JP2002221520A - Prediction method for immunology type - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for predicting a type of an immunoreaction derived by chemical matter. SOLUTION: This prediction method for an immunology type derived by the test matter predicts the type of the immunoreaction derived by the test matter on the basis of a ratio between an expression amount of a Th1 cytokine and an expression amount of a Th2 cytokine in a tissue of a Th1-predominance mouse administered with the test matter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for predicting an immune type induced by a chemical substance.

[0002]

BACKGROUND OF THE INVENTION Immunity induced by chemicals includes:
There are cell immunity and humoral immunity. Cellular immunity causes delayed type hypersensitivity and the like, and humoral immunity causes immediate type hypersensitivity and anaphylaxis. Such immunity is induced by helper T cells involved in sensitization by a chemical substance. Helper T cells are classified as Th0, Th1, or Th2 cells, and Th1 cells are involved in inducing cellular immunity, and Th2 cells are involved in inducing humoral immunity. Whether the cellular immunity or the humoral immunity is predominantly induced depends on the type of the chemical substance.

[0003]

If it is possible to determine whether the chemical substance induces cell immunity or humoral immunity, it is necessary to take safety measures before handling the chemical substance. Therefore, there is a need to develop a method for predicting the type of immune response induced by a chemical because it can help determine the course of treatment when sensitized to the chemical.

[0004]

Under such circumstances, the present inventors have conducted intensive studies and as a result, based on the ratio of the expression level of a specific cytokine in the tissue of a certain kind of mouse to which a chemical substance has been administered. Thus, the present inventors have found that the type of immune response induced by the chemical substance can be predicted, and have led to the present invention. That is, the present invention provides 1) the type of immune response induced by a test substance based on the ratio of the expression level of Th1 cytokine to the expression level of Th2 cytokine in the tissue of a Th1 dominant mouse to which the test substance has been administered. A method for predicting an immune type induced by a test substance, which is characterized by predicting; 2) (1) measuring the expression levels of Th1 and Th2 cytokines in the tissues of Th1 dominant mice to which the test substance has been administered; Determining the ratio of the expression levels of the two cytokines, and (2) administering the standard substance.
The ratio of the expression level of the Th1 cytokine to the expression level of the Th2 cytokine in the tissue of the Th1 dominant mouse is compared with the ratio determined in step (1) to predict the type of the immune response induced by the test substance. A method for predicting an immune type induced by a test substance, which comprises the step of: 3) a method for predicting an immunological type according to 1) or 2) above, wherein the expression level is an mRNA level. 4) The prediction method according to any one of 1) to 3) above, wherein the tissue is a local lymph node. 5) The Th1 dominant mouse to which the test substance is administered is compared with the Th1 dominant mouse in the negative control group. The prediction method according to any one of the above items 1) to 4), wherein the mouse has a node weight increased more than twice, 6) the Th1 cytokine is IL-12p40, and the Th2 cytokine is IL-4. 5) The prediction method according to any one of 7), 7) induced by the test substance based on the ratio of the expression level of Th1 cytokine to the expression level of Th2 cytokine in the tissue of a Th1 dominant mouse to which the test substance has been administered. It is intended to provide a system for predicting an immune type induced by a test substance, which is characterized by predicting the type of an immune response.

[0005]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A prediction method according to the present invention will be described below. Th1 dominant mice (Heinzel FP etc., J. Exp. Med, 169, 59-7) which can be used in the prediction method of the present invention.
2 (1989); Heinzel FP etc, J. Exp. Med, 177, 150
5-1509 (1993); Hocart MJ etc, J. Gen. Virol, 7
0, 2439-2448 (1989)), for example, C57BL / 6 mouse, CBA / CaH mouse and the like. Such a Th1 dominant mouse may be a young adult animal of about 6 to 15 weeks of age, and may be either male or female. As a breeding method, for example, there is a method in which a mouse is housed in a cage and a solid feed and filtered tap water are freely taken in through an automatic water supply device. A test substance is administered to Th1 dominant mice as described above. As the number of mice, one or more mice per test substance, preferably 2 to 3 mice per group are used. Examples of the method of administering the test substance include a method of applying a test substance solution to the depilated back and auricle, a method of applying only to the auricle, and a method of intradermal administration to a Foot Pad. The test substance solution is prepared by dissolving the test substance in an appropriate solvent. As the solvent, for example, a liquid in which the test substance is dissolved and which is not allergic or irritant can be used. Specifically, for example, a mixed solution of acetone and corn oil, ethanol, DMSO, physiological saline And the like.
The dose of the test substance includes, for example, the concentration and volume of the Th1 dominant mouse to which the test substance is administered, in which the local lymph node weight is increased by 2 times or more compared to the mice in the negative control group. be able to. Examples of the negative control group include a group to which only the solvent used for the preparation of the test substance solution was similarly administered instead of the test substance solution.

Next, the expression levels of Th1 cytokine and Th2 cytokine in the tissues of the Th1 dominant mouse to which the test substance has been administered are measured. Examples of the tissue include a local lymph node, a spleen, and the like. Examples of a method for collecting the tissue include, for example, a method in which the animal is sacrificed and collected after a certain period of time from the final administration.Th1 cytokine and Th2 cytokine are
Th1 type reaction (cellular immunity) and Th2 type reaction (humoral immunity)
Cytokines that are closely related to
30, 343-346 (1998); Buschenfelde CMZet al, Clin.
Exp. Immunol., 110, 378-385 (1997)]. Specifically, for example, Th1 cytokines include IL-12p40 and IL-18, and Th2 cytokines include IL-4 and IL-10. Examples of the method for measuring the expression level of the cytokine include a method for measuring the mRNA amount of the cytokine to be measured and a method for measuring the protein content of the cytokine to be measured. As a method of measuring the amount of mRNA,
For example, an RT-PCR method, a Northern blot method and the like can be mentioned.

The measured expression level of Th1 cytokine and Th2
The type of immune response induced by the administered test substance is predicted based on the ratio to the expression level of the cytokine (hereinafter, referred to as Th1 / Th2 ratio). Specifically, for example, the expression level of Th1 cytokine and T
The Th1 / Th2 ratio is determined from the expression level of the h2 cytokine, and the ratio is compared with the Th1 / Th2 ratio in the tissue of the Th1-dominant mouse to which the standard substance has been administered. As the standard substance, for example, a substance known to induce a Th1-type reaction or a Th2-type reaction dominantly can be used. Specifically, for example, as a standard substance that predominantly induces a Th1 type reaction, dinitrochlorobenzene (TNCB), trinitrochlorobenzene (TNCB), and the like can be mentioned.As a standard substance that predominantly induces a Th2 type reaction, anhydrous Trimellitic acid (TMA), diphenylmethane diisocyanate (MDI) and the like. Such a standard substance that induces a Th1 type reaction predominantly and a standard substance that induces a Th2 type reaction predominantly are each administered to a Th1 dominant mouse in the same manner as a test substance, and the expression amount of Th1 cytokine in tissues such as local lymph nodes. The Th1 / Th2 ratio is determined by measuring the expression level of the Th2 cytokine.
The Th1 / Th2 ratio determined for the test substance is compared with the Th1 / Th2 ratio determined for each of the standard substance that predominantly induces a Th1-type reaction and the standard substance that predominantly induces a Th2-type reaction. By determining whether they are close to each other, the type of immune response induced by the test substance can be predicted.

[0008]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the present invention is limited thereto. Example 1 Using a solution obtained by mixing acetone and corn oil at a ratio of 4: 1 as a solvent, 1% DNCB, 10% TMA, 0.4% 2,4-dinitrofluorobenzene (DNF)
B), 0.25% 4-ethoxymethylene-2-phenyloxazol-5-one (OXA), 0.4%
Toluene 2,4-diisocyanate (TDI) and 15% phthalic anhydride (PA) were prepared. Six-week-old male C57BL / 6 mice (Charles River Japan) were preliminarily reared for 7 days. The mice were housed in plastic cages (W290 × D340 × H170 mm, floor bedding: White Flake, CLEA Japan) and 6 to 10 mice / cage were kept. CRF-
1 The solid feed (Oriental Yeast Co., Ltd.) and filtered tap water were freely ingested. The breeding cage was replaced with a washed and sterilized cage three times a week. The environment of the breeding room is temperature 23 ± 2 ℃, relative humidity 55 ± 15%, ventilation rate 10
The lighting time was set to 12 hours (8:00 to 20:00) or more times / hour. The hair of the back of the C57BL / 6 mouse (7-week-old) after preliminary breeding was shaved, and the above compound solution or solvent (4: 1 mixture of acetone and corn oil) (100 μl) was applied thereto, and again 5 days later 100
μl each to the depilated back, and after 3 days 3
For two consecutive days, 20 μl was applied to both pinna once a day. Two mice were used as one group per compound. Twenty-four hours after the final application, the left and right auricular lymph nodes were collected, two animals per group were collected and weighed, and the lymph node weight per animal was calculated. Table 1 shows the results. In the compound administration group, a weight increase of about 3 to about 4 times was observed as compared with the solvent administration group.

[0009]

[Table 1] *: The lymph node weight of the vehicle administration group is represented as 1.0.

The mRNA levels of IL-4 and IL-12p40 in the collected auricular lymph nodes were quantified as follows. First, RNA was extracted from the auricular lymph node using Isogen (Nihon Shen) according to the method described in the attached manual of the reagent. From extracted RNA, SuperScript Reverse Transcr
Using iption System (Gibco BRL), cDNA was prepared according to the method described in the manual attached to the system. In addition,
Primers include oligo (dT) _12-18 primer (Gibco BR
L) was used. Next, using the obtained cDNA as a template, Takara
PCR was performed using Extaq DNA polymerase (Takara Shuzo). IL
In order to amplify the cDNA of -4, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 were used as primers, and after incubation at 94 ° C. for 3 minutes , 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute.
The cycle was repeated 24 times, and the temperature was kept at 72 ° C. for 7 minutes. In order to amplify the cDNA of IL-12p40, an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an oligonucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 were used as primers at 94 ° C.
After maintaining the temperature for 94 minutes, the temperature was maintained at 94 ° C. for 30 seconds, then at 60 ° C. for 30 seconds, and further at 72 ° C. for 1 minute as one cycle, and this was performed for 26 cycles, and further maintained at 72 ° C. for 7 minutes. . In order to amplify βactin cDNA as an internal standard, an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 5 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 6 were used as primers at 94 ° C.
After keeping the temperature for 94 minutes at 94 ° C. for 30 seconds and then at 55 ° C. for 30 seconds, the temperature was kept at 72 ° C. for 1 minute as one cycle, and this was repeated for 17 cycles, and further kept at 72 ° C. for 7 minutes. . Each of the DNAs obtained by the PCR is applied to a 2% agarose gel to which GelStar (Takara Shuzo) has been added to perform electrophoresis, and the fluorescence intensity of the DNA band is determined by a lumino image analyzer (LAS-1000plus, Image Gauge; Fuji Photo Film). )
Quantification was performed using IL-4 and quantified for each group
The amount of cDNA of IL-12p40 was corrected by the amount of cDNA of βactin, respectively, to obtain a numerical value representing the amount of mRNA of IL-4 and IL-12p40.
IL-4 to the amount of IL-12p40 mRNA determined for each group
Were plotted, and a quadrangle was created using the coordinates of DNCB as a standard substance for induction of Th1-type reaction and the coordinates of TMA as standard substance for induction of Th2-type reaction as an apex angle. In the created rectangle, the diagonal line connecting the diagonal angles other than the coordinates of DNCB and the coordinates of TMA as the boundary line, the coordinate side of DNCB as Th1 type region, the coordinate side of TMA as Th2 type region, and the coordinates plotted for each test compound Was determined in which area (FIG. 1). As a result, DNFB and OXA were Th1 type, TD
I and PA were determined to be Th2 type. In addition, regarding these compounds, DNFB (Garcia-Perez A et al; Contact Dermati
tis 4, 125-127 (1978)) and OXA (De Sausa, M et al; J
Exp Med 130, 671-690 (1969)) showed a Th1-type response, TDI (Bu
tcher BT et al; J Allergy Clin Immunol 28, 89-100
(1976)) and PA (Dearman et al; Clin Exp Allergy 22
(2) 241-50 (1992)) have been reported to cause Th2-type reactions, respectively.

[0011]

According to the present invention, it is possible to provide a method for predicting the type of immune response induced by a chemical substance.

[Sequence List Free Text] SEQ ID NO: 1 oligonucleotide primer designed for PCR SEQ ID NO: 2 oligonucleotide primer designed for PCR SEQ ID NO: 3 oligonucleotide primer designed for PCR SEQ ID NO: 2 4 oligonucleotide primer designed for PCR SEQ ID NO: 5 oligonucleotide primer designed for PCR SEQ ID NO: 6 oligonucleotide primer designed for PCR

[0013]

[Sequence list]

 <110> Sumitomo Chemical Company Limited <120> Method for prediction of immune responses <130> P152463 <160> 6 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 1 gaatgtacca gcagccatat c 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 2 ctcagtacta cgagtaatcc a 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 3 cgtgctcatg gctggtgcaa ag 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence < 220> <223> Designed oligonucleotide primer for PCR <400> 4 cttcatctgc aagttcttgg gc 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 5 gatgacgata tcgctgcgct g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 6 gtacgaccag aggcatacag g 21

[Brief description of the drawings]

FIG. 1 shows a graph for comparing the ratio of the expression level of Th1 cytokine to the expression level of Th2 cytokine in the tissue of a Th1 dominant mouse to which a chemical substance has been administered. The vertical axis is IL
-4 shows the expression level, and the horizontal axis shows the expression level of IL-12p40. Black circle; OXA-administered mouse, black triangle; DNFB-administered mouse, black square; TDI-administered mouse, open circle; PA-administered mouse, open triangle; DNC
B-administered mouse, open square; TMA-administered mouse.

Continued on the front page F term (reference) 2G045 AA40 CB01 DA12 DA13 DA14 FB05 FB12 GC15 JA01 4B024 AA11 CA04 CA12 HA11 4B063 QA01 QA05 QQ02 QQ53 QQ61 QR32 QR62 QR72 QS16 QS25 QX02

Claims (7)

[Claims] 1. Predicting the type of immune response induced by a test substance based on the ratio between the expression level of Th1 cytokine and the expression level of Th2 cytokine in the tissue of a Th1 dominant mouse to which the test substance has been administered. A method for predicting an immune type induced by a test substance, the method comprising: 2. (1) measuring the expression levels of Th1 cytokine and Th2 cytokine in the tissue of a Th1 dominant mouse to which a test substance has been administered, and determining the ratio of the expression levels of the two cytokines; And (2) comparing the ratio between the expression level of Th1 cytokine and the expression level of Th2 cytokine in the tissue of the Th1 dominant mouse to which the standard substance has been administered, with the ratio determined in step (1). A method for predicting an immune type induced by a test substance, comprising a step of predicting a type of an induced immune response. 3. The prediction method according to claim 1, wherein the expression level is an mRNA level. 4. The method according to claim 1, wherein the tissue is a local lymph node. 5. The Th1 dominant mouse to which a test substance has been administered,
The prediction method according to any one of claims 1 to 4, wherein the local lymph node weight is twice or more increased as compared with a Th1-dominant mouse in a negative control group. 6. The method according to claim 1, wherein the Th1 cytokine is IL-12p40 and the Th2 cytokine is IL-4. 7. Predicting the type of immune response induced by a test substance based on the ratio between the expression level of Th1 cytokine and the expression level of Th2 cytokine in the tissue of a Th1 dominant mouse to which the test substance has been administered. A system for predicting an immune type induced by a test substance, the system comprising: