American Journal of Pathology, Vol. 141, No. 6, December 1992 Copyright X) American Association of Pathologists

Isolation and Characterization of Granuloma Initiation Factor Maxwell A. Fung, Naoyoshi Sato, Toshihiro lida, Kimie Fukuyama, and William L. Epstein From the Department of Dernatology, University of California, San Francisco, San Francisco, California

A soluble component that transfers granulomatous tissue reaction was fractionated from Schistosoma mansoni egg-induced hepatic granulomas (SMHG) by

Sephacryl 5-300 column chromatography. The fractions separately bound to inert, Affi-Gel agarose beads were inoculated subcutaneously in naive mice. The low molecular weightfraction, consisting ofproteins 23 kd, 20 kd, and 16 kg produced organized granulomas 6 to 7 weeks after inoculation. This fraction was further purified by high-pressure liquid chromatography (HPLC) gelfiltration and gave three fractions eluting at retention times of 44, 46, and 48 minutes. Each fraction contained all low-molecularweight proteins in varying amounts and induced skin granulomas when inoculated subcutaneously. Amino acid sequence of the major 20-kd protein showed 11 N-terminal residues identical to those of cyclophilin Antisera raised to the protein with retention time of 46 minutes, reacted with cells in the granulomas but not surrounding liver tissue as detected by immunofluorescence microscopy. The findings indicate a low molecular weight soluble fraction of SMHG can induce new granuloma formation when injected in an immobilized form into skin of naive mice. The results suggest granuloma initiation factor is a homolog of the cyclophilin gene family. (Am JPathol 1992, 141:1445-1451)

Granulomatous inflammation is characterized by abnormal proliferation and differentiation of monocyte/ macrophages into organized tubercles of interdigitated epithelioid cells in response to the prolonged presence within tissue of certain foreign substances. These tissue changes are seen in such chronic and stigmatizing diseases as tuberculoid leprosy, sarcoidosis, and berylliosis. The causative factors for the tissue reaction remain unknown, however.

Using a model of murine schistosomiasis, we have shown that lyophilized Schistosoma mansoni egginduced hepatic granulomas (SMHG) can transfer the ability to form new organized granulomas to the skin of naive mice.1 These granulomas in turn can be isolated and when lyophilized can induce new granuloma formation for a second time in the skin of naive mice, suggesting the persistent granulomagenic stimulus is a tissue factor and not simply viable cells or an infectious agent. To biochemically isolate and characterize the stimulus, granuloma initiation factor (GIF), we have fractionated a crude extract of SMHG by gel filtration and ion exchange column chromatography. A high molecular weight (about 650 kd) macrophage chemotactic factor (MCF) was purified, but this fraction did not produce granulomas after inoculation into skin.4 In this article, we report on purification and characterization of low-molecular-weight proteins (16 to 23 kd) from an SMHG extract that have granuloma-inducing activity in mice.

Materials and Methods Mice and Infection Female C57BIJ6 mice 6 to 7 weeks old (Simonsen Laboratories, Gilroy, CA) were used. To produce SMHG, mice were subcutaneously infected with 75 cercariae of Schistosoma mansoni.

Purification of GIF Nine weeks after infection, mice were killed by cervical dislocation. The livers removed under sterile conditions were suspended in 20 mmol/l phosphate-buffered saline (PBS) pH 7.4 and homogenized in a Waring blender. Schistosoma mansoni egg-induced hepatic granulomas were isolated on a #50 stainless steel mesh and stored in Supported by NIH grant AR 31853 and UCSF Academic Senate "Opportunity" grant. Accepted for publication May 27, 1992. Address reprint requests to Dr. William L. Epstein, Department of Dermatology, University of Califomia, San Francisco, HSE-1092, Box 0536, San Francisco, CA 94143-0536. 1445

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3.0-g aliquots at -70°C. An aliquot of SMHG was homogenized in PBS (Pyrex glass homogenizer) and sonicated (Branson cell disrupter) for 4 minutes. The sample was centrifuged at 700g for 10 minutes to remove cell debris, and the supernatant was ultracentrifuged at 1 20,000g for 120 minutes. The supernatant was concentrated in an Amicon chamber using a YM-3 Diaflo membrane (Amicon Corp., Danvers, MA) to a final volume of 2 to 4 ml. The supernatant was applied to a gel filtration column (Sephacryl S-300, Pharmacia Fine Chemicals, Piscataway, NJ) at 4°C, and the eluent was divided into fractions according to molecular size (Fr 1 through Fr 5). The low-molecular-weight fraction (Fr 5) was concentrated with Amicon as above and further purified by highpressure liquid chromatography (HPLC) gel filtration on a Bio-Sil TSK-125 column (Bio-Rad, Richmond, CA). The resulting three protein peak fractions appeared at different retention times and were hand-collected in separate tubes (RT44, RT46, and RT48).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and N-Terminal Sequencing The proteins separated by Sephacryl S-300 and Bio-Sil TSK-1 25 HPLC gel filtration were analyzed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method of Laemmli.5 Proteins in the HPLC fractions were electroblotted onto a ProBlot membrane (Applied Biosyntems, Foster City, CA) using a MiniTransBlot tank (Bio-Rad). The blotted protein was stained with Coomassie Blue R-250 in 40% methanol/i % acetic acid. The 20-kd band was excised manually and submitted for N-terminal sequencing. Gas-phase N-terminal sequencing was performed by M. Shih, VAMC, Portland, Oregon. Micro-BCA (bicinchoninic acid) protein assay reagent (Pierce, Rockford, IL) was used to measure protein concentrations.6

Inoculation of GIF into Skin Five protein fractions from Sephacryl S-300 gel filtration and three fractions from HPLC gel filtration were incubated with a 1:1 mixture of Affi-Gel 10 and Affi-Gel 15 (Bio-Rad) agarose beads in a 15-ml polypropylene tube for 12 hours at 4°C. The resulting Affi-Gel protein complexes then were subcutaneously inoculated into the dorsal skin of anesthetized, healthy, noninfected mice. AffiGel beads (200 ,ul) bound to approximately 1 to 2 mg S-300 column purified protein or 10 to 20 ,ug HPLCpurified protein were used in each mouse. Protein concentrations were measured using the method outlined by Lowry et al.7 A sterile steel spatula was used to clear a

space in the subcutaneous plane of a 5-mm incision. The inoculation site was closed with Autoclip staples (ClayAdams, Parsipanny, NJ). Affi-Gel beads alone, or Affi-Gel coupled soluble proteins extracted from normal liver were inoculated into control mice under identical conditions as outlined above.

Light and Electron Microscopy Seven weeks later, inoculation sites were excised. The specimens were fixed in 10% formalin, embedded in paraffin, cut at 5 p, and stained with hematoxylin and eosin. Other portions of the biopsy specimens were fixed in 3% glutaraldehyde and postfixed in 2% 0S04. They were stained en bloc with uranyl acetate and embedded in epoxy resin. The blocks were cut at 0.5 to 1 ,u and stained with toluidine blue, or cut at 60 nm and stained with lead and uranyl acetate and viewed under Siemens Elmiskop 1A.

Antibody Production Antibodies against GIF were produced by subcutaneous and intradermal injection of 50 ,ug of RT46 protein mixed with Freund's incomplete adjuvant into a New Zealand White rabbit. A second injection of 100 p.g of RT46 protein was performed 17 days later and serum collected 5 days later.

Immunologic Studies Ouchterlony immunodiffusion was performed in 0.1% agarose gel on a glass slide for 20 hours at room temperature, with 80 ,ug protein/well and rabbit anti-RT46 serum. Frozen sections of liver of mice with schistosomiasis were cut at 5 p. thick and fixed in acetone for 5 minutes. They were reacted with the antiserum or preimmune rabbit serum for 1 hour. The reaction was stained with overlaid fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and viewed through a Zeiss fluorescence microscope.

Results

Purification of Proteins from SMHG A typical elution pattern of the 1 20,000g supernatant from SMHG by Sephacryl S-300 column chromatography is shown in Figure 1A. By SDS-12% PAGE, numerous pro-

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Figure 1. Purification of GIFactivity. A: Elution of SMHG proteins in 20 mmol/l PBS on SephacaylS-300 column protein concentration (-) through Fr5 were separatelypooled as indicated by arrows. Flow rate was 0.15 mlper minute. Molecular weight calibratedfrom standard proteins are: Fr 1, Mr > 300 kd; Fr 2, Mr (300 kd-100 kd); Fr 3, Mr (100 kd-40 kd); Fr 4, Mr (40 kd-20 kd) and Fr 5, Mr < 20 kd. B: SDS-12% PAGE of the Sephacryl S-300 fractions. The gels were stained with Coomassie brilliant blue R-250. Lane Mr contains molecular weight standards. Lanes 1-5 correspond to proteins (20 pg) in Fr 1-Fr5, respectively. Molecular weights of the 3 major protein bands in Fr 5 are marked. C: HPLC gelfiltration cbromatography of Fr 5 in 20 mmol/l PBS. Flow rate was 0.5 ml per minute. Protein peaks were detected at retention times of 44, 46 and 48 minutes. D: Electroblotted proteins of Fr 5 after HPLC gel filtration. Lane 1 shows molecular weight markers. Lanes 2, 3 and 4 are proteins ofRT44, 46 and 48 peaks, respectively. was monitored by 280 nm absorption and Fr 1

tein bands were shown in Fr 1 through Fr 4, whereas Fr 5 showed three major protein bands of 23 kd, 20 kd, and 16 kd (Figure 1 B). We attempted to separate the three proteins with different molecular weights by HPLC gel filtration, and three protein peaks with retention times of 44, 46, and 48 minutes were detected (Figure 1 C). However, SDS-1 0% to 20% gradient PAGE analysis showed that all HPLC-purified peaks consisted of the same protein bands of 23 kd, 20 kd, and 16 kd, although the proportions varied. Those protein bands were electroblotted on ProBlot membrane (Figure 1 D). N-terminal amino acid sequence of 20-kd protein bands excised from RT44 (Figure 1 D, lane 2) and RT46 (Figure 1 D, lane 3) were H2N-Val-Asn-Pro-Thr-Val-Phe-Phe-Asp-lle-Thr-Ala-, a sequence identical to the first 11 amino acid residues in

murine cyclophilin.8

Light and Electron Microscopy The inoculated Affi-Gel beads were readily detected in subcutaneous tissue. Different degrees of cellular response were seen around the beads coupled with fractions from Sephacryl S-300 gel filtration. The Fr 5 reaction was primarily granulomatous in nature (Figure 2A, B). The beads were surrounded by tightly attached layers of mononuclear cells/macrophages. Some cells exhibited large nuclei characteristic of epithelioid cells, but scattered eosinophils and giant cells also were observed. Those mixed with Fr 1 showed diffuse infiltration of large numbers of cells, as we previously reported,4 whereas those coupled to Fr 2, 3, and 4 caused minimal tissue reactions with scattered mononuclear cells (Figure 2C). Similar protein fractions prepared from normal liver ex-

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Figure 2. Light microscopy of skin sites inoculated with A4ff-Gel beads bound to Fr 5 protein or controls. A: Low-power shows collections of macrophages, giant cells and eosinophils forming dense concentric layers about individual beads (C. B: High power view reveals several layers of cells around the beads and many of the cells have large oval or oblong nuclei and abundant cytoplasm. The smaller dark staining cells are mainly eosinophils. C: A control infected with Affi-Gel bound to Fr 4 protein. At most one layer of cells formed around the beads andthe matrix between beads was looselyftirillar withfew cells. D: Another control inoculated with protein extractfrom normal liver bound to Affi-Gel. One layer of cellsformed about the beads and the matrx between beads containedfewv cells. Magnification A: X340, B: X850,

C and D: X900.

tract did not elicit granulomatous reaction (Figure 2D). The tissue reaction caused by separate inoculation of the three fractions from HPLC gel filtration column was es-

sentially the same as that due to Fr 5 (Figure 3A through C). Toluidine blue staining of plastic-embedded RT46induced lesions showed accumulation around the Affi-

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Figure 3. Light microscopy ofgranulomatous tissue reaction elicited by inoculation ofAffi-Gel beads bound to HPLC purified protein RT46. A: Low power reveals accumulation of mononuclear cells and eosinophils, as well as giant cells. B: High power demonstrates macrophages forming concentric layers around beads O). C: Epon-embedded section gives more details of cell ipes in the lesions including macrophages and epithelioid cells. The darker multilobulated cells are eosinophils. Magnification A: X250; B and C: x 1220.

Gel of predominantly macrophages and epithelioid cells with large oval nuclei and abundant light-staining cytoplasm (Figure 3C). Electron microscopy of the granulomas showed prominent nucleoli in typical epithelioid cell nuclei, the close association of the cells with one another, and the characteristic dilated rough endoplasmic reticulum (RER) in the cytoplasm of epithelioid cells with electron dense material within the RER lumina (Figure 4A, B).

Immunologic Studies A single precipitin line was formed between wells containing Fr 5 and rabbit anti-RT46 serum (Figure 5A). By immunofluorescence microscopy, concentration of the antigen over SMHG was detected, whereas surrounding hepatic cells showed no reaction (Figure 5B). Reaction of the preimmune serum on a consecutive section was negative (Figure 5C).

Discussion This study provides evidence that in vivo granulomatous inflammation can be induced by low-molecular-weight proteins partially purified from SMHG. The experimental model was facilitated by use of Affi-Gel agarose beads, which are inert and do not elicit an inflammatory reaction alone. Other beads, such as Sepharose beads, are toxic to the tissue in our hands. Immobilization of the soluble proteins has been found essential to elicit a chronic gran-

ulomatous response that evolves slowly over a period of weeks. Also, we noted that small amounts of GIF bound to Affi-Gel take longer to produce granulomas. Thus, 10 jig of HPLC-purified GIF requires 7 to 8 weeks to give a complete reaction, whereas 20 ,ug GIF causes a comparable response in 5 to 6 weeks (data not shown). These proteins, designated GIF, are products of the cells in the granulomatous infiltrate as shown by the immunofluorescence study with rabbit antisera. They differ from granulomagenic factors that have previously been described in the literature, such as soluble egg antigen (SEA). Soluble egg antigen was obtained initially as a phosphate buffer soluble extract of schistosome eggs and when bound to inert beads and injected intravenously in sensitized mice produced short-lived pulmonary granulomas.9 10 Many investigators have purified and characterized the more than 20 proteins in crude SEA.11 The major antigenic proteins thought to be associated with granuloma formation were glycoproteins of molecular mass, ranging from 70 kd to more than 200 kd,11 and apparently require both protein and carbohydrate moieties for their action.12 Clearly, these proteins are different from GIF, and one would be very surprised if any SEA persisted in the freeze-dried tissue used for second passage granuloma formation.2 The only lowmolecular-weight protein purified from SEA (w1 antigen) was hepatotoxic for T-ce--deficient mice.13 Recent studies have implicated cytokines, such as interleukin 1 beta (IL-1 p) and tumor necrosis factor alpha (TNF-a), in granuloma reactions in vitro14,15 and in vivo.1618 In general, however, the experiments, using IL-1 ,-coated beads, have been short term, lasting only a

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Figure 4. Electron microscopy of cells accumulated in the skin site inoculated with RT46. Three epithelioid cells show tightly interdigitated plasma membranes. They demonstrate active Golgi vesicles and dilated rough endoplasmic reticulum in the cytoplasm. Magnification X8000. Inset: Part of the cytoplasm of an epithelioid cell, Magnification X9000.

few days,1417 and it is unclear whether the aggregated macrophages were organized epithelioid cells. Crude GIF did not contain IL-1, or TNF-a by bioassay, kindly performed by Dr. Crissie, Cetus Corp., Berkeley, Califor-

nia, and the N-terminal sequence obtained from purified GIF was totally different from either cytokine. Nevertheless, in a preliminary unpublished study, we found that IL-1-coated beads induced GIF in the skin lesions after 5

Figure 5. Immunological studies. A: Ouchterlony immunodiffusion shows reactivity between rabbit anti-RT46 and Fr 5 (Well 5). Well 1, Fr 1; Well 2, Fr 2; Well 3, Fr3; Well 4, Fr 4. 80 pgprotein/well. Wells on the right side each contained rabbit anti-serum. B: Immunofluorescence microscopy demonstrates reactivity ofanti-RT46 serum with cellular components around SMHG, parasite egg C). C: Normal rabbit serum shows no reaction, parasite egg (9.

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weeks, suggesting that cytokines may act as granulomagenic agents by inducing GIF. The N-terminal sequence analysis of the major 20-kd protein in GIF showed identical homology for the first 11 amino acids with mouse cyclophilin.819'20 It appears to have a molecular weight larger than cyclophilin, however, and is likely to be a homolog of the emerging cyclophilin gene family.20 The potential functions of this class of cyclosporine A binding protein with cis-trans isomerase activity also seem to be broadening,21 and one can question whether these proteins might act as intracellular transducers of the inflammatory response. In the case of lymphocytes, the binding of the immunosuppressant, cyclosporine, would fit this supposition. And some unpublished evidence is accruing that cyclophilin may display chemotactic activity (Handschumacher RE, personal communication, 1991). In any event, it should now be possible to isolate cDNA clones of GIF and deduce its full amino acid sequence, and the highly specific antisera will allow determination of its tissue distribution.

8. 9.

10.

11.

12. 13.

14.

Acknowledgment The authors thank Dr. D. T. Lau for production of antibody. 15.

References 1. Okamoto M, Epstein WL, Suya H, Kanazawa K, Fukuyama K: Transfer of granulomatous inflammation with nonviable preparations of schistosome granulomas in naive mice. Exp

Cell Biol 1987, 55:73-178

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2. lida T, Nozaki Y, Fukuyama K, Epstein WL: An improved infectious murine skin model of organized granulomatous

inflammation. Experientia 1991, 47:273-277 3. lida T, Sato N, Fukuyama K, Lau DT, Epstein WL: Immunogenetic influences on skin granuloma formation in mice. Exp Mol Pathol 1991, 54:172-180 4. Sasaki Y, lida T, Sato N, Fukuyama K, Epstein WL: Macrophage chemotactic factor partially purified from granulomatous inflammation. Cell Immunol 1991, 134:171-179 5. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T5. Nature 1969, 227:

680-4685 6. Smith PK, Crohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk BC: Measurement of protein using bicinchoninic acid. Anal Biochem 1985, 150:76-85 7. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein

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measurement with the folin phenol reagent. J Biol Chem 1951, 193:265-275 Hasel KW, Sutcliffe JG: Nucleotide sequence of a cDNA coding for mouse cyclophilin. Nucl Acids Res 1990, 18: 4019 von Lichtenberg F, Smith TM, Lucia HL, Doughty BL: New model for schistosome granuloma formation using a soluble egg antigen and bentonite particles. Nature 1971, 229:199200 Boros DL, Warren KS: Delayed hypersensitivity-type granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J Exp Med 1970,132:488-507 Boros DL: Immunopathology of Schistosoma mansoni infection. Clin Microbiol Rev 1989, 2:250-269 Weiss JB, Aronstein WS, Strand M: Schistosoma mansoni: Stimulation of artificial granuloma formation in vivo by carbohydrate determinants. Exp Parasitol 1987, 64:228-236 Dunne DW, Lucas S, Bickle Q, Pearson S, Madgwick L, Bain J, Doenhoff MJ: Identification and partial purification of an antigen wl from Schistosoma mansoni eggs which is putatively hepatotoxic in T-cell deprived mice. Trans R Soc Trop Med Hyg 1981, 75:54-71 Shikama Y, Kobayashi K, Kasahara K, Kaga S, Hashimoto M, Yoneya I, Hosoda S, Kazuhiko S, Ide H, Takahashi T: Granuloma formation by artificial microparticles in vitro. Am J Pathol 1989,134:1189-1199 Sato IY, Kobayashi K, Yamagata N, Shikama Y, Kasama Y, Kasahara K, Takahashi T: Modulation of granuloma formation in vitro by endogenous mediators. Immunopharmacology 1991, 221:73-82 Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Soezima K, Ide H, Takahashi T: Direct evidence for granuloma inducing activity of interleukin-1. Am J Pathol 1988, 130:1189-1199 Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Soezima K, Ide H, Takahashi T: The role of monokines in granuloma formation in mice: The ability of interleukin 1 and tumor necrosis factor-a to induce lung granulomas. Clin Immunol Immunopathol 1989, 51:419-425 Kindler V, Sappino A-P, Grau GE, Piguet P-F, Vassalli P: The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 1989, 56:731-740 Harding MW, Handschumacher RE, Speicher DW: Isolation and amino acid sequence of cyclophilin. J Biol Chem 1986, 261:8547-8555 Hasel KW, Glass JR, Godbout M, Sutcliffe JG: An endoplasmic reticulum-specific cyclophilin. Mol Cell Biol 1991, 34843491 Schreiber SL: Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 1991, 251:

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Isolation and characterization of granuloma initiation factor.

A soluble component that transfers granulomatous tissue reaction was fractionated from Schistosoma mansoni egg-induced hepatic granulomas (SMHG) by Se...
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