JPH05222081A - Lymphotoxin factor - Google Patents

Abstract

PURPOSE:To provide the subject new factor originated from the peripheral blood of a patient of chronic rheumatism, effective in clarifying the mechanism of systemic tissue disorder in chronic rheumatism and useful as a treating agent or diagnostic for systemic tissue disorder in chronic rheumatism. CONSTITUTION:The objective factor is derived from a fraction having a molecular weight of 900kd by gel-filtration fractionation method. It inhibits the DNA synthesis of normal peripheral blood monocyte and non-specifically damages the normal peripheral blood monocyte and human cell strain. It is stable to the heat-treatment at 56 deg.C for 30min or at 85 deg.C for 5min and is free from protease activity and collagenase activity. The factor can be prepared e.g. by collecting and clotting the peripheral blood of a patient of chronic rheumatism, separating the blood serum by centrifugal precipitation, fractionating the serum by high-performance liquid chromatography, etc., concentrating the obtained fraction having a molecular weight of 900kd by ammonium sulfate precipitation, etc., dialyzing with PBS and purifying the obtained crude product by ion-exchange chromatography.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cytotoxic factor derived from peripheral blood of a patient with chronic rheumatism. The cytotoxic factor of the present invention is used in fields such as treatment of chronic rheumatism and clinical examination.

[0002]

BACKGROUND OF THE INVENTION In patients with chronic rheumatism (RA), not only joint destruction but also various systemic tissue injuries such as anemia, osteoporosis and fibrinoid necrosis found in various organs are observed. It recognized. With respect to the cause of local destruction, there are some reports that causative substances have been analyzed by analyzing the mechanism such as invasion of synovial tissue into articular cartilage. For example, IL-1 activity, P in the bone marrow near the affected joint for local tissue injury of RA
Polymorphonuclear cell factor (PMN facto
r)) It is reported that the activity is extremely high and can cause joint destruction. However, although various investigations have been conventionally made on systemic tissue injury in RA patients, the cause thereof has not been clarified at present.

It is also known that the DNA synthesis of stimulated peripheral blood mononuclear cells is suppressed in vivo by various mechanisms. However, it has not been reported so far that peripheral blood serum of RA patients reduces the number of viable cells of autologous peripheral blood mononuclear cells and causes nonspecific cell destruction. In addition, cytokines such as TNF-α and lymphotoxin (Lymphotoxin) have been used as substances with cytotoxicity.
(TNF-β)) and the like are known, but these cytokines do not destroy normal cells but only pathological cells such as tumor cells, causing systemic tissue damage in RA patients. It is different from the causative substance. Therefore, it has been required to search for a causative agent of systemic tissue injury in RA patients and to develop a therapeutic agent or diagnostic agent using it.

[0004]

Means for Solving the Problems As a result of intensive studies on the cause of the above-mentioned systemic tissue injury, the present inventors have found that
It causes non-specific cell destruction in unfractionated serum of severe RA patients with strong systemic tissue injury, along with rapid and severe destruction of all joints of the whole body, and even viable cells of their own peripheral blood mononuclear cells. It was also found that there is an injurious factor (hereinafter referred to as a cytotoxic factor), and further that this cytotoxic factor is a substance obtained from a fraction having a molecular weight of about 900 kd.

Furthermore, the present inventors further confirmed that this factor is a cytokine such as tumor necrosis factor (TNFα), a substance of the complement system, and a protease, a collagenase or a phospholipase A ₂ (phospholipase).
It is a specific factor different from degrading enzymes such as ase A ₂ (PLA ₂ )), and has the effect of nonspecifically damaging normal peripheral blood mononuclear cells and various human cell lines at physiological concentrations. The present invention has been completed and the present invention has been completed.

That is, the gist of the present invention is to provide a cytotoxic factor derived from the peripheral blood of a patient with chronic rheumatism and having the following characteristics. (1) Obtained from a fraction having a molecular weight of about 900 Kd by the gel filtration fractionation method. (2) Damage to DNA synthesis of normal peripheral blood mononuclear cells. (3) Nonspecific damage to normal peripheral blood mononuclear cells and human cell lines. (4) Stable against heat treatment at 56 ° C. for 30 minutes or 85 ° C. for 5 minutes. (5) It has no protease activity and collagenase activity.

In the present invention, the cytotoxic factor of the present invention is derived from peripheral blood of a patient with chronic rheumatism, and the above (1)
The substance having the characteristics (5) to (5) means not only a purified product as a pure product but also a preparation containing the substance. Hereinafter, the cytotoxic factor of the present invention will be described in more detail.

(A) Molecular weight: Obtained from a fraction having a molecular weight of about 900 Kd by gel filtration fractionation method. (b) Color: colorless. (c) Water-soluble: Poorly soluble in distilled water, and easily soluble in physiological saline and phosphate buffer (pH 7.4).

(D) Damage to DNA synthesis of normal peripheral blood mononuclear cells. Normal peripheral blood mononuclear cells (normal PBMC) obtained from healthy subjects
Phytohemagglutinin (PHA),
Stimulated by Wellcome Diagnostics Inc.), was added the cytotoxic agent of the present invention, cultured in microtiter plates, tritium thymidine ⁽³ H-thymidine in a few hours before the completion of the culture ⁽³ H-TdR )) Is added and the uptake is measured with a liquid scintillation counter. In this case, ³ by adding the cytotoxic agent of the present invention
Reduced uptake of H-TdR, ie DN of normal peripheral blood mononuclear cells
A synthetic injury is observed.

(E) Non-specifically damaging normal peripheral blood mononuclear cells and human cell lines. The cytotoxic factor of the present invention is a normal PBM obtained from a healthy person.
C and various human cell lines such as Burkitt lymphoma (Bu
Raji cells, which are lymphoblast-like cell lines established by rkitt's lymphoma, and acute lymphoblastic leukemia.
CCRF-CEM cells, which are T lymphoblastoid cell lines established from the peripheral blood of patients, HL-60, which is a promyelocytic cell line, and histiocytic cells. It shows non-specific cytotoxicity against monocyte-like cell line U-937 etc. established by lymphoma).

In the present invention, the term "cytotoxicity" means that the number of viable cells is reduced by adding the cytotoxic factor of the present invention to various cells treated with mitomycin C and culturing the cells for about 24 hours. The following method is used to evaluate the number of viable cells. 1) Calculation of the number of viable cells by trypan blue staining After culture, trypan blue staining is performed and the number of viable cells is calculated. The number of viable cells at this time is evaluated by the ratio (cell viability) to the number of cells before the start of culture. 2) Tetrazolium salt dye, 3- (4,5
-dimethylthiazol-2,5-diphenyltetrazolium bromide
(MTT)) for rapid colorimetric
assay) (Mosmann T., J. Immunol. Methods, 65 , 55-63,
(1983), Green LM, et al, J. Immunol., 137, 3488-34.
93, (1986)) MTT is a mitochondrial dehydrogenase (mitocondorial).
However, only viable cells can decompose MTT. Therefore, after culturing with the addition of the cytotoxic factor of the present invention, the absorbance (OD) was determined by a rapid colorimetric method using MTT. Can be measured to assess the number of viable cells.

(F) Heat resistance: The fractionated serum containing the cytotoxic factor of the present invention is treated at 56 ° C. for 30 minutes or 85 ° C. for 5 minutes at pH 7.4.
It is stable and does not lose its cytotoxicity even when it is immediately cooled with ice after being heat-treated at.

(G) It has no protease activity or collagenase activity. The cytotoxic factor of the present invention is a Protease Substrate Gel Kit (Bio-
Rad) (Bierrum OJ, et al, Scand.J.Immunol.Sup
pl. , 2 , 81-88, (1975)), it shows no protease activity. In addition, collagenase activity is used as a substrate for fluorescein isothiocyanate.
isothiocyanate (FICT)) A solution method (Terato K. et al, Biochim. Bioph) using labeled type I collagen (Koragen Gijutsu Kenshukai) as a substrate.
ys.Acta, 445 , 753-762, (1976)). In this case, when the FICT-labeled collagen decomposed and released in the supernatant was excited at 495 nm and the emission fluorescence intensity at 520 nm was measured to evaluate the enzyme activity, the cytotoxic factor of the present invention does not show collagenase activity.

(H) It has almost no PLA ₂ activity. The cytotoxic factor of the present invention is obtained by the method of Tojo and Ono (To
jo et al, J. Biol. Chem., 263 , 5724-5731, (1988), Tojo
et al, Methods in Enzymology, 197 , 390-399, (1991))
When PLA ₂ activity is measured by, the activity is hardly shown. That is, the fatty acid liberated by PLA ₂ was derivatized with 9-anthyldiazomethane, separated by reverse-phase high performance liquid chromatography, and detected by ultraviolet absorption at 254 nm, and then margaric acid (margaric acid) was detected. ) Is used as an internal standard to quantify the amount of each fatty acid. The reaction system contained 5 mM CaCl ₂ and 0 as a substrate.
8 mM 1-palmitoyl-2-oleyl-phosphatidylglycerol (1-palmitoyl-2-oleoyl-phosphatidylg
lycerol, Avanti Polar Inc. Co.), 5 mM sodium cholate, 0.1 M NaCl, 0.1 M Tris-HCl (Tris-HCl (pH 8.5)) was added to the sample. PLA ₂ activity is evaluated by the amount of oleic acid released per minute.

The cytotoxic factor of the present invention having the above-mentioned properties is a cytokine known as a cytotoxic substance, such as TNF-α and Lymphotoxin (T
It is a substance that is clearly different from NF-β)). This is because the cytokine does not destroy normal cells, but only pathological cells such as tumor cells, as will be shown in Comparative Examples below. Further, the cytotoxic factor of the present invention is 56
It is also different from the substance of the antigen-antibody-complement system because it exhibits heat resistance such that it is not inactivated even by heat treatment at 30 ° C for 30 minutes. In addition, the cytotoxic factor of the present invention has no protease activity, collagenase activity, and almost no PLA ₂ activity, and therefore has a cytotoxic activity different from that of conventionally known cytotoxic substances. Judged as something.

The cytotoxic factor of the present invention has a molecular weight of about 0.1 from the serum of peripheral blood of RA patients by a known method such as gel filtration.
It is obtained by separating and purifying a 900 kd fraction. The purification method is not particularly limited, and various methods commonly used for protein purification are appropriately combined and applied. An example will be described below.

First, the peripheral blood of a patient is collected, coagulated and then spun down to separate serum. The obtained serum is fractionated using a conventional fractionation method such as a high performance liquid chromatography system. The molecular weight of each obtained fraction is about 900k
Fractions d are for example ammonium sulfate precipitated, YM5 ultrafiltration membrane (YM5 ultrafiltratio).
n membrane) and the like, and dialysis with PBS is carried out to prepare a crude liquid containing the cytotoxic factor of the present invention. For purification from the obtained crude liquid, a purified product can be prepared by a conventional purification means such as ion exchange chromatography or chromatofocusing.

The cytotoxic factor of the present invention is used for investigating the mechanism of systemic tissue injury in rheumatoid arthritis, as well as a therapeutic method for the systemic tissue injury of rheumatoid arthritis, development of a therapeutic agent, and chronic joint injury. It is used in the diagnosis of rheumatism and the diagnosis of medical conditions. As a therapeutic method, for example, plasma exchange can be performed to selectively remove the cytotoxic factor of the present invention from patient plasma. As the therapeutic agent, for example, it can be used as a sensitizing therapeutic agent which sensitizes a patient with a trace amount of the cytotoxic factor of the present invention. In addition, as a diagnostic agent, for example, the activity of the cytotoxic factor of the present invention in the serum of a patient can be measured and used for the determination of disease state or severity.

[0019]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. Example 1 (1) Target Patients Seven patients with rheumatoid arthritis (RA), aged 37 to 64 years old (mean 47 ± 8.3 years old) who are undergoing medical treatment, were targeted.
All patients were diagnosed by the American College of Rheumatoid Arthritis Rheumatoid Arthritis Diagnostic Criteria (1987) (Arnett et al, Arthritis
Rheum., 31 , 315-324, (1988)). Also,
Those who did not receive any steroids within 6 months of this study were considered. As a normal control, healthy subjects aged 38 to 47 years without inflammatory disease or immune abnormality 4
Cases (2 males, 2 females, average age 43 ± 4.7 years) were targeted.

(2) Preparation of Peripheral Blood Mononuclear Cells Peripheral blood of the above 11 subjects was collected as sample serum, coagulated, and then centrifuged at 800 × g for 15 minutes to separate serum. The collected serum was frozen and stored at −80 ° C. until the experiment. Peripheral blood of the 11 cases was collected by adding heparin and
Peripheral blood mononuclear cells (PBMC) were separated by ll / Paque (Pharmacia) specific gravity separation method. Cells are phosphate buffered (phosph
ate-buffered saline (PBS)) three times and then 100
The suspension was suspended in a culture medium RPMI1640 containing U / ml penicillin, 100 μg / ml streptomycin, and 5% fetal bovine serum.

(3) DNA Synthesis by Autologous Peripheral Blood Mononuclear Cells 0.25 PBMCs from 11 patients obtained from each patient and healthy volunteers were obtained.
% Phytohemagglutinin (PH
A), manufactured by Wellcome Diagnostics), and 1 × 10 ⁵ / well (200 μm) in a 96-well microtiter plate.
The culture was carried out at 37 ° C. and 5% CO ₂ for 72 hours each at the concentration of 1). At this time, 50% autologous serum added group of each patient and healthy person (PHA-stimulated autologous PBMC 1 × 10 ⁵ cells / well (200 μl)
To 100 μl (50%) of each autologous serum,
Non-added group (PHA stimulated self PBMC 1 × 10 ⁵ cells / well (200
μl) alone was prepared). 1μ in 4 hours after completion of culture
Ci / well tritium thymidine ( ³ H-TdR) was added, and the uptake was measured by a liquid scintillation counter. The results of the measured values are as shown in FIG. 1 (the results of the measured values are all expressed as mean ± standard deviation).

(4) Effect of Autologous Serum on DNA Synthesis of PBMC In all healthy subjects (4 cases), ³ H-Td was added by addition of serum.
Increased R uptake was observed. On the other hand, it showed an increase of ³ H-TdR incorporation in 4 cases out of RA 7 patients, observed the bright kana reduced uptake of ³ H-TdR in 3, inhibition of DNA synthesis was observed. From this, it was considered that the serum of these three RA patients contains a substance that suppresses cell proliferation and / or a substance that acts on cytotoxicity.

Example 2 (1) Size-exclusion chromatography
Fractionation of Serum Components by Chromatography) The sera of 1 healthy subject and 3 RA patients with reduced ³ H-TdR uptake in Example 1 were subjected to size exclusion chromatography to obtain No. 31 to No. Up to 40 fractions. That is, 10 ml of serum of each sample was used for the high-performance liquid chromatography system (high-performance liq
uid chromatographic gel filtration system, TSK ge
Fractionation was performed using a G4000 SW, manufactured by Tosoh Corporation. The eluent is 0.01M phosphate buffer containing 0.5M NaCl.
buffer (pH 7.4)) and a flow rate of 3 ml / min, and each fraction was collected every minute. Fractions from each fraction were collected using YM5 ultrafiltration membrane (Amic
on company) to 1.8 ml, dialyzed against PBS, and adjusted to 2.0 ml.

(2) Effect of serum fraction components on DNA synthesis of normal peripheral blood mononuclear cells Normal PBMC obtained from healthy subjects were stimulated with PHA,
After adding 20 μl of each serum fraction prepared in the above (1) at a concentration of 1 × 10 ⁵ / well (200 μl), 37 ° C,
Cultured in 5% CO _{2 for} 72 hours, PHA-stimulated normal PBMC
Was investigated for its effect on DNA synthesis. DNA synthesis was measured by the same method as in Example 1.

That is, 20 μl of PHA-stimulated normal PBMC
The fractionated serum of healthy subjects or the fractionated serum of RA patients was added and cultured for 3 days, and the ³ H-TdR uptake was measured. As a control, ³ H-TdR uptake was measured when the medium was cultured alone. The results of the measured values are as shown in FIG. 2 (the results of the measured values are all expressed as mean ± standard deviation). A to F in FIG. 2
Indicates a molecular weight marker, (A) blue dextran 2000, (B) thyroglobulin, (C) ferritin (ferriti).
n), (D) catalase, (E) ovalbumin, (F) ribonuclease A (ribo)
nuclease A).

Uptake of ³ H-TdR when PHA-stimulated normal PBMC was cultured in medium alone was 128,030 ± 3,327 dp.
It was m. When the fractionated serum of healthy subjects was added, ³ H-TdR
Uptake of 116,520 dpm in all fractions
As described above, inhibition of DNA synthesis was not observed. R
Uptake of ³ H-TdR when the serum fraction component of patient A was added was suppressed by two fractions (fraction No. 42 (average molecular weight 900 kd) and fraction No. 56 (average molecular weight 280 kd)). It was Especially in Fraction No. 42, strong D was observed in all 3 RA patients.
Inhibition of NA synthesis was observed (mean ³ H-TdR; 1,486 ± 2,14
4 dpm). In fraction No. 56, RA patient 2
In some cases, mild inhibition of DNA synthesis was observed (mean ³ H-TdR
; 80,462 ± 98,020 dpm). No suppression was observed in the remaining 1 RA patient.

Example 3 Measurement of cytotoxicity The cytotoxicity of each serum fraction component to cells was determined by (1) counting the number of viable cells by trypan blue staining, and (2) high-speed using tetrazolium salt (MTT). Colorimetric assay (arapid col
orimetric assay). First, MTT at a concentration of 5 μg / ml is dissolved in PBS, and 20 μl of this is added to each well of a microplate and incubated at 37 ° C. for 4 hours. After this, sodium lauryl sulfate containing 0.01N hydrochloric acid (so
50 μl of dium laurylsulfate) was added for solubilization and immunoreader NJ-2001 (Immuno Reader NJ-2001) (Nippon I
The absorbance (OD) at 570 nm was measured with an nterMed KK company.

As target cells for knowing the cytotoxicity of the specimen, normal PBMC obtained from healthy subjects and various human cell lines, such as lymphoblast-like cell line (lymphoblast-like cell).
Raji cells which are L line), CCRF-CEM cells which are T lymphoblastoid cell line,
HL, a promyelocytic cell line
-60 and a monocyte-like cell line U-937 were used.

The cytotoxicity was measured as follows. 0.5 μg / ml of mitomycin C (M
MC)) in 100 μl medium, normal PBMC is 2 × 1
For other cell lines at a concentration of 0 ⁶ cells / well,
The cells were cultured at 37 ° C. for 8 hours at a concentration of 2 × 10 ⁵ cells / well. After that, 40 μl (20%) of each serum fraction component and 60 μl of culture solution were added, and the mixture was further cultured for 20 hours. Here, cell viability was calculated by trypan blue staining. At the same time, the cytotoxicity was evaluated as a percentage of the number of damaged cells by a high-speed colorimetric method using MTT, and calculated as follows. Cytotoxicity (number of damaged cells) (%) = {OD
(Control sample) -OD (sample) / OD (control sample)} x 1
00

Example 4 Measurement of cytotoxicity of fraction No. 42 The effect of fraction No. 42 on cells treated with mitomycin C was examined. Add 40 μl of Fraction No. 42 to each well and add 24
After culturing for a period of time, trypan blue staining and cell viability (cell
viability (%)) was calculated. That is, the number of viable cells at this time was evaluated by the ratio to the number of cells before the start of culture. The number of viable cells is normal PBMC, Raji cell, CCRF-CEM cell,
53.9 ± 6.1 in HL-60 cells and U-937 cells, respectively
%, 24.5 ± 8.4%, 11.1 ± 10.1%, 84.3 ± 7.2%, 46.1
Both decreased to ± 6.4% (Table 1). Therefore, fraction N
It was revealed that o.42 causes cell damage rather than suppression of DNA synthesis.

[0031]

[Table 1]

Furthermore, the cytotoxicity of fraction No. 42 against various human cell lines was examined by a rapid colorimetric assay using MTT. Cytotoxicity is normal P
BMC, Raji cells, CCRF-CEM cells, HL-60 cells, and U-
45.2 ± 6.4%, 77.6 ± 3.6%, 9 in 937 cells, respectively
The values were 4.6 ± 0.7%, 10.4 ± 1.6% and 40.6 ± 6.1% (Table 1). Fraction No. 42 non-specifically injured all cells. Also, treated with mitomycin C by a microscope.
After culturing CCRF-CEM cells for 24 hours, the cells were cultured only in the medium and the cells cultured by adding 40 μl of fraction No. 42 were compared and observed. It was found that the cells were significantly destroyed and the number of cells was obviously reduced.

Next, this most sensitive CCRF-CEM
When the concentration dependency of the cytotoxicity of fraction No. 42 was examined using cells as target cells, the cytotoxicity increased in a volume-dependent manner in the range of 5 μl to 40 μl (FIG. 3).

Comparative Example 1 TNF-α Cytotoxicity Among cytokines, TNF-α is known to have cytotoxicity against certain tumor cells. Therefore, we also examined the cytotoxicity of TNF-α using MTT in the same manner as in Example 4. The reported in vivo TNF-α activity, including RA synovial fluid, is at most 25 U-ml. In this test, 10 times as much TNF-α (250U / ml) was used. As shown in Table 1, 250U / ml of recombinant human was used.
The cytotoxicity of TNF-α (Genzyme) was 2.0 ± 2.4 for normal PBMC, and almost no cytotoxicity was observed for CCRF-CEM cells.

Example 5 Cytotoxic heat resistance of Fraction No. 42 Fraction No. 42 was treated at 56 ° C. for 30 minutes or at 85 ° C. for 5 minutes at pH 7.4.
Immediately after the heat treatment in (3), it was cooled with ice, and it was measured whether or not the cytotoxicity was lost. Therefore, it was considered that the cytotoxicity of fraction No. 42 was not due to the substance of the complement system. Further, no decrease in cytotoxicity was observed even after treatment at 85 ° C. for 5 minutes, and it was found that the cytotoxicity of fraction No. 42 was stable to heat.

Example 6 Measurement of Protease Activity and Collagenase Activity Protease activity
It measured using the gel kit. This gel kit is 1%
If the sample contains agar and bovine casein and the sample contains protease, a transparent circle is formed around it and the activity is measured from the diameter. In addition, this gel kit allows serum plasmin (serum plasmi
n), trypsin, α-chymotrypsin (α-
It has been proved that chymotrypsin) and pronase activity can be measured. The minimum measurement sensitivity of this gel kit was 34 unit / ml in terms of trypsin.

Collagenase activity can be determined by using fluorescein isothiocyanat as a substrate.
e (FICT) labeled type I collagen
n) (Koragen Gijutsu Kenshukai) was used as a substrate for a solution method. The enzyme activity was evaluated by exciting FICT-labeled collagen decomposed and released in the supernatant at 495 nm and measuring the emission fluorescence intensity at 520 nm. As a result of the evaluation, no protease activity or collagenase activity was detected in any of the samples including the fraction No. 42.

[0038] Measurement PLA ₂ activity of Example 7 PLA ₂ activity, Dongcheng and Ono method (Tojo et al, JB
iol.Chem., 263 , 5724-5731,1988, Tojo et al, Methods
in Enzymology, 197 , 390-399, (1991)). That is, the fatty acid liberated by PLA ₂ was derivatized with 9-anthyldiazomethane and separated by reverse phase high performance liquid chromatography to obtain 254 nm
It was detected by ultraviolet absorption. Margaric acid
acid) was used as an internal standard to quantify the amount of each fatty acid. The reaction system was 5 mM CaCl ₂ , and 0.8 mM 1-palmitoyl-2-oleyl-phosphatidylglycerol (1-palmitoyl-2-oleoyl-phosphatidylglycerol, A as a substrate).
vanti Polar Inc. Co.), 5 mM sodium cholate (so
dium cholate), 0.1M NaCl, 0.1M Tris-HC
1 (pH 8.5)) to which the sample was added. PLA ₂ activity was expressed as nmoles, the release of oleic acid per minute.

As a result of the evaluation, PLA ₂ activity was as high as 137.4 ± 28.8 nmol / min / ml in the original serum,
Fraction No. 42 showed a low activity of 2.1 ± 0.8 nmol / min / ml, which was a very low activity of 1.53% of the total serum, and it was contaminated with substances other than the factor of the present invention. It was thought to be due to.

Comparative Example 2 Measurement of Cytotoxicity of PLA ₂ on Various Human Cell Lines [Tojo et al, Methods]
in Enzymology , 197 , 390-399, (1991), Pruzanski et
al, J. Rheumatol., 12 , 211-216, (1985)) extracted PLA ₂
Was used at a 100-fold higher activity than fraction No. 42, and the same experiment as in Example 4 was carried out, but no cytotoxicity was shown. Therefore, it was determined that the cytotoxicity of fraction No. 42 was not due to PLA ₂ .

[0041]

EFFECT OF THE INVENTION By providing the cytotoxic factor of the present invention, it becomes possible to investigate the mechanism of systemic tissue damage in chronic rheumatism, and to develop therapeutic agents and diagnostic agents for systemic tissue damage in chronic rheumatism. Became possible.

[Brief description of drawings]

FIG. 1 shows the effect of autoserum addition on PBMC DNA synthesis.

FIG. 2 shows the effect of each serum fraction component on DNA synthesis of normal PBMC.

FIG. 3 shows the dose-dependent cytotoxicity of fraction No. 42 containing the cytotoxic factor of the present invention.

Claims (1)

[Claims] 1. A cytotoxic factor derived from peripheral blood of a patient with chronic rheumatism and having the following characteristics. (1) Obtained from a fraction having a molecular weight of about 900 Kd by the gel filtration fractionation method. (2) Damage to DNA synthesis of normal peripheral blood mononuclear cells. (3) Nonspecific damage to normal peripheral blood mononuclear cells and human cell lines. (4) Stable against heat treatment at 56 ° C. for 30 minutes or 85 ° C. for 5 minutes. (5) It has no protease activity and collagenase activity.