey, Anal. Biochem, 117:307 (1981)). Preparation and analysis of anti-FsF-1 antibodies. Initial (preimmune) serum samples were obtained from two female NZW rabbits (3 to 4 kg, Buckshire Corp., Perkasie, PA) which were then immunized with purified FSF-1 by repeated intradermal injections on the back. Ten ml of unfractionated granuloma supernatant (approximately 10 mg of protein by Bradford assay; Bradford Anal. Biochem. 72:248, 1976)) was processed by gel filtration and heparin affinity chromatography (batch elution procedure) as described above. Purified FsF-1 was concentrated to a volume of 1 to 1.5 ml (approximately 1 to 2 μg; fluorescamine protein assay (Bohlen et al., Biophys. 155:213 (1973)) by ultrafiltration using a 6-8 kDa nominal exclusion cellophane dialysis bag suspended in a slurry of polyethylene glycol (molecular mass = 8 kDa, Sigma) . The concentrate was emulsified in an equal volume of CFA (Sigma) . The rabbits were boosted at 4- to 6-wk intervals by introdermal injections of the same amount of purified FSF-1 in IFA (Sigma) . Aliquots of serum obtained routinely 6 to 8 days after each booster injection were stored at either -20 or -70°C, IgG from preimmune or immune serum was prepared by protein -A Sepharose (Sigma) chromatography (Goding, J. Immunol. Methods 20:241 (1978)).

The specificity of the anti-FSF-1 antibody was assessed by dot-blot ELISA as described in Hawkes et al.. Anal. Biochem. 119:142 (1981). Briefly, purified FSF-1 (10 to 15 ng) , and purified acidic FGF (100 mg, 200 ng) , PDGF (40 ng, 100 ng) , and purified human plasma fibronectin (2, 20. 100 ng (prepared as described in Wyler et al., J. Immunol. 138:1581 (1987)) were adsorbed

to ntirocellulose paper (Bio-Rad) in a volume of 1 to 5 ml. The nitrocellulose was then washed overnight in blocking buffer (PBS containing 5% w/v Carnation nonfat powdered milk) . The nitrocellulose was then incubated at room temperature for 1 h with anti FSF-1 IgG antibody (1:50 final dilution); including, anti-FsF-1 IgG (1:50 final dilution) : rhIL-2 (2ng, 12 ng, 20 ng; Genzyme, Boston, MA); rmIL-3 (2.5 ng, 7.5 ng; Genzyme) rmIL-4 (0.7 ng, 4 ng, 7 ng; Collaborative Research, Bedford, MA) ; rmIL-6 (2.5 ng, 15 ng, 25 ng; Biosource International, Westlake Village, CA) ; rh IL-7 (0.75 ng, 4.5 ng, 7.5 ng; Biosource International); rm GM-CSF (0.25 ng, 1.5 ng; 2.5 ng; Genzyme) . After extensive washing with blocking buffer, the nitrocellulose was incubated for 0.5 to 1 h at room temperature in alkaline phosphatase-conjugated . goat anti rabbit IgG (Promega Corporation Madison, WI) . The development of the blot was carried out by incubation with substrate (containing nitroblue tetrazolium chloride and 5-bromo-4 chloro-3i odyl phosphate, p-toluidine salt (Promega)) dissolved in alkaline phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl ₂ , pH 9.5) : 33 ml nitroblue tetrazolium chloride and 16.5 ml 5-bromo-4 chloro-3-imodyl phosphate p-toluidiήe salt was used for every 5 ml of the buffer. The reaction was stopped with deionized water.

Antibody neutralization of biologic activity. Purified acidic FGF, PDGF or heparin-Sepharose purified FSF-1 (approximately 10-20 ng in 100 μl) or culture supernatant of granerlama-derived CD4 ⁺ lymphocytes) were combined with preimmune IgG (2.5 μg in 100 μl) or anti- rsF-1 rabbit IgG (2.5 μg in 100 μl) and the mixture was incubated at 37°C for 3 to 4 h in polypropylene culture tubes that had been pretreated with BSA. The samples were then filter sterilized and tested in the fibroblast proliferation assay. Alternatively, the samples were

incubated with immobilized protein A-Sepharose to remove Ag-antibody complexes; the samples were centrifuged at 1000 x g for 10 min and the supernatant was tested for fibrogenic activity. Amino acid analysis of FSF-1. Amino acid composition of FSF-1 was determined by analysis of heparin eluate bound to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA (LeGendre et al., Biotechniques 6:154 (1988)). The amino acid composition was determined by the standard method of Waters PICO-TAG. Reagents. Murine rlL-3, 1L-4, and recombinant mouse granulocyte-macrophage CSF were obtained from Genzyme, Boston, MA. Murine, rlL-6, human rlL-7, human rlL-8 were obtained from Bio-Source International, Westlake Village, CA. Human PDGF, a-endothelial cell growth factor, goat anti-human PDGF (IgG) were purchased from Collaborative Research. Bovine acidic FGF used for the dot blot ELISA assay and neutralization studies and rabbit antibovine acidic FGF fragment (Leu 60-Leu 98) , polyclonal IgG was obtained from UBI, Lake Placid, NY. Biosvnthetic labeling of FsF-1 CD4+ cells from granulomas were cultured for 24 h in methionine and cysteine- free RPMI 1640 medium (SeTectamine®Kit, GIBCO, Grand Island, NY) to which was added 75μCi Tran ³⁵ S-Label® ( ³⁵ S E. coli hydrolysate labeling reagent containing 70% L-methionine [ ³⁵ S] and 15% L-cysteine [ ³⁵ S] ; sp. act. 1181 Ci/mmole; ICN Biomedical, Irvine, CA) . The cell-free culture supernatants were collected at 24h by centrifugation (200 g x 10 min) and 200 μl of the supernatant was first incubated (1-2 hr; 4°C) with NRIgG (5 or 15 μg in 200 μl) and the mixture was then incubated (1 hr; 4°C) with protein A-Sepharose (50-100 μl; Sigma) . Two hundred microliters of supernatant of this mixture was retrieved following centrifugation (12,000 g x 5 min) and retreated in a similar manner, this time with anti

FsF-I IgG (5 or 15 μg in 200 μl) and protein A-Sepharose. The beads were pelleted (12,000 g x 5 min) , washed twice in phosphate-buffered 0.15 M NaCl and then boiled for 5 min in sample buffer (lOmM Tris-HCl; 2% sodium dodecyl sulfate; 5% glycerol; 2% dithiothreitol; 0.05% pyronin Y; pH 6.8). Supernatant from these treated beads were subjected to electrophoresis in a 70 x 70 x 0.5 ui slab gel of 10% acrylamide (BioRad Labs, Richmond, CA) using standard method (Laemmli, Nature 227:680 (1970)). Following electrophoresis, proteins were transferred electrophoretically (l-2h; 70 volts) onto nitrocellulose paper (BioRad) using standard procedures. The nitrocellulose paper was then exposed to X-ray film (X- OMAT, AR; Kodak, Rochester, NY) for 72 h at -70°C. RESULTS

Purification of FSF-1. Gel filtration chromatography of granuloma culture supernatant resolves the fibroblast growth factor in fractions with apparent molecular mass 25 to 28 kDa (Fig. 1) . Each fraction was tested at different dilutions (from 1/10 to 1/50) ; maximum stimulation of [ ³ H]-thymidine incorporation was obtained with a dilution of 1/20 of the most active fraction.

A pool of two or three consecutive active fractions obtained by gel filtration was subjected to heparin-affinity chromatography. When the elution was performed with a linear gradient of NaCl (0.2.5 M) , peak mitogenic activity (containing 85 to 90% of the total activity) eluted from the affinity column with 1.25 to 1.5 M NaCl (Fig. 2). Accordingly, we used 1.5 M NaCl to elute the fibroblast mi ogen in subsequent experiments. By this procedure, most of the biologic activity was retrieved in the adsorbed fraction; negligible activity was present in the unadsorbed fraction. Maximum fibroblast stimulation was achieved when the active

fraction was tested to a dilution of 1/100 and 1/1000. Nonspecific binding of the fibroblast mitogen to heparin- free Sepharose 4B beads was not detected in control experiments (data not shown) . Heparin-Sepharose fractionated material was subjected to anion-exchange FPLC chromatography (Fig. 3) . Fibroblast mitogenic activity eluted from the column with 1.2 to 1.5 M NaCl in a single active peak; maximum activity was present in fractions eluting with 1.5 M NaCl. These fractions were active at a concentration of 1/100 to 1/1000. Only fractions 17 and 18 detectably adsorbed uv light (280 nm) . Commercial heparin (from porcine intestinal mucosa, Sigma, cat. no. H-3125) when applied to a mono Q column under the same conditions also eluted in these two factors.

The biologically active fraction that eluted from heparin-Sepharose was analyzed by silver stain of SDS- PAGE (Fig. 4) and under reducing conditions revealed in single band with molecular mass - 60 kDa. This band corresponds to the migration position of one of the major proteins detected in electropherograms of crude granuloma supernatant. Rabbit IgG produced in response to immunization with heparin-purified FsF-1 reacts by dot- blot ELISA with heparin-purified FsF-1 and neutralizes its biological activity. Anti FsF-1 IgG but not pre¬ immune IgG also identifies in Western blot of crude granuloma supernatant FsF-1 and two of its degredation products (Figure 5) .

Identity of FSF-1. The heparin affinity of FSF-1, as revealed in the above experiments^ suggested that the mitogen might be identical to a previously-defined HBGF. Such factors are classified in part according to their anodic or cathodic migratory behavior during IEF (Lobb et al., J. Biol. Che . 261:1924 (1986)). We established that the granuloma-derived mitogen has pl~ 6.2 (Wyler et

al. , J. Immunol. 129:1706 (1982)), thus FSF-1 has a characteristic of class 1 (acidic) HBGF.

We determined the amino acid composition of FSF-1 and compared it with that of bovine acidic FGF. At least 6 of the 15 amino acids analyzed different (by - 50%) in their mole percent contract (Table 1) . The possibility that the FSF-1 preparation might have been contaminated ^• with heparin places into question the accuracy of the mole-percent determination of serine and glycine (Folkman et al.. Science 235:442 (1987)). Nonetheless, the extent of the differences in amino acid composition of FSF-1 and FGF indicates their molecular distinctiveness. Inasmuch as the structure of FGF is highly conserved in evolution (Lobb et al. , Anal. Biochem. 154:1 (1986)). In addition, peptide of purified FSF-1 were generated using standard techniques and the amino acid sequence of one of the peptides was determined to be: Leu-Gln-Thr-Glu-Ala-Tyr-Lys-Glu- (SEQ. ID NO: 2) This sequence is unique from other heparin-binding growth factors, thus providing evidence that FSF-1 is a novel protein.

We also conducted a series of experiments to assess the potential similarity of FSF-1 to acidic FGF, the prototype class 1 HBGF molecule (Lobb et al., supra). We also compared FSF-1 with PDGF, a group of closely related mesenchymal cell mitogens (LeGendre et al. , supra) .

Anti-FSF-1 antibody detected FSF-1 in both purified form and in crude granuloma supernatant in a dot-blot ELISA assay (Fig. 5) . The antibody did not detect acidic FGF or PDGF, mitogens that were detected with the appropriate homologous antibodies (Fig. 5) .

Anti-FSF-1 antibody inhibited the proliferative responses of fibroblasts to FSF-1 (Fig. 7A; p < 0.005; comparison by Student's t-test of mean fibroblast

responses to FSF-1 in the presence of normal rabbit IgG or anti FsF-1 IgG in four separate experiments) . Under identical conditions, the antibody preparation did not significantly (p > 0.4) affect the mitogenic activity of acidic FGF. Furthermore, anti-PDGF, which abrogated the mitogenic effects of PDGF, had no effect on FSF-1 (Fig. 7B) .

A characteristic feature of class 1 HBGF is their ability to stimulate endothelial cell proliferation (Folkman et al., supra) . As shown in Figure 8A, fibroblasts and endothelial cells proliferate in response to acidic FGF. Inasmuch as the magnitude of the response of endothelial cells to the lower (<2 ng/ml) concentration of acidic FGF is greater than that of fibroblasts, these cells appear to be the more sensitive to this mitogen. In contrast, FSF-1 in concentrations in the range of 1 to 40 ng/ml, did not induce endothelial cell proliferation (Fig. 8B) .

Furthermore, anti FsF-1 did not detect plasma fibronectin or acidic FGF in a dot-blot ELISA; nor did anti-fibronectin antibody react with FsF-1. Crude franuloma supernatant had no biological activity characteristic of TNF in an L929 cytotoxicity assay and purified TNF was not mitogenic in our assay which utilizes serum-free conditions. We detected no 1L-2 activity in granuloma supernatants (Wyler et al. , J. Immunol. 129:1706 (1982)), and detected no fibroblasts mitogenic activity in rIL-2 (S. Praka^h, unpublished observations) . Finally, we detected no biological activity in the following commercially-prepared cytokines; rIL-3 (0.2-200 U/ml) , rIL-4 (0.05-20U/ml) rhIL-5(1-1000 U/ml) , rIL-6 (0.002-lOOU/ml) , IL-7 (0.02- 100 U/ml) rIL-8 (0.02-100 U/ml) or GM-CSF (0.3-20 U/ml). The lack of relevant biological activity in these cytokines implies that FsF-1 is unique.

Flow cytometry of granuloma cells

We analyzed granuloma cells by flow cytometry after they were treated with anti FsF-1 IgG (or preimmune IgG) and FITC-conjugated anti IgG with or without subsequent treatment with phycoerythrin-conjugated anti CD4 antibody (Figure 9 and 10) . In the mouse, CD4 is express on a subpopulation of lymphocytes but not on macrophages (Crocker et al., . Exp. Med. 166:613 (1987) ) . Using one-and two-color flow cytometric analysis, we determined that approximately 20-25% of the CD4+ lymphocytes also stained specifically with anti FsF- 1.

We next employed FACS to obtain a highly-enriched (99% pure) population of CD4 ⁺ granuloma cells that we then incubated at a density of 0.5-1.0 x 10 ⁶ cells/ml for 24 h in serum-free medium. The conditioned medium from these cultures stimulated fibroblast proliferation (Table 2) . In contrast, culture supernatants of CD4 ⁺ lymphocytes isolated from spleen cell suspensions prepared from uninfected mice contained no such biological activity. This indicates that the treatment of cells with anti-CD4 antibody in the course of their purification did not trigger the secretion of FsF-1. Biosvnthetic labeling of FsF-1 in CD4+ lymphocytes The foregoing results suggested that egg granuloma-derived CD4+ lymphocytes produce FsF-1. To confirm this conclusion more precisely, and exclude the possibility that FsF-1 was merely bound to the CD4+ lymphocytes and subsequently released, we incubated these isolated granuloma cells with [ ³⁵ S]-methionine/cysteine for 24h and processed the culture supernatants by immunoprecipitation with anti FsF-1. Autoradiographs of the electroblots of SDS-PAGE preparations of untreated culture supernatant disclosed in excess of 10-15 distinct bands (Figure 11) . In contrast, immuno-precipitation of

the CD4+ lymphocyte supernatant with anti FsF-1 IgG revealed a singled 60 kDa band (Figure 11) . On the basis of its Rf, this band corresponds to the major labelled product of the isolated lymphocytes and to that of heparin-affinity purified FsF-1 from granuloma supernatant (Prakash et al., supra) . As noted by Western analysis, preimmune IgG does not react with this protein (Fig. 5) .

Heparin affinity chromatography has proven to be a valuable purification procedure in the isolation of certain mesenchymal growth factors and angiogenic factors (Lobb et al., supra) . An advantage of this technique is that the relatively high affinity binding of these factors to heparin is uncharacteristic of most proteins (Lobb et al., supra) . The simplicity of the scheme we were able to devise for purifying FSF-l from culture supernatants is a consequence of its heparin-binding property. Early in our studies, we noted that an initial gel filtrations step enhances the efficiency of the subsequent affinity chromatography step, perhaps by removing fibronectin (another heparin-binding protein that is a constituent of these supernatants (Wyler, Rev. Infect. Pis. 9(Suppl:5391 (1987)) from the crude granuloma supernatants. The final anion exchange FPLC (Fig. 3) confirmed the homogeneity of biologic activity in the purified fractions, and the single brand (or occasionally a doublet) detected on silver-stained SDS- PAGE gels (Fig. 4) supports the conclusion that purification was achieved. Furthermore, we estimate that the purification scheme resulted in approximately 10,000- to 50,000-fold enrichment in specific activity. We base this estimate on our measurement of the total protein concentration of crude granuloma culture supernatant («1 mg/ml; Bradford assay (Bradford, Anal. Biochem. 72:248 (1976)), our detection of protein in the heparin-

Sepharose eluate near the lower limit of sensitivity of the fluorescamine assay (-200 ng/ml (Bohlen et al., supra) ) , and the dilution of material yielding maximum proliferative responses being 1:10 to 1:20 crude material and 1:100 for purified material.

The apparent molecular mass -60 kDa of FSF-1 revealed by SDS-PAGE conflicts with our estimates of molecular mass »25 to 28 kDa by gel filtration chromatography (Fig. 1) . By Western blot analysis of crude granuloma supernatants probed with polyclonal anti FSF-1 antibody, we detected a single band in the range of 55 to 58 kDa and at times also a doublet at 30-kDa band. The basis for this apparent discrepancy in m.w. determination by gel filtration and by SDS-PAGE remains to be elucidated. However, a number of factors are known to affect migration in gel filtration. In contrast, only the 60 kDa protein was identified by immunoprecipitation of metabolically-labeled CD4 ⁺ lymphocyte-derived proteins (Fig. 11). This indicates that the minor bands are apparently products of degredation of FSF-1 most likely generated by granuloma-derived proteases present in culture supernatants (but not in CD4 ⁺ lymphocyte culture supernatants) . One possibility is that FSF-l forms aggregates, and that such aggregates formed during heparin-af inity chromatography are resistant to dissociation under the conditions we used in conducting SDS-PAGE.

In addition to providing for a convenient purification method, the fact that FSF-1 is heparin- binding has important implications in establishing its molecular identity. We previously determined that the granuloma-derived mitogen is a protein with pi -6.2(7). These properties suggest that FSF-1 might be a member of the acidic heparin-binding growth factor class of proteins (class 1 HBGF) , mitogenic proteins of diverse

cellular origin that are structurally identical or closely related, and highly conserved between mammalian species (Harper et al., Biochemistry 25:4097 (1986)). The class 1 HBGF, exemplified by acidic FGF, are all potent mitogens for fibroblasts as well as endothelial cells. Our indicator endothelial cells from bovine aorta responded in a characteristic manner to bovine FGF but not FSF-1 (Fig. 8) . It is unlikely that the lack of response to FSF-1 is due to species differences, because the HBGF are structurally and functionally conserved

(Burgess et al., Annu. Rev. Biochem 58:575 (1989)), and because we found that another cytokine from egg granulomas with molecular characteristics distinct from FSF-1 could stimulate proliferation of bovine aortic endothelial cells, but not fibroblasts (Wyler et al., J. Infect. Pis. 155:728 (1987)). This suggests that FSF-1 probably is distinct from FGF and sensu stricto is not a class 1 HBGF. Supporting this conclusion are our observations that antibodies prepared against FSF-1 neither reacts with FGF in a dot-blot ELISA nor neutralize its biologic activity, whereas it does both to FSF-1 (Figs. 6 and 7) . Finally, the amino acid content of FSF-1 and FGF (acidic and basic) reveal differences indicating that these molecules are not identical (Table 1) , and the amino acid sequence of the peptide derived from FSF-1 is distinct from other known proteins. The antibody preparations permitted us to distinguish FsF-1 from other heparin-binding growth factors (Prakash et al., supra) and to determine by flow cytometry that a subpopulation (20-25%) of CD4+ lymphocytes in granuloma cell suspensions apparently express FsF-1 on their surface (Figure 9 and 10) . The fact that culture supernatants of CD4+ lymphocytes isolated from egg granulomas contained fibroblasts mitogenic activity that was neutralized with anti FsF-1

IgG indicated that these cells secrete the mitogen (Table 2) . The definitive evidence that the CD4+ lymphocytes produce and secrete FsF-1 was obtained in experiments involving biosynthetic labelling and immunoprecipitation of CD4+ proteins (Figure 4) . Although the results of the present studies do not exclude the possibility that other granuloma cells also might be potential sources of FsF-1, our prior studies indicate that FsF-1 is not secreted by granuloma macrophages (Wyler et al. , supra) . Furthermore, since S. mansoni egg granulomas from mice treated with anti IL-5 antibodies lack eosinophils (Sher et al. _r Proc. Natl. Acad Sci. USA 87:61 (1990)) but nonetheless secrete fibrogenic activity, eosinophils are an unlikely source of FsF-l. On the other hand, egg granulomas from S. mansoni-infected. congenitally athymic mice (which lack mature T lymphocytes and do not develop hepatic fibrosis) produce no fibroblast mitogen (Prakash et al. , J. Immunol. 144:317 (1990)). This observation is consistent with our conclusion that FSF-1 is a lymphokin .

We conclude that the granuloma CD4+ lymphocytes are stimulated in vivo to produce FsF-1, and that this production is not induced artificially during isolation of the lymphocytes. Several points support this conclusion. First, in contrast to many of the studies that have examined production of fibrogenic proteins by chronic inflammatory cells (lymphocytes and macrophages; for example, see Wahl et al., J. Immunol 121:942 (1978); Wahl et al. , Lvmphokines 2:179 (1981), we do not add antigens, mitogens, or other nonspecific stimuli to our granuloma or cell cultures. Second, the cell sorting methods we used did not trigger lymphocytes to produce a fibroblast mitogen (see above) . Third, fibroblast mitogenic activity can be detected in extracts of recently isolate egg granulomas (Wyler et al. , J. Infect.

Pis. 144:254 (1981)) and is detectable in the cell-free supernatants of isolated egg granulomas within a few hours of their in vitro incubation (Wyler et al. , supra) . Fourth, unfractionated splenocytes and splenic CP4+ lymphocytes isolated by flow cytometry fail to spontaneously secrete fibroblast mitogens (Wyler et al., Infect. Immun. 38:103 (1982); and the present study). On the other hand, sensitized splenic lymphocytes stimulated with an aqueous extract of schistosoma eggs (concanavalin-binding fraction of soluble egg antigen) do secrete a fibroblast mitogen, presumably FsF-1 (Wyler et al. , supra) .

It is noteworthy that FsF-1 detected by immunoprecipitation corresponded to a prominent 60 kDa protein produced by isolated granuloma CD4 ⁺ lymphocytes . (Figure 5) . Our results suggest that FsF-1 might be a major protein produced by this subpopulation of granuloma cells. We have observed that sensitized splenic lymphocytes from S. mansoni-infected mice, when stimulated with an aqueous extract of schistosoma eggs secrete a fibroblast mitogen, presumably FsF-1, Wyler et al._. Infect. Immun. 38:103 (1982). It therefore seems likely that CD4 ⁺ cells are stimulated ^"" in vivo to produce FsF-1 in response to egg antigens. Based on our current observation, such production may continue at least briefly when the lymphocytes are dissociated from the eggs and antigen-presenting cells. The notable finding that FsF-1 not only is secreted but also can remain associated with the surface of CD4 ⁺ lymphocytes suggests that in addition to the action of the secreted cytokine, direct contact between membrane-associated FsF-1 positive lymphocytes and fibroblasts might stimulate fibroblast growth.

A number of cytokines (1L-1, TNF, TGFB) , some of which were originally identified on the basis of the

other biological activities they possess, also have been shown to exhibit fibrogenic activity in vitro (for example, see Schmidt et al. , J. Immunol. 128:2177 (1982); Vilcek et al. , J. Exp. Med. 163:632 (1986); Sugarman et al.. Science 230:943 (1985); Massague, J. Biol. Chem.

260:7059 (1984); Leof et al. , Proc. Natl. Acad. Sci. USA. 83:2453 (1986)). In addition, fibroblast growth factors, some of which were purified from other sources and were recognized for this biological property (PDGF [Ross et al.. Cell 46:155 (1986)], FGF [Gospodarowicz et al. , J. Biol. Chem. 250:2515 (1975); Gospodarowicz et al. , J. Biol. 253.:3736 (1978)]), and heparin-binding epidermal growth factor [HB-EGF;Higashiyama et al. , Science 251:936 (1991)]) have been found to be produced by macrophages. However, based on its biochemical composition, physicochemical properties, and antigenicity, FsF-1 is distinct from these fibroblast growth factors. Moreover, the fact that FsF-1 is a lymphokine is an additional distinguishing characteristic. Indeed, because we have not detected fibroblast mitogenic activity in several purified and recombinant lymphokines, and since avid heparin-binding is not a known characteristic of most lymphokines, we believe that FsF-1 is a previously unidentified lymphokine. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

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