WO2012144676A1 - Chemokine expression regulator - Google Patents

Abstract

Provided is a method for regulating the expression of chemokine materials using Fas ligands. The chemokine materials may be one or more kinds selected from the group consisting of MIP-1, RANTES, and IP-10. Since the chemokine materials participate in an inflammatory response, a technique for regulating the inflammatory response using Fas ligands is also provided.

Description

 【Specification】

 [Name of invention]

 Chemokine Expression Modulator [Technical Field]

 A method of controlling the expression of chemokine-like substances using Fas ligand is provided. The chemokine-like material may be at least one selected from the group consisting of MIP-l, RANTES and IP-10. Since the chemokine-like substance is involved in the inflammatory reaction, a technique for controlling the inflammatory response using the Fas ligand is also provided.

[Background technology]

 Fas Ligand (FasL, CD95L, CD 178, Apo-1) is one of Type II membrane proteins, along with TNF, CD40L, 4-1BBL, and Tumor necrosis factor (TNF). It belongs to the lineage and is mainly expressed in immune privileged sites such as activated T cells, NK cells, tumor cells and eyes. Fas ligand has a homotrimer structure and is known to perform apoptosis of target cells through trimerization with its receptor Fas.

 Fas ligand can be divided into membrane Fas ligand and soluble Fas (sFas) ligand. Because acetosis is induced by cell-cell contact, the membrane fas ligand is responsible for atoptosizing the cells by forming a death inducing signaling complex (DISC) along with Fas. Soluble Fas ligand is a truncated form of the membrane Fas ligand cut by serine matrix metalloproteinase -3 or -7 (serine matrix metalloproteinase, MMP-3 or MMP-7). Contrary to the membrane Fas ligand function, it is known to inhibit the aptosis of target cells or to act as a chemoattractant depending on the cell microenvironment.

Few reports of the role of Fas ligand in inflammatory diseases, particularly Rheumatoid arthritis (RA), have been shown to increase the amount of soluble Fas ligand in RA patients compared to patients with osteoarthritis. this is The only report is to reduce VEGF secreted from synovial fibroblasts and to inhibit angiogenesis. In addition, in relation to membrane Fas ligand, apoptosis through Fas-Fas ligand in CIA (collagen induced arthritis) model suppresses the production of autoreactive cells in the early stages of RA, thereby relieving RA. It is reported that the key plays a role.

 Considering that chemokines play an important role in inflammatory reactions, the importance of Fas ligand's role in chemokine expression is growing, but the results have been insignificant.

【Detailed Description of Invention】

 [Technical problem]

 Accordingly, the present inventors have completed the present invention by finding that Fas ligand is an important regulator in the expression of chemokine-like substances.

 Thus, one example of the present invention provides a method of controlling the expression of chemokine-like substances using Fas ligand.

 Another example provides a method for inhibiting an inflammatory reaction by inhibiting Fas ligand and an anti-inflammatory reaction composition comprising a Fas ligand inhibitor as an active ingredient.

 Another example provides methods for screening chemokine expression modulators by targeting Fas ligands.

Technical Solution

 A method of controlling the expression of chemokine-like substances using Fas ligand is provided. The chemokine-like material may be at least one selected from the group consisting of MIP-1, RANTES and IP-10. Since the chemokine-like substance is involved in inflammatory reactions, a technique for controlling inflammatory reactions using Fas ligand is also provided.

The present inventors have completed the present invention by confirming that expression of chemokine-like substances, in particular Mip-la, RANTES, and / or IP-10 is increased when Fas ligand is cultured with Fas ligand. We also tested the role of soluble Fas (sFas) ligands in rheumatoid arthritis using the K / BxN serum transfer model, so that lpr mice deficient in sFas induce rheumatoid arthritis as in B6 mice, but lack sFas ligand. gld mice were found to inhibit the development of rheumatoid arthritis. This finding is meaningful because it means that the Fas ligand is involved in the development of rheumatoid arthritis, apart from the apoptosis effect by the Fas ligand interaction with Fas.

 In addition, the present inventors confirmed that there is no difference in the incidence of rheumatoid arthritis even when the Fas ligand, as previously known, is treated with a caspase inhibitor capable of inhibiting aptosis caused by the interaction with Fas. When soluble Fas ligand was injected into gld mice deficient in sFas ligand, it was observed that rheumatoid arthritis was induced again. In addition, when the developmental stage of rheumatoid arthritis was divided to block the activation of Fas ligand at each of the early, middle, and late stages, it was observed that rheumatoid arthritis was suppressed and alleviated by blocking activation of the Fas ligand regardless of the onset stage. Could. This is contrary to the previous report that apatosis by the interaction of Fas and Fas ligand plays a role in alleviating rheumatoid arthritis by inhibiting the production of autoreactive cells in the early stages of rheumatoid arthritis. The present invention reveals for the first time that the Fas ligand (sFas ligand) independently acts as a trigger for arthritis, particularly rheumatoid arthritis, irrespective of the aptosis caused by the Fas ligand interacting with Fas.

 Taken together, the sFas ligand increases the expression of one or more chemokine-like substances selected from the group consisting of Mip-la, RANTES, and IP-10, thereby acting as an important factor in inflammatory reactions. In addition, it has been confirmed that it is one of the important causes of rheumatoid arthritis, it is useful as an effective target molecule not only for the prevention of inflammation, especially arthritis, but also for the therapeutic level.

Based on these findings, one example of the present invention is a chemokine-like substance using sFas ligand, such as Mip-la (macrophage inflammatory protein-1 α, accession number: NP_035467, AAI_11444, CAI25136, ABY66390, EDL15715, etc.), Mip- lp (accession number: NP_038680, AAI 19258, AAI 19260, CAI 25137, EDL 15716), RANTES (Chemokine (CC motif) ligand 5, CCL5, accession number: CAJ18523, AAH33508, AAU43779, EDL 15710, etc.) and IP-10 (Chemokine (CXC motif) ligand 10, CXCLIO, accession number: AAH 30067, EDL 05256, NP_067249, etc.) provides a method for controlling the expression of one or more chemokine-like substances selected from the group consisting of. Specifically, one or more chemokines selected from the group consisting of Mip-la, RANTES, and IP-10 by administering Fas ligand to the subject or enhancing Fas ligand activity (e.g., treating Fas ligand activity enhancer on the subject). Methods of increasing the expression of a substance are provided. Also provided is a method of inhibiting Fas ligand (eg, subjecting Fas ligand activity inhibitor to subject), thereby reducing the expression of one or more chemokine-like substances selected from the group consisting of Mip-la, RANTES, and IP-10. .

 In another aspect, there is provided a chemokine-like substance regulator comprising an sFas ligand as an active ingredient. More specifically, there is provided an expression enhancer of at least one chemokine-like substance selected from the group consisting of Mip-la, RANTES, and IP-10 comprising an sFas ligand or an sFas ligand activity enhancer as an active ingredient. In addition, there is provided an expression inhibitor of at least one chemokine-like substance selected from the group consisting of Mip-la, RANTES, and IP-10 comprising an sFas ligand inhibitor as an active ingredient.

 In another aspect, there is provided a use for the regulation of chemokine-like substances of sFas ligand. More specifically, the use of the sFas ligand or the sFas ligand activator for Mip-la, RANTES, and IP-10 or at least one chemokine-like substance selected from the group is provided for use in the production of the expression promoting agent. In addition, there is provided a use of the sFas ligand inhibitor for use in the suppression of the expression or preparation of expression inhibitors of at least one chemokine-like substance selected from the group consisting of Mip-la, RANTES, and IP-10.

 In an embodiment according to the invention, the sFas ligand or its activity enhancer may be administered to the subject, or the sFas ligand may be incubated together to increase chemokine expression, or the sFas ligand inhibitor may be administered to the subject to reduce chemokine expression. have.

 In addition, the sFas ligand may itself act as a chemokine to directly act as an inflammatory cell influx and / or to induce inflammation (see Example 8).

Such chemokine-like substances are known to be involved in inflammatory reactions Therefore, in another aspect of the present invention there is provided a technique for inhibiting the sFas ligand to suppress inflammatory reaction. In particular, techniques for preventing, treating, alleviating and / or alleviating inflammation associated with one or more chemokine-like substances selected from the group consisting of Mip-ΐα, RANTES, and IP-10, such as arthritis, in particular rheumatoid arthritis Can be provided.

 Thus, one embodiment of the present invention provides a method for preventing and / or treating arthritis, comprising administering an sFas ligand inhibitor to a patient with arthritis, preferably rheumatoid arthritis. The method of preventing and / or treating arthritis may further comprise identifying a patient with arthritis, preferably rheumatoid arthritis, prior to administering the sFas ligand inhibitor.

 In another example, a composition for the prophylaxis and / or treatment of arthritis, preferably rheumatoid arthritis, comprising an sFas ligand inhibitor is provided. In another example, there is provided a use of the sFas ligand inhibitor for use in the prevention and / or treatment of arthritis, preferably rheumatoid arthritis, or for the preparation of prophylactic and / or therapeutic agents.

As described above, since the _sFas ligand itself serves as a chemokine, another example of the present invention provides an inflammation-inducing composition comprising a soluble Fas (sFas) ligand as an active ingredient. Another example provides the use of soluble Fas (sFas) ligands as chemokines. Yet another example provides a method for inducing innovation comprising administering or contacting the soluble Fas ligand to a mammal or tissue or cell isolated from the mammal. When the mammal is used as a living body, the mammal may be interpreted as excluding humans.

 In particular, the sFas ligand according to the present invention is characterized in that the chemokine expression control and inflammatory reaction regulatory activity such as arthritis occurs regardless of binding to Fas and apoptosis.

 In addition, we also found that sFas ligands are related to NF-k such as pAKT (P31750, NP_033782, AAH 66018, AAI 15584), pSyk (AAA87462, NP_035648, AAH 65121), pERK (NP_033257, NP_001033752, NP_036079, AAH_58258, AAH_06708B) and the like. Increasing the expression of the protein was also confirmed.

As used herein, 'Fas ligand, unless otherwise indicated, means a soluble Fas (sFas) ligand. In addition, the sFas ligand in the present invention may be of mammalian origin, preferably of rodent or human origin, For example, it may be an accession number AAA 19778 (SEQ ID NO: 1), or AAC50071 (SEQ ID NO: 2).

 As used herein, 'enhancing the activity of the sFas ligand' refers to all the actions of increasing the expression of the sFas ligand as well as enhancing the activity of the sFas ligand. sFas ligand activity enhancers are all substances that enhance the activity of sFas ligand by inducing the activation of cells expressing or secreting the sFas ligand. For example, GPI (Glucose-6) contained in K / BxN serum used to induce arthritis -Phosphate Isomerase) protein, anti-GPI antibody, or GPI-anti GPI antibody complex, and the like, but are not limited thereto.

 In addition, as used herein, the inhibition of sFas ligand, as well as reducing or eliminating the activity of the Fas ligand, as well as reducing, eliminating, and reducing the action of the sFas ligand, including expression inhibition, elimination and inactivation of the sFas ligand, and And / or any action that blocks. The Fas ligand inhibitor refers to any substance that reduces the expression of Fas ligand, decreases, eliminates, and / or blocks the action, such as Fas ligand blocking antibody, Fas ligand solubilization inhibitor, for example,

Nl-hydroxyl-3-isobutyl-2-methyl-N4- (2- (methylamino) -2-oxo-l-phenylethyl) succinam ide), MMP (matrix metalloprotease) -3 inhibitor (Stromelysin-1 inhibitor; e.g.,

C ₂₇ H ₄₆ N ₁₀ O ₉ S, etc.) may be one or more selected from the group consisting of. The subject may be a mammal, preferably a human or rodent, or a cell or tissue isolated therefrom. Preferably, the subject has a pathological condition caused by one or more expressions selected from the group consisting of chemokines such as Mip-ΐα, RANTES, and IP-10, such as patients suffering from inflammation, or such pathological symptoms such as inflammation It may be isolated from a living body, such as induced cells, tissues, or body fluids. In one embodiment of the present invention the inflammation may be arthritis, the inflammation-induced cells are arthritis-inducing cells, more specifically immune cells and / or synovial cells infiltrated in the arthritis-induced joints, peripheral blood of a patient with arthritis Cells and the like.

 The dose of the Fas ligand inhibitor may be appropriately adjusted according to the condition of the patient, the degree of inflammation, and the like, for example, 0.1 to 100 ug / kg (body weight), preferably 1 to 1, based on the active ingredient content. It may be 50 ug / kg (weight), but is not limited thereto.

As shown in the examples below, inflammation according to the invention, in particular The prevention and / or treatment method of arthritis has the advantage that the effect of alleviating and treating the disease is excellent not only in the prevention of the onset and in the early stage of the onset, but also in the middle and late stages of the progression.

 In another example, a method for screening a chemokine expression inhibitor targeting a Fas ligand is provided. The screening method

 Reacting the candidate with the sample; And

 Measuring Fas ligand activity of the sample

 Including,

 If the activity of the Fas ligand in the sample treated with the candidate material is removed or decreased than the Fas ligand activity in the sample not treated with the candidate material, the candidate material may be characterized as being determined as a chemokine expression inhibitor. .

 The chemokine may be one or more selected from the group consisting of Mip-ΐα, RANTES, and IP-10. Since the chemokine-like substance is known to be involved in inflammatory reaction, the chemokine expression inhibitor selected by the screening method can be used as a prophylactic and / or therapeutic agent for inflammation, especially arthritis such as rheumatoid arthritis. Thus, the screening method may be a method of screening for inflammation inhibitory and / or therapeutic agents for arthritis (eg, rheumatoid arthritis).

 The sample may be a cell, tissue or organ obtained from an animal, preferably a mammal, and may preferably comprise an immune privileged site, more preferably, a T cell, an NK cell, a tumor. Cell or eye.

The Fas ligand activity can be measured by conventional methods, which can be easily understood by those of ordinary skill in the art. For example, when a soluble FasL is injected into an animal to induce arthritis, a change in the thickness of the joint is measured by about 1 to 20 days, preferably about 8 to 12 days, for example, 10 days, compared to an individual not injected. The degree of activation can be assessed. Or, for example, chemokines expressed or secreted in the group incubated with soluble sFasL after inducing arthritis-induced immune cells and incubated with soluble sFasL, were subjected to realtime PCR, Cytoflowmetry, fluorescence activated cell sorter (FACS), or ELISA. By measuring by, the degree of activity can be evaluated. For example, if you do not process sFasL By comparing the expression of chemokines in groups, if the treated group had increased expression of chemokines than the untreated groups, it can be determined that the expression of chemokines increased according to the activity of the candidate compounds upon contact of the candidate compounds.

Realtime PCR compares the relative amounts between groups, ELISA confirms the absolute amount, and FACS (fluorescence activated cell sorter) is advantageous for measuring the degree of activation per cell.

 The candidate includes all synthetic or natural compounds, polypeptides, polynucleotides, and the like.

In this manner the screening chemokine expression control ^Ze has the advantage that inflammation, in particular, as well as prevention of the onset and early onset of arthritis treatment effects, illness that has progressed to some extent the middle and superior mitigation illness and treatment in the end. The chemokine expression control technology using the Fas ligand, the prevention and / or treatment of arthritis such as inflammation, in particular rheumatoid arthritis using the Fas ligand presented in the present invention, and the search technology of novel chemokine expression inhibitors using the same, can effectively prevent the occurrence of arthritis In addition to being able to achieve excellent therapeutic effects even after some progress, it is a very useful technique for treating many patients suffering from arthritis.

[Brief Description of Drawings]

 La to lc shows the degree of expression of chemokine according to the Fas ligand concentration, la shows the expression level of ΜΙΡ-1α, lb shows the expression level of RANTES, lc shows the expression level of IP-10.

 Figure 2a shows the thickness and the clinical index of the joints in three kinds of mouse models induced arthritis by K / BxN serum transfer according to Example 2,

 2b is a picture of the joints of the three kinds of mice,

 2c shows the expression level of IFNY, IL-4 and TGFp in the three kinds of mice.

 3 shows joint thickness and clinical index in z-VAD treated mice according to Example 3. FIG.

 Figure 4a shows the joint thickness and clinical index measurement results of the mouse of Example 4,

4b shows the result of measuring the joint thickness and clinical index of the mouse of Example 5. To show,

 4c shows the expression level of IFN ^ IL-4 and TGF | in the mouse joint of Example 5.

 Figure 5a shows the results of measurement of the joint thickness and clinical index of the early arthritis mice of Example 6,

 5b shows the results of measurement of the joint thickness and clinical index of the arthritic middle mouse of Example 6,

 5c shows the results of measuring joint thickness and clinical index of the terminal arthritis terminal mice of Example 6,

 5d to 5f represent the early, middle and end mouse joints of Example 6, respectively.

It shows the expression level of IFNy, IL-4 and TGFp.

 Figure 6 shows the joint thickness and clinical index when the production of soluble Fas ligand in vivo is suppressed.

 Figure 7a shows the number of inflammatory cells moved to the bottom chamber after 24 hours with inflammatory cells in the upper chamber and soluble Fas ligand in the bottom chamber, 7b shows the number of cells in the upper chamber and bottom chamber after 24 hours, respectively will be.

[Mode for Invention] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples. Example l: Effect test for increasing chemokine expression of Fas ligand

Serum from K / BxN mice (KRN TCR transgenic mouse wave NOD mice obtained from Drs.D.Mathis and C.Benoist of Harvard medical school, Boston, MA, obtained from the normal mice). Arthritis was induced by injection. After 10 days of observation, infiltrated and synovial cells in the joint were isolated and in vitro with soluble Fas ligand (accession number AAA 19778 (SEQ ID NO: 1), lg, 5 μξ, 10 μg (R & D system), respectively). Incubated. Culture medium was 10% Fetal bovine serum (Gibco), 1% penicillin streptomycin (Gibco), DMEM (Dulbeccos' s Modified Eagle's Medium) (WelGene) Was used, the 12 well culture plate well / ml in the culture medium of 1 for producing from SPL science, was dispensed a well / 10 ⁶ cells.

 After culturing in a 37 ° C., 5% C02 incubator for 24 hours, the cells and the culture medium were separated by centrifugation, and RNA was extracted from the cells. RNA extraction was carried out in the order provided using the RNasy mini prep kit produced by Qiagen. Of these, 3 g of RNA was again produced in promega.

The MMLV reverse transcription kit was used to synthesize cDNA. The expression of chemokines Mip-ΐα, RANTES, and IP-10 was measured using Realtime PCR ABI 7500 manufactured by Applied Biosystem on the obtained cDNA. Chemokine primers were also prepared by Applied Biosystem, and the rate of chemokine expression was normalized to GAPDH, a type of house keeping gene that shows a high level of expression without any change in external conditions.

 The results thus obtained are shown in FIGS. La to lc. As can be seen from FIGS. La and lc, the expression of all three chemokines increased when treated with soluble Fas ligand, and generally increased in proportion to the treatment concentration. Can be. Example 2: Rheumatoid Arthritis Induction Test in Animal Model 1

 To determine the role of Fas ligand in the antibody-induced arthritis model, gld mice with overlapping Fas ligand and lpr mice lacking Fas, and control (normal) B6 with both Fas and Fas ligands RA was induced in mice. First, blood in naturally induced arthritis-induced Κ / ΒχΝ mice (crossed by KRN TCR transgenic and NOD mice from Drs.D.Mathis and C.Benoist from Harvard medical school, Boston, MA) Serum was collected. B6 (orient bio), lpr (central laboratory animals) and gld mice (central laboratory animals) as described above were injected with 150 μ1 of K / BxN serum obtained above intraperitoneally (ip) on days 0 and 2. It was. Joint thickness was measured with a caliper (Manostat, Switzerland).

 The Clinical index is referenced as follows:

 0: no joint swelling,

 1 ： swelling of one finger joint,

 2: mild swelling of wrist or ankle,

3: severe swelling of wrist or ankle. The thickness and clinical index of the joint measured as described above are shown in FIG. 2A. As can be seen in Figure 2a, B6 mice and lpr mice were injected with serum and the joints began to swell from day 4, the highest index was observed at 8-9 days. However, in the gld mice deficient in Fas ligand, the joints did not swell almost 10 days after the serum injection, and the clinical index remained at 3-4 points. In addition, the joint photograph of the test mouse is shown in Figure 2b. As can be seen in Figure 2b, in the gld mice it was observed that almost no swelling of the joints.

 In the mRNA of the K / BxN serum transfer model, the expression of cytokines, which are important in controlling arthritis, was identified. Serum transfer was performed and after 10 days of observation, the mice were euthanized and the joints were cut out. RNA was isolated from the joint tissues using the RNeasy kit (Qiagen), and all procedures were performed according to the manual provided by the manufacturer of the RNeasy kit (Qiagen). CDNA was synthesized from the isolated RNA 3 using Reverse Transcription System (Promega Corporation, Madison, Wisconsin). Expression of each cytokine (IL-4, IFNy, and TGF | 3) was measured using the Applied Biosystems 7500 sequence detection system (Perkin-Elmer Biosystems) and all results were compared with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) expression. Averaged. Each of the primers used was as follows, and synthesized in Applied Biosystem (Foster City, CA).

 TGF-β 1 (forward): 5'-GC AAC ATGTGGAACTCTACC AGAA-3 ',

 TGF-βΙ (reverse): 5'-GACGTCAAAAGACAGCC ACTCA-3 ',

 TGF-βΙ specific probe:

FAM-ACCTTGGTAACCGGCTGCTGACCC-TAMRA

 The results obtained above are shown in FIG. 2C. As shown in Figure 2c, the expression of IL-4 and IFNY was increased in g6 mice in B6, and lpr mice, whereas the expression of TGF (3 was increased in gld mice. Gand plays an important role in inducing joint inflammation in the K / BxN serum transfer model, and this role is independent of Fas.

The representative function of the Fas ligand, which has been known so far, is to bind to its receptor, Fas, to induce apoptosis of the target cell. If a Fas ligand acts on joint inflammation through apoptosis by interaction with Fas, Fas Lpr mice deficient and gld mice deficient in Fas ligand should show the same arthritis-induced behavior. However, as shown in Fig la-lc, Fas-deficient lpr mice and Fas ligand-deficient gld mice showed different patterns in the induction of arthritis. This suggests that the Fas ligand does not contribute to the pathogenesis of arthritis by induction of apoptosis through binding with Fas, but may be related to another mechanism. Example 3: Rheumatoid Arthritis Induction Test 2 in Animal Models

 In order to show that the Fas ligand is involved in arthritis induction as a separate mechanism, rather than through apoptosis by binding to Fas, the following test was further performed.

 B6 mice, such as used in Example 2, were treated with a caspase inhibitor (caspase inhibitor, z-VAD, Calbiochem) that was able to inhibit apoptosis intraperitoneally with an amount of 50ug or lOOug the day before, followed by K / BxN serum was injected to induce arthritis. For the arthritis-induced mice, the thickness and clinical index of the joint were measured in the same manner as in Example 2, and the results are shown in FIG. 3.

 As confirmed in FIG. 3, it was observed that arthritis scores (joint thickness and clinical index) were increased in the same manner in both the z-VAD and the non-treatment cases. From the above data, it was confirmed that apatosis was not involved in arthritis induction through K / BxN serum transfer. Example 4: Rheumatoid Arthritis Induction Test 3 in Animal Models

 In order to further confirm the role of the Fas ligand, the test was performed by inducing arthritis by immunotransmitting splenocytes of B6, lpr, and gW mice to gld mice, respectively.

First, B6, lpr, and gld mice were euthanized and spleens were collected, homogenized with 1 ml of red blood cell (RBC) lysis buffer (Qiagen), and then RBC was removed _: lymphocytes were obtained. After filtration with a sterile 40μπι cell strainer (BD bioscience), 1 x 107 splenocytes per gld mouse the day before serum transfer were injected intravenously (iv) with sterile PBS at a total volume of 300 μΐ. As described above, the joint thickness and the clinical index of the mice to which the splenocytes were transferred and the mice to which the splenocytes were not transferred as the control were measured in the same manner as in Example 2, and the results are shown in FIG. 4A. As can be seen in Figure 4a, it was observed that the arthritis score, which is expressed by joint thickness and clinical index, was increased in gld mice that transferred splenocytes of B6 and lpr mice compared to non-transferred gld mice. On the other hand, gld mice, which transfer splenocytes of gld mice, showed little induction of arthritis, similar to non-transferred gld mice. The data confirmed that even if Fas is deficient, the presence of a Fas ligand may cause arthritis. Example 5: Rheumatoid Arthritis Induction Test 4 in Animal Models

 Next, gs mice were injected with sFas ligand (soluble Fas ligand) and then transferred to K / BxN serum to test whether arthritis was induced.

2 μg or 10 of the mouse sFas ligand (R & D) were injected into the gld mouse intraperitoneally on the day before the serum transfer, and then the joint thickness and clinical index were measured by the same method as in Example 2, and the results are shown in FIG. 4B. As shown in FIG. 4B, the injection of 2 μ _{β in} sFasL showed no significant change in the joint thickness and clinical indices. However, the injection of sFasL 10 increased the joint thickness and clinical indices to the same extent as wild-type B6 mice. . Also it is shown in Figure 4c to measure IFNy, expression of IL-4 on the joint and TGFJ mR A in sFasL 10 μ _β a gld mouse injection. As will be confirmed in Fig. 4c, sFasL 10 μ _β of the joint mRNA on the expression of IFNy and IL-4 in the injection gld mice increased while, TGF (expression was reduced. Example 6: through the Fas ligand inhibition Arthritis treatment test

These results confirm that Fas ligand is one of the important etiologies for inducing arthritis in the K / BxN serum transfer model. The results indicate that the blocking of Fas ligand activation plays an effective role in the prevention and treatment of arthritis. To present. To confirm this hypothesis, the onset of arthritis was divided into early, mediated, and late phases to block the activation of Fas ligand and to observe the arthritis pathology. Fas ligand blocking antibody (BD bioscience) was mixed with PBS in an amount of Ι μ ^ and injected into the blood vessel (iv) of Β6 mice at a total volume of 300 μΐ. Fas ligand blocking antibodies were treated from one day prior to serum transfer to 9 days after serum transfer to block the role of Fas ligand in the early stages of arthritis development. Joint thickness and clinical index of the animal model were measured in the same manner as in Example 2, and are shown in FIG. 5A. As can be seen in Figure 5a, it was confirmed that the onset of arthritis is significantly suppressed in mice treated with Fas ligand blocking antibody.

 In addition, the Fas ligand blocking antibody was mixed with PBS in an amount of H ^ g or 20 from 5 days to 9 days after the serum transfer, which is considered to be an intermediate stage of joint inflammation, and injected intravascularly into Β6 mice at a total volume of 300 μΐ, and the joint thickness. And clinical indices were measured and shown in FIG. 5B. As shown in FIG. 5B, arthritis was suppressed even when the Fas ligand blocking antibody was treated during the onset of arthritis, and this inhibitory effect was more significant as the concentration of the Fas ligand blocking antibody was increased. It was shown.

 Finally, from 8 days after serum transfer, when the arthritis score (joint thickness and clinical index) is highest, to 11 days of observation, Fas ligand blocking antibody was mixed with PBS in an amount of 20 μg to adjust the total volume to 300 μΐ, and the blood vessels of Β6 mice Internal (Lv) was injected. For the mouse, the joint thickness and clinical index were measured in the same manner as in Example 2, and the results are shown in FIG. 5C. As shown in FIG. 5C, it was confirmed that arthritis was rapidly suppressed when Fas ligand blocking antibody was treated in the early and middle stages of arthritis.

In addition, the expression levels of cytokines (IL-4, IFNy and TGFP) on the joint mRNA of the serum-transferred B6 mouse model were measured at the early, middle and late stages of arthritis, respectively, and the results are shown in FIGS. 5D to 5F, respectively. . As shown in FIGS. 5D to 5F, expression of IL-4 and IFNY was decreased and expression of TGF (3) was increased in mice injected with Fas ligand blocking antibody in the early, middle and late stages of arthritis. Example 7: Arthritis-induced Experiment When Inhibiting the Production of Soluble Fas Ligand In fact, it was tested whether the induction of arthritis is inhibited when the production of soluble Fas ligand which promotes the induction of arthritis is inhibited in vivo. In wild-type B6 mice, Fas deficient lpr mice, and Fas ligand deficient gld mice, the matrix metalloproteinase-3 inhibitors (MMP-3 inhibitor, Calbiochem, Ac-Arg-Cys-Gly-Val-) the day before and after the first serum transfer. After injecting Pro-Asp-NH ₂ ) \ 00μg into the abdominal cavity, the joint thickness and clinical index were measured in the same manner as in Example 2, and the results are shown in FIG. 6. As shown in FIG. 6, when the production of sFas ligand was inhibited in wild-type B6 mice, joint thickness and clinical index were decreased, and such effects could not be observed in lpr and gld mice. Through the data, it was confirmed that the arthritis treatment effect of the MMP inhibitor is obtained through the inhibition of sFas ligand production, the production of sFas ligand is an important factor promoting the induction of arthritis. Example 8: Identification of direct chemokine role of solubleFas ligand Based on the above data, the generation of sFas ligand promotes chemokine expression, which induces arthritis. Next, we tested whether the sFas ligand could act as a chemokine that promotes the influx of inflammatory cells, rather than simply as a chemokine expression regulator. After inducing arthritis in wild-type B6 mice in the same manner as in Example 2, and obtaining the inflammatory cells introduced into the joint on the 8th day having the highest joint thickness and the highest clinical index, the upper chamber of the 80μηι transwell plate (BD Falcon) was 300μ1 culture solution. Inflammatory cells were added to (RPMI 1640, 10% Fetal bovine serum, 1% antibiotics) and Ong, 50ng and 100ng of sFas ligand were added to 700μ1 culture medium. After 24 hours, the ratio of the number of cells moved from the upper chamber to the bottom chamber according to the sFas ligand in the total upper chamber is shown in FIG. 7A. After 24 hours, the number of cells in the upper chamber and the bottom chamber, respectively, is shown in FIG. 7B.

As can be seen in Figures 7a and 7b, the number of cells moved to the bottom chamber of the inflammatory cells in the upper chamber increased in proportion to the sFas ligand concentration. Based on the above results, the sFas ligand was able to confirm that sFasL itself acts as a chemokine as well as regulates chemokine expression. Taken together, the Fas ligand activity blockade suppresses chemokine expression and suppresses inflammatory reactions, and it is confirmed that the Fas ligand activity blockade is effective in suppressing arthritis symptoms as well as in the early stages of inflammation as well as after the onset of arthritis. These results could also provide breakthrough information in the development of prevention and treatment of inflammation, including arthritis. Furthermore, it provides a new use of sFasL itself as a chemokine.

Claims

[Range of request] 1. Composition for inhibiting the expression of at least one chemokine selected from the group consisting of Mip-ΐα, RANTES, and IP-10, containing a soluble Fas (sFas) ligand inhibitor as an active ingredient. 2. The composition of claim 1, wherein the soluble Fas ligand inhibitor is at least one selected from the group consisting of Fas ligand blocking antibody, Fas ligand solubilization inhibitor, and MMP-3 inhibitor. 3. Inflammatory composition comprising a soluble Fas (sFas) ligand as an active ingredient. 4. A composition for preventing or treating inflammation containing a soluble Fas (sFas) ligand inhibitor as an active ingredient. 5. The composition of claim 4, wherein the soluble Fas ligand inhibitor is at least one selected from the group consisting of Fas ligand blocking antibody, Fas ligand solubilization inhibitor, and MMP-3 inhibitor. 6. The composition of claim 4 or 5. wherein the inflammation is arthritis. 7. The composition of claim 6 wherein the arthritis is rheumatoid arthritis. 8. Mip-ΐα, RANTES, and of Soluble Fas (sFas) Ligand Inhibitors Use for use for the inhibition of expression of one or more chemokines selected from the group consisting of IP-10. 9. Use according to item 8, wherein the soluble Fas ligand inhibitor is at least one selected from the group consisting of Fas ligand blocking antibody, Fas ligand solubilization inhibitor, and MMP-3 inhibitor. 10. Use of soluble Fas (sFas) ligand as chemokine. 11. Use for the prevention or treatment of inflammation of soluble Fas (sFas) ligand inhibitors. 12. Use according to item 11, wherein the soluble Fas ligand inhibitor is at least one selected from the group consisting of Fas ligand blocking antibodies, Fas ligand solubilization inhibitors, and MMP-3 inhibitors. 13. Use according to 11 or 12, wherein the inflammation is arthritis. 14. Use according to point 13, wherein the arthritis is rheumatoid arthritis. 15. A method comprising administering a soluble Fas ligand inhibitor to a patient in need of reduced chemokine expression or to a cell that is overexpressed with chemokine, A method of reducing the expression of one or more chemokines selected from the group consisting of Mip-ΐα, RANTES, and IP-10. 16. The method of claim 15, wherein the soluble Fas ligand inhibitor is at least one selected from the group consisting of Fas ligand blocking antibody, Fas ligand solubilization inhibitor, and MMP-3 inhibitor. 17. A method of inducing inflammation comprising administering the soluble Fas ligand to a mammal or tissue or cell isolated from the mammal. 18. A method comprising administering a soluble Fas ligand inhibitor to an inflammatory patient or to a cell that has caused inflammation.  How to prevent or treat inflammation. 19. The method of preventing or treating inflammation according to item 18, wherein the soluble Fas ligand inhibitor Fas ligand blocking antibody, Fas ligand solubilization inhibitor, and MMP-3 inhibitor are at least one member selected from the group. 20. The method for preventing or treating inflammation according to item 18 or 19, wherein the inflammation is arthritis. 21. The method for preventing or treating inflammation of paragraph 20, wherein the arthritis is rheumatoid arthritis. 22. Refusing a candidate substance to the sample; And  Measuring Fas ligand activity of the sample  Including  The group consisting of Mip-la, RANTES, and IP-10 when the activity of the Fas ligand in the sample treated with the candidate substance was removed or decreased than the Fas ligand activity in the sample not treated with the candidate substance. A method for screening chemokine expression inhibitors, characterized in that determined by at least one chemokine expression inhibitor selected from. 23. Refusing the candidate substance to the sample; And  Measuring Fas ligand activity of the sample  Including,  When the activity of the Fas ligand in the sample treated with the candidate substance is removed or less than the Fas ligand activity in the sample not treated with the candidate substance, the candidate substance is determined as an inflammatory therapeutic agent, Screening method. 24. The method of screening 23, wherein the inflammation is arthritis.