JPH055813B2 - - Google Patents

Description

[Detailed description of the invention]

FIELD OF INDUSTRIAL APPLICATION The present invention is in the field of anti-inflammatory compounds, in particular:
The present invention relates to the use of a polypeptide called cartilage-inducing factor (CIF) and polypeptides functionally related thereto as factors to inhibit the progression and inhibition of inflammation, including lymphohistiocytitis, granulitis and acute inflammation. (Prior art) CIF-A and CIF-B are disclosed in jointly filed Japanese Patent Laid-Open No. 61-36223 (published on February 20, 1986).
Two bovine bone-derived CIFs have been described. In both cases, the molecular weight by SDS-PAGE is approximately
It is a dimer with 26000 daltons. When examining the production of cartilage-specific proteoglycans (PGs) in an agarose gel culture model using rat fetal mesenchymal cells, each of these was found to be in vitro.
shows chondrogenic activity. However, both in
In vivo, it has no chondrogenic activity by itself. Amino acid sequencing of CIF-A reveals that it is a type-β transforming growth factor (TGF-β).
It was shown to have the same partial (30 amino acid) N-terminal sequence as reported for a polypeptide from human placenta called . CIF
The partial N-terminal sequence of -B is different from that of TGF-β. Both CIFs show activity in the TGF-β assay (ability to induce fixation-independent proliferation of normal rat kidney cell colonies in soft agar). TGF-β derived from bovine kidney, human placenta or human platelets has been granted international patent application number PCT/US83/
01460 (Published March 29, 1984, Publication number WO84/
01106) and EPA84450016.5 (Published December 1984)
19th, publication number 0128849). These applications show that when such TGF-β binds to EGF or TGF-α, it (1) promotes cell proliferation in the soft agar culture assay described above, and (2) inhibits cell proliferation and protein deposition in a rat soft tissue wound healing model. provides data showing that it promotes According to these applications, TGF-β is characterized as a dimer with a molecular weight of approximately 26,000 by SDS-PAGE. Disclosure of the Invention The present invention is based on the discovery that the two bovine CIFs described above exhibit anti-inflammatory activity. Evaluation of CIF-containing implants has shown that CIF has activity both locally and systemically in suppressing acute and/or chronic inflammation. Because of the similarity (or perhaps identity, in the case of CIF-A) of these polypeptides to TGF-β, it is believed that TGF-β also has these newly discovered activities. These activities appear to be independent of the presence of active agents or cofactors and are different from previously reported chondrogenic and cell proliferation activities. For convenience, this section and the claims use the term CIF as a general term encompassing CIF-A, CIF-B, TGF-β, and functional equivalents thereof. Subsequently, it was discovered that CIF localizes to hematopoietic and lymphopoietic sites and suppresses thymocyte responses to interleukin-1 (IL-1), indicating that CIF acts on erythrocytes and/or lymphocytes. This study shows that it may also be effective in the treatment of functional disorders or malfunctions in differentiation. Therefore, the present invention provides two new uses of CIF. One is a composition used for the treatment of inflammation, which contains a cartilage-inducing factor. Both acute and chronic types of inflammation are treated hereby. Treatment may also be systemic, or CIF may be administered locally to treat inflammation at a given site. The other is a composition used for the treatment of hematopoietic or lymphopoietic dysfunction or insufficiency, which composition contains a chondrogenic factor. The present invention will be explained in detail below. As used herein, the term "inflammation" refers to both acute reactions (i.e., reactions in which inflammation is actively progressing) and chronic reactions (i.e., reactions that are slow to progress and in which new connective tissue is formed). shall be included. Chronic and acute inflammation may be distinguished by the type of cells involved. Acute inflammation often contains polymorphonuclear neutrophils, whereas chronic inflammation is usually characterized by a lymphohistiocytic and/or granulotic reaction. Special types of inflammation include, for example, diffuse inflammation, focal inflammation, croup inflammation, interstitial inflammation, obstructive inflammation, parenchymal inflammation, reactive inflammation, specific inflammation, toxic inflammation and traumatic inflammation. be. As used herein, the word "treat" shall mean prevention or attenuation of an existing condition. Thus, in the case of inflammation, the method of the invention may be used to prevent inflammation or to reduce existing inflammation. The term "functionally equivalent" as used in the description of a polypeptide refers to a polypeptide that has the same amino acid sequence as the polypeptide being referred to, whether natural or artificial, and regardless of species or origin. or are substantially homologous (i.e., at least 90% identical in amino acid sequence) but differ in amino acid sequence such that this difference does not have an adverse effect on anti-inflammatory activity. It shall be. CIF-A, CIF-B and TGF-β are Methods
for Preparation of Media, Supplements, and
Sub−strate for Serum−Free Animal Cell
Culture (1984) pp181−194, Alan R. Liss, Inc.
It shows activity in the TGF-β assay described in .
This assay examines cell colony formation in soft agar to determine the ability to induce fixation-independent proliferation in non-neoplastic normal rat kidney (NRK) fibroblasts. From platelets, placenta and kidney tissue
The procedure for obtaining TGF-β is described in International Patent Publication WO84/
No. 01106 and EPA Publication No. 0128849. Briefly, this procedure involves extracting the source material with acid-ethanol, fractionating the extract by gel filtration, and extracting TGF-β from the filtrate by high performance liquid chromatography (HPLC).
This includes separating the The procedure for isolating CIF from bovine bones is described in Japanese Patent Application Laid-open No. 1986-
No. 36223 (published on February 20, 1986). To do this, extracts of demineralized bone (DMB) that solubilize non-fibrous proteins (e.g. ≧4M
guanidine hydrochloride, 8M urea), and this extract was subjected to gel filtration to obtain a fraction smaller than 30Kd.
This fraction is chromatographed on carboxymethyl cellulose (CMC) at PH 4.5-5.5, preferably PH 4.8, the fraction adsorbed to CMC is eluted with a NaCl gradient, and approximately 150-250 mM NaCl
This includes purifying the eluted portion of the protein by RP-HPLC or gel electrophoresis. CIF isolated from natural sources to date
A, CIF-B and TGF-β are polypeptide dimers with molecular weights of approximately 25 to 26 Kd as determined by SDS-PAGE. Nature (1985) 316:701~
705, the nucleotide sequence and deduced amino acid sequence of human TGF-β derived from tuberculoid platelets are reported. Human TGF-β derived from mature platelets is characterized as a homodimer consisting of a 112 amino acid long monomer. Platelet/placenta/kidney derived TGF-β, CIF-A and CIF-B are non-species specific for TGF-β activity. Therefore, these polypeptides are highly conserved among animal species (i.e., the amino acid sequence of a given polypeptide from different mammalian species may vary, including additions, deletions, etc. of one or more amino acid residues). or by substitution, which does not adversely affect the non-species-specific activity of the molecule), and is considered to have cross-species functionality. Therefore, CIF-A, CIF-B and
TGF-β may be derived from cells or tissues of various animal sources, or may be recombinant.
It may also be obtained by DNA engineering. Correlating with this,
CIF (TGF-β) from one vertebrate species can be used to treat other vertebrate species. CIF
The most common use of (TGF-β) as an anti-inflammatory agent is in the treatment of humans, domestic animals such as cows, sheep and pigs, and competition or pet animals such as dogs, cats and horses. CIF-A and CIF-B are preferably used in the method of the invention. EXAMPLES The following examples illustrate specific embodiments of the invention. They do not limit the invention in any way. 1. Preparation of CIF from Bone A. Preparation of Demineralized Bone Fresh bovine metatarsals were freshly obtained from a slaughterhouse and transported on dry ice. The bones were cleaned to remove bone marrow and non-bone tissue, broken into pieces smaller than 1 cm in diameter, and crushed in a mill at 4°C. The crushed bones were washed twice with 9.4 g of double distilled water per kg of bone for approximately 15 minutes each and then with 0.01 N HCl overnight at 4°C. Washed bones were treated with 3 x 3 volumes of ethanol, followed by 3
Degreased with x3 volumes of diethyl ether. Each wash was performed for 20 minutes, all at room temperature.
The resulting defatted bone powder was then diluted with 0.5N HCl (25
/Kg defatted bone) and demineralized at 4°C. Decant the acid and wash the resulting DMB until the wash PH is higher than 4, and
DMB was dried by suction filtration. B Extraction of non-collagen protein DMB prepared in section A was added at 3.3 kg/kg.
4M guanidine hydrochloride, 10mM ethylenediaminetetraacetic acid (EDTA) (PH6.8), 1m
M PMSF, and 10mM NEM for 16 hours. The suspension was filtered with suction and the insoluble material was extracted again for 4 hours. The soluble fractions were combined and concentrated at least 5 times by ultrafiltration using an Amicon ultrafiltration (10K) unit, and the concentrate was dialyzed against 6 changes of 35 volumes of chilled deionized water over 4 days; It was then freeze-dried. All operations in this section are performed at 4°C.
It was held at However, freeze-drying was performed under standard freeze-drying conditions. C. Gel filtration The extract of section B redissolved in 4M guanidine hydrochloride, 4M guanidine hydrochloride, 0.02% sodium azide, and 10mM EDTA (PH
Separation was performed on a Sephacryl S-200 column equilibrated with 6.8). Fractions were analyzed by absorbance at 280 nm and fractions were combined as shown in FIG. Fraction F2 in Figure 2 is low molecular weight (LMW,
10,000 to 30,000 daltons), which was dialyzed against six changes of 180 volumes of deionized water and lyophilized. All operations except dialysis drying and dialysis (4°C) were performed at room temperature. D Ion exchange chromatography Fraction F2 of section C was mixed with 6M urea, 10mM
It was dissolved in NaCl, 1mM NEM, and 50mM sodium acetate (PH4.8), and centrifuged at 10,000 rpm for 5 minutes. The supernatant was separated on a CM52 (commercially available CMC) column equilibrated with the same buffer. Bound proteins were dissolved in the same buffer using a 10 to 400 mM NaCl concentration gradient in a total volume of 350 ml and a flow rate of 27 ml/h.
eluted from the column. Three major fractions, named CM-1, CM-2 and CM-3, were collected as shown in FIG. CM-2 and
CM-3 eluted at approximately 150-250mM NaCl. Each fraction was dialyzed for 4 days against 6 changes of 110 volumes of deionized water and lyophilized. All of the above operations were performed at room temperature except for dialysis (4°C). E RP-HPLC Lyophilized fraction CM-2 combined in section D
and CM-3 were each dissolved in 0.1% trifluoroacetic acid (TFA), and a portion of the solution was
Vydac C18 RP-HPLC column (4.6mm ID×
25 cm) and 1 using 0.1% TFA.
Washed at ml/min for 5 minutes. Elution solvent is 0.1%
A concentration gradient of 0% to 60% acetonitrile in TFA was used at a rate of 2%/min. RP- of combined CM-2 and CM-3
From HPLC, peak A at about 29.5 minutes and about
Two peaks were obtained, peak B at 31.2 minutes. The proteins of these peaks are the subject of the aforementioned US Patent Application No. 630,938. These are each
They are named CIF-A and CIF-B. Proteins were stored at -20°C in 0.1% TFA/acetonitrile eluent until use. F Characterization of CIF-A and CIF-B Table 1 below shows the partial amino acid composition of CIF-A and CIF-B.

【table】

Table: SDS-PAGE analysis of CIF-A and CIF-B shows that both have molecular weights of approximately 26,000 Daltons. Both proteins were tested for TGF-β derived from human platelets, human placenta, or bovine kidneys in the TGF-β assay described above.
It showed activity comparable to that reported in . The N-terminal amino acid sequences of the first 30 amino acids of CIF-A and CIF-B were determined as follows.

【table】

[Table] The N-terminal amino acids of CIF-A are the same as those reported for platelet-derived human TGF-β (see Nature, below). 2. Anti-inflammatory activity of CIF A. Formalization of implants containing CIF-A/CIF-B Collagen in solution (CIS, 1-3 mg protein/
ml; From Collagen Corporation
(sold under the registered trademark VITROGEN100) and bone collagen powder (BCP, a freeze-dried solid of bone collagen) such that the minimum final concentration of CIS-derived collagen is 10%.
A collagenous carrier was prepared. CIF-A and
Mixture with CIF-B in a weight ratio of 2:1 (0.1%
(in TFA) to the carrier at a weight ratio of 1:1200, 1:
4500, 1:6000, 1:8000 and 1:20000
Added with. These formulations were stirred at 4° C. for 1-2 hours and either directly lyophilized or lyophilized after dialysis against water. The carrier alone was used as a control. B Histological Evaluation of Section A Implant Formulation 1 Implantation The lyophilized formulation was rehydrated with 2 parts by weight of chilled sterile water and mixed into a homogeneous paste. The rehydrated material was formed into tightly packed pellets (80-100 mg wet weight).
The pellets were implanted subcutaneously in the abdominal thoracic region of young male rats. Each rat received bilateral implants. Explanted tissues were collected on days 3, 10, and 14 after transplantation and evaluated histologically. 2. Histological evaluation The explanted tissue was fixed with 10% neutral formalin and embedded in paraffin using a conventional method. Sections were then stained with either hematoxylin-eosin or Gomori triple staining. 3 Results The results of the histological evaluation are summarized below. <3 days after transplantation> 1. Carrier only On the third day after transplantation, the graft was mostly non-cellular. Sparse neutrophils were the most obvious cell type. 2 CIF-Carrier The grafts were still relatively acellular at day 3. However, fibroblasts from adjacent muscles and surrounding subcutaneous tissues were clearly activated. These fibroblasts were cytoplasmic and often orthochromatic, indicating that they were highly activated. Fibroblast infiltration began at the edges of the graft. <10 days after transplantation> 1. Carrier only The inflammatory profile changed significantly up to 10 days after transplantation. The graft contained a diffusely mixed inflammatory cell infiltrate, which was primarily populated by lymphocytes and histiocytes. Focal areas of granulocytes (neutrophils and eosinophils) and giant cells were clearly seen around some bone collagen particles. 2 CIF-Carrier At this point there were almost no inflammatory cells associated with the graft. There were many proliferating fibroblasts throughout the graft. A matrix of collagenous connective tissue was clearly seen around and around the bone collagen end particles. <14 days after transplantation> 1. Carrier only By 14 days after transplantation, most of the bone collagen particles were isolated by granulomas consisting of lymphocytes, histiocytes, and giant cells. Graft-associated fibrosis was clearly observed, along with multifocal areas of eosinophils. 2 CIF-Carrier There was negligible graft-related inflammation compared to control grafts. A dense collagenous connective tissue matrix was clearly observed throughout the graft. Morphologically, fibroblasts appeared to be less metabolically active than at earlier time points. These histological observations indicate that CIF suppresses inflammatory cell function in vivo.
The absence of polymorphonuclear neutrophils, lymphocytes, and histiocytes at CIF-containing graft sites suggests that CIF may act as a potent anti-inflammatory agent. CIF to carrier weight ratio of 1:8000 and 1:
20,000 grafts had significantly reduced graft-associated inflammation compared to carrier-only grafts. Grafts with higher CIF to carrier weight ratios developed a dense collagenous connective tissue matrix throughout the graft. At all CIF levels, graft-related inflammation was negligible compared to no CIF. In a similar in vivo experiment, rats were given bilateral grafts with and without CIF-containing extracts, but inflammation was reduced or absent in the grafts away from the CIF-containing grafts. This was noticed. These observations are
CIF has been shown to act both locally and systemically. CIF when used as a topical anti-inflammatory agent
(and/or TGF-β) is usually formulated in an effective amount with a pharmaceutically acceptable carrier in a weight ratio of 1:1000 to 1:20000. If tissue deposition is undesirable at the site, reduce the level of CIF to the carrier below the level that promotes tissue deposition (e.g., approximately 1:6000 for collagen carriers).
(to a lower weight ratio). In addition to being formulated as injectables, CIF can also be incorporated (dispersed) into solid osmotic implants such as collagenous soft or hard tissue grafts, prostheses, sponges, wound dressings, and sutures. ) can alleviate local inflammatory reactions to these solid bodies. Because such implants are made of permeable materials, CIF can diffuse out of the implant and exert its anti-inflammatory properties. If it is desired to minimize other activities of CIF (cell proliferation, tissue deposition), CIF is incorporated without active agents or cofactors, preferably at levels below levels that promote tissue deposition. When used to treat inflammation locally in the body, formulations containing CIF or TGF-β can be locally injected or inhaled, depending on the particular formulation and the area where inflammation control is desired. ,
Surgically placed or otherwise administered. For systemic administration, CIF may be formulated with conventional carriers used with water-soluble proteins for injection into the circulation. Alternatively, if required by therapeutic instructions, it may be formulated as a sustained release implant formulation. The dosage of CIF (TGF-β) for the treatment of inflammation depends on the patient, the inflammatory condition being treated, and the mode of administration. Generally, the adult human dose is approximately
It will range from 0.1 to 1000 μg. When administering CIF locally, lower amounts in this range will usually be used, typically between 0.1 and 10 μg.
Correspondingly, amounts for systemic administration will typically range from 10 to 1000 μg. CIF may be particularly effective in treating inflammation involving the respiratory system. In this application, CIF may be administered by inhalation with a suitable aerosol. In this type, these factors are used in the treatment of widespread interstitial diseases of the lungs, such as asbestosis, silicosis or coal miner's pneumoconiosis; in the respiratory tract, such as rheumatoid arthritis, erythematous scars or Gutspasture syndrome. and in the treatment of granulitis of the lungs and pulmonary ducts, such as Wegener's granulomatosis and eosinophilic granulomatosis. These anti-inflammatory peptides may be coupled to carriers in the form of salves, ointments, or other topical formulations, and thus may be useful in the control of dermatitis upon topical application. Such prescriptions are especially
It may be useful in the topical treatment of plaque psoriasis, contact dermatitis, skin ulcers, and acute or chronic eczematous dermatitis. CIF may be used alone or combined with slow-release carriers and injected into or around joints, bones or muscles to control inflammation associated with various diseases. Some examples include myositis (viral, bacterial, parasitic, fungal or autoimmune), myasthenia gravis, osteomyelitis, osteoarthritis and rheumatoid arthritis. Since CIF molecules have been shown to be stable at low PH and resistant to enzymatic digestion, these factors may be administered systemically by ingestion. These properties make these factors particularly useful in controlling inflammation of the gastrointestinal tract. In particular, it may be useful in the treatment of gastric and duodenal ulcers, granulating gastritis, esophagitis (of many causes); enteritis (of many causes); and colitis (of many causes). 3. Immunohistochemical site determination materials and methods for CIF-A (TGF-β) A. Synthesis of synthetic polypeptide Synthetic polypeptide (designated as A1/30),
It was constructed to have the same N-terminal amino acid sequence (residues 1 to 30) as CIF-A and TGF-β. Peptide A1/30 was synthesized by solid phase method. The peptide was collected on p-methylbenzhydramine resin, cleaved from the resin, deprotected by two-step hydrogen fluoride treatment, and purified by reversed-phase liquid chromatography on octadecyl silica. Peptide A1/
30 was determined to be homogeneous by RP-HPLC and thin layer chromatography, and its amino acid sequence was confirmed by gas phase continuous analysis. B. Radioiodination Purified CIF-A and CIF-B were radioiodinated with Na ¹²⁵ I using the last peroxidase method. Specific activity is 0.8~1.0×
It was 10 ⁷ cpm/μg protein. C. Immunization of New Zealand white rabbits with peptides
Immunized with A1/30. From 250 per injection
Using 500 μg of peptide A1/30, starting from 6
Multiple intramuscular injections were given every two weeks for 10 weeks. The first immunization was with Freund's complete adjuvant, followed by booster injections.
It was performed with Freund's Incomplete Ajiyu Band.
Ten days after the last booster injection, the rabbits were exsanguinated by puncturing their hearts. Blood was allowed to clot for 4 hours at 22°C and overnight at 4°C.
Serum was collected and stored at -70°C. D Purification of antibodies Serum IgG was purified using Sepharose Protein-A
It was purified using Briefly, serum was diluted with an equal volume of 0.01M Tris (PH7.2) containing 0.15M NaCl.
It was diluted with Antibodies were precipitated at 4°C using an equivalent volume of saturated ammonium sulfate adjusted to pH 8.0, and
Collected by centrifugation at 100,000×g for 30 minutes. The protein pellet was resuspended in a minimal volume of PBS at pH 7.2 and dialyzed against PBS. The retentate was clarified by centrifugation and the supernatant applied to a 10 ml column of Sepharose Protein-A. 0.1M bound IgG
Elution was performed with glycine-HCl (PH2.0). Antibodies were immediately neutralized (4.0 M Tris), dialyzed against PBS, and lyophilized. E. Enzyme-linked immunosorbent assay (ELISA) The reactivity of the antiserum with peptide A1/30, CIF-A, or low molecular weight bone extract was evaluated by ELISA. Peptide A1/
30 was solubilized in PBS, whereas purified CIF
-A or partially purified bone extract is 0.01N
Solubilized in HCl. Dilute the antigen with 0.01M carbonate buffer (PH9.6), 400n in 100μ volume.
g of protein was added to the wells of a Mylotiter plate. Peptide A1/30 and CIF-A were air dried on the wells overnight. Plates containing partially purified extracts were sealed and placed at 4°C overnight. Contains 1% (w/v) BSA before use
Incubate with PBS for 1 hour and ELISA
Nonspecific protein binding to the plate was blocked. Serial 2-fold dilutions of antiserum were applied to each well.
Add 100 μl each and leave for 1 hour. Prepare the plate with 0.05% (w/v) Tween-20 and 1%
Wash with PBS containing (w/v) BSA and peroxidase-conjugated goat F(ab′) ₂ anti-rabbit.
IgG was added and left for 2 hours. 5 plates
Washed eight times and added peroxidase substrate. The substrate was prepared with 2,2'-azino-di-(3-ethylbenzthiazoline) sulfonic acid (ABTS, 0.03% w/v) and 0.03% (v/v) in 0.1 M citrate buffer (PH4.0). v)
Consisting of H ₂ O ₂ . The color was allowed to develop for 30 minutes, and the absorbance at 414 nm was measured. F. Antibody Competition ELISA An antibody competition ELISA was used to determine whether the antibodies detected by the ELISA were antigen specific. Various concentrations (1 to 100 ng) of soluble competitive antigen peptide (A1/30) were added to the wells to compete the binding of the antibody to the synthetic polypeptide adsorbed on the synthetic resin; in this study, the antiserum was :5000, the antibody was washed away, and bound antibody was detected by ELISA. G Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE of purified and partially purified proteins
It was separated by The concentration gel was made of 4% polyacrylamide, and the separation gel was made of 15% polyacrylamide. Gels were either silver stained or electrophoretically transferred to nitrocellulose. In some cases, the gel is “spiked” with ¹²⁵ I-labeled CIF-A as an internal standard,
-A or blot protein was identified. H Two-dimensional acetic acid-urea PAGE Partially purified low molecular weight bone extracts were evaluated by two-dimensional polyacrylamide gel electrophoresis. Protein (20 to 40 μg) was added to a glass tube (2
x 4 x 125 mm) containing 2.5M urea and acetic acid
Run one-dimensionally in 15% polyacrylamide at 160V (constant voltage) for 5-6 hours, then run two-dimensionally in a 15% SDS-polyacrylamide slab gel (depending on its size and charge). separated. Gels were silver stained or proteins were transferred to nitrocellulose (described below). In some cases, the gel
Factors were identified on silver-stained gels by "spiking" with ¹²⁵ I-labeled CIF-A and CIF-B, followed by autoradiography. I. Immunoblotting Samples were first separated by one-dimensional or two-dimensional electrophoresis. After separation, 25mM Tris (PH
Proteins were transferred to nitrocellulose using a Transprot apparatus filled with 8.3) and 192 mM glycine (PH 8.3) and 20% (v/v) methanol. Proteins were transferred at 170 mA (constant current) for 18 hours. After transfer, 0.05%
(v/v) Tween-20 and 1% (w/v) BSA
Nonspecific protein binding was blocked for 1 to 2 hours using a PBS buffer containing .
The protein blots were then incubated for 2 hours in a 1:100 dilution of rabbit anti-A1/30 serum. Wash the blot with the same buffer for 30 min.
Then 200000 cpm/ml [ ¹²⁵ I] protein-
A: Incubated for 1 hour. Wash the immunoblot, air dry, and dry the immunoblot.
1~ at -80℃ using XAR-5 film
I put it on autoradio roughie for 18 hours. J Immunohistochemical staining Cell association and distribution of CIF-A were determined by immunohistochemical staining. Fetal bovine tissue was collected from one cow at 6 months of gestation. Tissues were fixed in 10% neutral formalin. The hard tissue was demineralized with 10% formic acid.
Dehydrate the tissue and use Fisher Histomatic Tissue
Paraffin embedding was performed using a Processor model 166A (150 hour cycle). Sections were deparaffinized in xylene and containing 0.15M NaCl.
Endogenous peroxidase was blocked with a solution of 0.01M Tris and 0.1% hydrogen peroxide (15 minutes). Next, the sections were treated with a solution containing 1 mg/ml testicular hyaluronidase in an aqueous solution of 0.1M sodium acetate (PH5.5) at 37°C for 30 minutes. Non-specific protein binding was determined using a solution containing 0.5% (w/v) BSA in Tris-saline.
Blocked for a minute. The sections were diluted optimally (1:
50) Incubate for 1 hour with rabbit (IgG) anti-A1/30 or nonimmune rabbit IgG, wash with Tris-saline, and peroxidase-conjugated goat F(ab') ₂ anti-rabbit IgG.
and incubated for 1 hour. Slides were treated with diaminobenzidine substrate buffer.
This substrate buffer contains 0.1% hydrogen peroxide
It consists of 0.5mg/ml diaminobenzideine dissolved in 0.05M Tris (PH)-saline solution. section
Counterstained with Mayer's hematoxylin. Results A Antibody reactivity Antiserum titers were A1/30 and CIF-A.
It was 1:10,000 for CIF-A, and 1:1,000 for partially purified bone extract containing CIF-A. The antibody also reacted with platelet TGF-β, as expected.
This is because the N-terminal sequences are the same. A competitive ELISA method was used to determine whether the antibodies detected by ELISA were antigen-specific antibodies. In this experiment, we used
Binding of antiserum optimal dilution (1:5000) to 400 ng of peptide A1/30 was performed using various amounts (1 to 5000).
100 ng) of soluble synthetic polypeptide. Competition for antibody binding showed a linear dose-response pattern, with 100 ng of soluble competitor competing more than 80% of the binding. The linear titration properties of antibodies and linear and nearly perfect competition for binding suggest that antibodies are antigen-specific. The specificity of the antibody was also confirmed by immunoblotting, in which the antibody was isolated from peptide A1/30.
Similarly, both non-reduced CIF-A and CIF-A reduced with β-mercaptoethanol had immunoreactivity. Since antibodies against peptide A1/30 were determined to be immunoreactive with CIF-A, they were used to determine whether other molecular weight species of CIF-A were present in bone crude extracts. did. High-molecular weight and low-molecular weight bone extracts were prepared, and component proteins were separated by gel electrophoresis. Silver-stained gels and double immunoblots were prepared. Tests showed that only proteins with the same molecular weight as CIF-A were detected by antibodies against peptide A1/30 in both high and low molecular weight bone extracts. B Cell Association and Tissue Distribution Staining specifically labeled osteocytes within the femoral cancellous bone. Furthermore, articular chondrocytes, especially cells strongly associated with cartilage canals, were strongly stained. Chondrocytes within the epiphyseal cartilage were not labeled by the antibody. Kidney tissue and platelets were examined. Epithelial cells lining the renal calyx were specifically stained, whereas surrounding stromal and parenchymal cells were stained with anti-A1/30.
Not labeled with antibody. I also checked the bone marrow,
Platelet-producing megakaryocytes were specifically labeled with the antibody. Some mononuclear bone marrow cells were also stained with antibodies. Staining was also performed to determine whether CIF-A is also associated with hematopoietic and lymphopoietic centers. Although the cytoplasm of hematopoietic stem cells in the fetal liver was strongly stained, hepatic parenchymal cells and interstitial cells were not labeled. Hematopoietic stem cells in bone marrow were specifically stained with antibodies. The thymus was examined and specific staining of the thymus corpuscles and some thymus medullary cells was observed.
No specific staining was observed in the more undifferentiated thymic cortex cells. Further staining studies were performed to confirm the thoracic localization of CIF-A. CIF−
The antibody against A specifically stained reticular epithelial cells and epithelial cells containing thoracic bodies in the thoracic medulla. Epithelial cells in the cortex and the surrounding capsule were not stained with antibodies. There was no detectable staining in subcapsular cortical or medullary thymus cells. Along with the aorta, other tissues including the thyroid gland, adrenal glands and mandibular salivary glands were examined. CIF-A was not detected by staining techniques in any of these tissues. The localization of CIF-A/TGF-β in hematopoietic centers (bone marrow and liver) and lymphopoietic centers (thoracic line) suggests that this molecule may regulate erythroid and/or lymphocyte differentiation in vivo. It suggests. Therefore, CIF refers to congenital thoracic hypoplasia,
It will be used in the treatment of indications associated with dysfunction or dysfunction in hematopoiesis and/or lymphopoiesis, such as severe combined immune deficiency, hereditary hemolytic anemia and acquired hemolytic anemia. 4 Activity of CIF on developing T lymphocytes Since it has been established that CIF is inherently associated with developing T lymphocytes, the following tests were performed to determine the activity of CIF on mitogen responses of thymocytes.
The effectiveness of CIF was determined. A. Preparation of cell culture A single cell suspension of Murine (C3H/HeJ) thymocytes was prepared. Cells were suspended in Eagle's minimal essential medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine and 5×10 ⁻⁵ M 2 -mercaptoethanol. Cell morphology and cell viability by trypan blue exclusion were determined. B IL-1 Assay Thymocytes (1×10 ⁶ ) were plated in flat-bottomed 96-well microtiter plates. cells with optimal concentration of phytohemagglutinin (PHA)
and IL-1 (2U IL-1 and PHA). IL-1 or recombinant IL-1 (rIL-1) derived from endotoxin-free purified human mononuclear blood cells was used. IL-1 increases the proliferative response of thymocytes to half its maximum potential
The amount of 1 unit of specific activity was defined as 1 unit of specific activity. Different amounts of CIF-A (TGF-β) or CIF-
B was added to the thymus cell culture (see Figures 4-7). 5% in a moist incubator
Cells were cultured for 72 hours at 37°C in _CO2 . Prior to harvest (24 hours), cultures were pulsed with 0.5 μCi of ³ H-thymidine. Cultures were collected using a semi-automatic cell harvester and ³ H-thymidine incorporation was determined by standard liquid scintillation methods. Results Figures 4 to 7 show the test results in graphs. As shown in Figure 4A, for TGF-β at concentrations of 1.9 x 10 ^-11 M and 1.5 x 10 ^-10 M, 2 U
³ H-thymidine uptake by thymocyte cultures treated with 40 U of rIL-1 was inhibited (86% to 92% of control). Culture concentration 1.9 x 10 ^-12 M
TGF-β inhibited the proliferative response by 49% to 59% at each concentration of rIL-1 tested. Similarly, 1.9×
10 ^-11 M CIF-B is the rIL at all concentrations tested.
-1 versus thymocyte proliferation (90% of control)
inhibited. 1.5× compared to control untreated cultures.
50 for thymus cell cultures containing 10 ^-10 M CIF-B.
% to 60%, whereas 1.9×
Cultures treated with 10 ^-12 M CIF-B showed 17% to 26% inhibition. TGF-β or CIF-B
The cell viability of cultures treated with was determined by cell morphology and trypan blue exclusion.
It was comparable to untreated cultures. The range of effective amounts of TGF-β and CIF-B is shown in FIG. The effective range for both factors is
It was between 10 ^-12 M and 10 ^-10 M. Maximal inhibition of both factors at a culture concentration of approximately 2 × 10 ^-10 M (control
90%). The concentration that shows half maximal inhibition is
6× each for TGF-β and CIF-B
10 ^-12 M and 4×10 ^-12 M. The results shown in FIGS. 4 and 5 show that when TGF-β or CIF-B is added to the culture at the same time as IL-1 and PHA, the proliferation response is inhibited in a dose-dependent manner. Figure 6 shows
We demonstrate the effects of these factors on thymocytes previously stimulated with mitogens. In this experiment, TGF-β or CIF-B (1.9×
2U of thymus cell culture before adding 10 ^-10 M)
Stimulation was performed with IL-1 and PHA for 1 to 48 hours.
The results in Figure 6 indicate that cultures previously stimulated with mitogens for 1 to 6 hours showed that exogenous
It is shown that addition of TGF-β or CIF-B significantly (86% of the control) inhibits the inhibition.
Thymocytes prestimulated for 24 hours were partially stimulated by TGF-β or CIF-B treatment (from 47% of controls).
63%) inhibited. Thymocytes prestimulated for 48 hours were unaffected or only slightly affected by TGF-β or CIF-B treatment.
(27%) was only inhibited. As shown in Figure 6, when cells were exposed to TGF-β or CIF-B during the early induction stage, these factors most effectively inhibited the mitogen response. It can be seen from the results shown in FIG. 7 whether continuous exposure to these factors is necessary to maintain the inhibitory effect. In these experiments,
Thymocytes were pretreated (1 to 4 hours) in culture tubes containing different concentrations of TGF-β or CIF-B. Pretreated cells were extensively washed to remove exogenous factors and then transferred to microculture with 2U IL-1 and PHA for an additional 72 hours. For control cultures, TGF-β or
The cells were treated in the same manner as in the pretreatment culture except that CIF-B was added directly to the microculture wells. The results are TGF-β or CIF-B
Cells pretreated with (10 ^-10 M) for 4 hours show less responsiveness (50% of control) to subsequent mitogen stimulation than untreated control cultures. 10 ^-10 M TGF-β or
Cells pretreated with CIF-B for 1 to 2 hours were mitotic and only slightly responsive to stimulation (20% to 30%, respectively). Cells pretreated with low concentrations (10 ^-12 M and 10 ^-11 M) of the factors for 1 to 2 hours were not inhibited in their response to IL-1 induction. The results of Example 4, in conjunction with the immunolocalization results, suggest that TGF-β (and possibly CIF-B) may serve a physiological function by controlling the clonal expansion of developing T lymphocytes. It suggests. Furthermore, it is suggested that cells with Hassal bodies are probably the cell type that contributes to TGF-β production in the thymus. This concept is supported, in part, by studies that radiolabel the whole animal. This study shows that thymus cell proliferation is mostly confined to the subcapsular and cortical regions of the thymus, and that thymocyte maturation is compartmentalized within the medullary region of the thymus. . Although the mechanism by which TGF-β and CIF-B inhibit the proliferative response of thymocytes is unknown, it is likely that IL-1 induced by IL-1-treated thymocytes
The explanation may be that production and IL-2 receptor expression are perturbed by these factors. If cells fail to produce either this growth factor or receptor, T lymphocytes will fail to be activated. 5 Activity of CIF on Mature Lymphocytes Materials and Methods A. Purification of Peripheral Blood Lymphocytes Peripheral blood mononuclear cells were isolated from heparinized blood of healthy adult blood donors on a Ficoll-Hypaque density gradient. A pale yellow membrane containing no red blood cells was collected from the water-ficoll interface. Monocytes were removed by incubation on tissue culture plastic in RPMI 1640 medium containing 10% fetal calf serum (FCS) for 1 hour at 37°C. 0.25%
using sheep red blood cells (E-rosette) at 37°C.
T-lymphocytes were isolated from non-adherent cells by the rosette method for 2 hours. Ficoll−Hypaque
Centrifuge at 500 x g for 30 to 40 minutes.
T-lymphocytes that formed E-rosettes were separated from B-lymphocytes that did not form rosettes. The pellet was >95% E-rosette-forming cells. Sheep red blood cells were hemolyzed with 0.83% ammonium chloride. B lymphocytes that did not form rosettes were collected from the Ficoll-water interface. B T cell mitogen assay T lymphocyte proliferation was evaluated by mitogen microassay. The assay consists of seeding 0.2×10 ⁵ T cells in a 96-well microassay plate. cells, L-glutamine,
Penicillin-streptomycin and 10%
Cultured in RPMI 1640 medium supplemented with FCS.
This culture was mixed with concanavalin-A (from 6.2).
mitogen stimulation with 24 μg/ml). In this microculture, CIF-A or CIF-B
was added between 0.01 and 40 ng/ml. The final culture volume was adjusted to 250 μ and the mixture was incubated for 5 days at 37° C. in a 5% CO ₂ atmosphere in a humidified incubator. Cultures were harvested 18 hours before harvesting with an automated cell harvester.
Pulsed with 1.0 μCi of ³ H-thymidine (specific activity 25 Ci/mmol). Inhibition of ³ H-thymidine incorporation by various concentrations of CIF-A or CIF-B was determined from triplicate cultures using the following formula. ^% inhibition = (1 - mitogen treatment with CIF (cpm) / mitogen treatment without CIF (cpm) x 100 CB cell activation assay 10 ⁵ T lymphocytes were incubated with 2.5 Lymphocyte cultures were created by mixing with derived B lymphocytes. Cells were incubated in 1 ml of RPMI 1640 medium supplemented with L-glutamine, penicillin-streptomycin, and 10% FCS, 5% _CO2 , 37
Cultured at ℃. This culture was placed in a 12 x 75 mm culture tube. Pokeweed mitogen 1/10
Added to culture at 0 dilution. In these cultures,
CIF-A or CIF- at various concentrations (1.9×10 ^-12 M, 1.9×10 ^-10 M or 1.5×10 ^-9 M)
Added either B. Cells were cultured for 7 days. At the end of the culture period, the cell-free culture supernatant was analyzed for IgM by sandwich ELISA.
Alternatively, IgG was assayed. flat bottom polyvinyl chloride microtiter plate,
Approximately 120 to 500 μg of affinity-purified goat anti-human IgM or IgG in phosphate buffered saline (PBS) was incubated overnight at 4°C. To remove unbound antibody, plate in PBS containing 0.05% Tween-20.
(PT buffer). Culture supernatant, 0.05
It was diluted to 1/10 or 1/100 with PBS (PTB buffer) containing % Tween-20 and 0.5% bovine serum albumin. Purified human IgG or IgM at various concentrations (2 to 10 ng) for standard curve preparation.
was added to appropriate wells. After 2 hours at room temperature, the plates were washed with PT buffer. 1/5000
Diluted peroxidase-conjugated goat anti-human
100μ of IgG or anti-human IgM was added to each well. After incubating for 1 hour, plates were washed with PT buffer and 100 μ of freshly prepared substrate was added. This substrate is 0.03% (w/v) in 0.1M citrate buffer (PH4.0).
It consists of ABTS and 0.03% (v/v) hydrogen peroxide. The color was developed for 30 minutes and the absorbance at 414 nm was determined. The concentration of IgG or IgM was calculated from a standard curve prepared at the same time as this assay. IgG production by B cells or
Inhibition of IgM production can be achieved by CIF-A or CIF-
Calculated by comparing cultures treated with B to control cultures not treated with CIF.
The following formula was used. % inhibition of antibody = (1-ng/ml Ig (with CIF)/ng/ml Ig (without CIF)) x 100 Results The results of the T cell proliferation assay are plotted in FIG. As you can see from this figure, CIF-A and CIF
-B was found to have suitable activity against T lymphocyte proliferation. ^3H -thymidine uptake is approximately 38×10 ^-12 M of CIF-A or
It was inhibited by 78% to 80% by CIF-B. CIF
-A, half the maximum activity was obtained at concentrations lower than 1×10 ⁻¹² M. The concentration at which half of the maximum activity was obtained for CIF-B was estimated to be approximately 1.9×10 ⁻¹² M. These results clearly demonstrate that both CIFs are potent inhibitors of human T lymphocyte proliferation. The results of the B cell activation assay are summarized in Table 2 below. Data shown are expressed as mean ± SD for three experiments.

[Table] As shown in Table 2, 1.9×10 ^-12 M of CIF-A produced
The amount of IgM decreased by 33% and IgG decreased by 16%. 1.9×
CIF-A at a concentration of 10 ^-10 M or 1.5 x 10 ^-9 M
IgM production was completely inhibited in cultures containing .
Similar concentrations of CIF-A reduced IgG production by 54% to 56
% inhibited. It was found that CIF-B is comparable to CIF-A. As shown in Table 2 ^, CIF-
Cultures containing B contain no IgM and no IgG.
It was only 54%. CIF-B at a concentration of 1.5×10 ^-9 M
Cultures treated with 100% had no measurable IgM and only little (98% inhibition) or no IgG. These results indicate that CIF-A
and CIF-B have both been shown to be very potent inhibitors of antibody production by plasma cells. These in vitro T cell proliferation and B cell activation experiments raise the possibility that lymphocyte regulation is one mechanism involved in the previously observed in vivo lymphohistiocytic anti-inflammatory activity of CIF. Suggests. The results of these tests were published by Kehrl,
This confirms the study reported by JH, et al, Clinical Research (1985) 610A. 6. Using CIF in a Rodent Arthritis Model
Vivo studies Experimental collagen-induced arthritis (ECIA)
can be induced by injecting natural collagen (bovine or rat) emulsified in Freund's incomplete adjuvant into the skin of rats. Inbred Lewis rat strain LEW
(RT1 ¹ ), peripheral polyarthritis is known to occur within 14 to 30 days after immunization. Extensive mononuclear cell infiltrates occur in the cartilage of the joints and ears. Sensitized rats develop circulating antibodies against native collagen in response to an intradermal skin challenge test. Twelve LEW rats (12-15 weeks old) are divided into 3 groups of 4 rats each. The first group and the second group are
Intracutaneously inject 2.5 ml/Kg body weight of bovine collagen in Freund's complete adjuvant to sensitize it to bovine collagen. Group 1 will serve as a non-sensitized control. Measurements of the ankle and subjective grading of arthritis (from 0 to +4) are performed before sensitization and periodically during the study. Arthritis grading finger,
This is done based on the degree of inflammation and swelling of the feet and ankles. Groups of rats that showed a significant inflammatory response were treated at 1, 3, 5, and 7 days after the response was observed.
On day 1, treat by injecting CIF (500 ng dissolved in 0.05 ml sterile PBS) directly into the joint. A group of rats with a similar response will be treated similarly by injecting PBS without CIF. Before and after treatment, serum is collected from the rats and assayed for antibodies against type collagen by ELISA. Rats are subjected to an intradermal skin challenge test using 50 μg of type collagen on the abdomen 3 days before sacrifice. After the challenge
Measure erythema and induration at 24, 48 and 72 hours. At 72 hours, the challenge site will be biopsied and examined histologically. Five to seven days after the final injection, rats are sacrificed, exsanguinated, and their paws are fixed in 10% neutral formalin for histological examination. The results of these studies show that treatment with CIF significantly reduces inflammation compared to the untreated control group. The CIF-treated group has a decreased response to the type collagen skin challenge test when compared to the untreated control group. Furthermore, antibody titers against type collagen are significantly reduced in the treated group. These results suggest that administering CIF to diseased joints not only controls inflammation locally, but also acts as a systemic immunosuppressant for the treatment of arthritis. (Summary of the invention) Acute and/or chronic inflammation can be treated with CIF (TGF).
-β). CIF can be administered locally or systemically, depending on the indication, and does not require co-administration of active agents or cofactors for efficacy.

[Brief explanation of drawings]

FIG. 1 is the amino acid sequence of human TGF-β monomer derived from platelets. Figure 2 is Example 1 (section C)
It is a graph of the absorbance (absorption) (280 nm) of the gel filtration fraction of . FIG. 3 is a graph of the absorbance (280 nm) of the elution fraction of preliminary ion exchange chromatography in Example 1 (section D). Figures 4A and 4B show different amounts of recombinant IL-1 (rIL-1).
against stimulation-induced proliferation of murine thymocytes.
The effects of CIF-A (TGF-β) and CIF-B are shown graphically (Example 4). Figure 5 shows TGF-
Figure 4 is a graph showing dose-dependent inhibition of stimulated murine thymocyte proliferation by β and CIF-B (Example 4). FIG. 6 is a graph showing the effects of TGF-β and CIF-B on thymocyte cultures prestimulated for different times with LI-1 (Example 4). Figures 7A and 7B graphically illustrate the effect of TGF-β and CIF-B pretreatment on the responsiveness to IL-1 proliferation (Example 4).
FIG. 8 is a graph of the results of the T cell proliferation assay described in Example 5 (section B).

Claims (1)

[Claims] 1. A composition used for inflammation treatment, which contains cartilage-inducing factor CIF-A or CIF-B.