KR101769122B1 - Chemokine expression regulator - Google Patents

Abstract

A method of modulating the expression of chemokine analogues using a Fas ligand is provided. The chemokine analogues may be at least one selected from the group consisting of MIP-1, RANTES and IP-10. Since the chemokine-like materials are involved in the inflammatory reaction, techniques for controlling the inflammatory reaction using the Fas ligand are also provided.

Description

Chemokine Expression Regulator {CHEMOKINE EXPRESSION REGULATOR}

A method of modulating the expression of chemokine analogues using a Fas ligand is provided. The chemokine analogues may be at least one selected from the group consisting of MIP-1, RANTES and IP-10. Since the chemokine analogues are involved in the inflammatory response, techniques for controlling the inflammatory response using Fas ligands are also provided.

Fas ligand (FasL, CD95L, CD178, Apo-1) is one type II membrane protein and is associated with TNF, CD40L, 4-1BBL, and Tumor necrosis factor And is expressed in immune privileged sites such as mainly activated T cells, NK cells, tumor cells and eyes. The Fas ligand has a homotrimer structure and is known to perform apoptosis of target cells through trimerization with its receptor Fas.

Fas ligands can be divided into membrane Fas ligands and soluble Fas (sFas) ligands. Since apoptosis is induced by cell-cell contact, the membrane Fas ligand plays a role in apoptosis of cells with Fas and a death inducing signaling complex (DISC). Soluble Fas ligand is a cleavage form of membrane Fas ligand truncated by Serine matrix metalloproteinase (MMP-3 or MMP-7). In contrast to membrane Fas ligand function, it is known to inhibit target cell apoptosis or to function as a chemoattractant depending on the cell microenvironment.

The role of Fas ligand in inflammatory diseases, particularly rheumatoid arthritis (RA), has not been extensively reported, and the amount of soluble Fas ligand in RA patients is increased compared to patients with Osteoarthritis, It is the only report that it plays a role in suppressing angiogenesis by decreasing secretion of VEGF from synovial fibroblast. In addition, in relation to membrane Fas ligand, it has been reported that Fas-Fas ligand-mediated apoptosis in the collagen induced arthritis (CIA) model plays a role in inhibiting RA by inhibiting the production of autoreactive cells at the early stage of RA have.

Considering that chemokines play an important role in the inflammatory response, research on the role of Fas ligand in chemokine expression is becoming increasingly important, but the results are limited.

Thus, the present inventors have found that Fas ligand is an important regulator of expression of chemokine-like substances, thus completing the present invention.

Thus, one example of the present invention provides a method of modulating expression of a chemokine analogue using a Fas ligand.

Another example is a method for suppressing inflammatory reaction by inhibiting Fas ligand and a composition for inhibiting inflammation comprising Fas ligand inhibitor as an active ingredient.

Another example provides a method for screening a modulator of chemokine expression targeting a Fas ligand.

A method of modulating the expression of chemokine analogues using a Fas ligand is provided. The chemokine analogues may be at least one selected from the group consisting of MIP-1, RANTES and IP-10. Since the chemokine-like materials are involved in the inflammatory reaction, techniques for controlling the inflammatory response using the Fas ligand are also provided.

The present inventors completed the present invention by confirming that the expression of chemokines, especially Mip-1α, RANTES, and / or IP-10, is increased by culturing inflammatory cells with Fas ligand in Fas ligand.

We also tested the role of soluble Fas (sFas) ligand in rheumatoid arthritis using the K / BxN serum transfer model to show that in spr-deficient lpr mice rheumatoid arthritis is induced as well as B6 mice, but deficient in sFas ligand gld mice were found to inhibit the development of rheumatoid arthritis. This finding is significant because it is known that the Fas ligand is involved in the development of rheumatoid arthritis independently of the apoptotic action by interaction with Fas.

In addition, the present inventors confirmed that there is no difference in the extent of rheumatoid arthritis even when a known Fas ligand is treated with a caspase inhibitor capable of inhibiting apoptosis caused by interaction with Fas, When soluble fas ligand was injected into gld mice deficient in sFas ligand, rheumatoid arthritis was re-induced. It was also observed that the activation of Fas ligand inhibited and alleviated rheumatoid arthritis regardless of the stage of onset, when the activation phase of Fas ligand was blocked at the early stage, middle stage and late stage of onset of rheumatoid arthritis I could. This is in contrast to the previous reports that the interaction of Fas and Fas ligand with apoptosis inhibits the production of autoreactive cells at the initial stage of the invention of rheumatoid arthritis and thereby alleviates rheumatoid arthritis. Regardless of the apoptosis caused by the interaction of Fas with the ligand, the Fas ligand (sFas ligand) has been shown to function independently as an inducer of arthritis, especially rheumatoid arthritis.

In summary, the sFas ligand serves to increase the expression of one or more chemokines selected from the group consisting of Mip-1α, RANTES, and IP-10, thereby acting as an important factor in the inflammatory response. It has also been identified as one of the major causes of rheumatoid arthritis and is useful as a target molecule not only for the prevention of inflammation, especially arthritis, but also for therapeutic purposes.

On the basis of this finding, an example of the present invention relates to a method for screening a chemokine-like substance such as Mip-1? (Macrophage inflammatory protein-1 ?, accession number: NP_035467, AAI_11444, CAI25136, ABY66390, EDL15715, (Accession number: NP_038680, AAI 19258, AAI 19260, CAI 25137, EDL 15716), RANTES (CXC motif) ligand 10, CXCL10, accession number: AAH 30067, EDL 05256, NP_067249, and the like). Specifically, one or more chemokines selected from the group consisting of Mip-1 alpha, RANTES, and IP-10 may be administered to the subject by administering a Fas ligand to the subject or by enhancing Fas ligand activity (e.g., treating the subject with a Fas ligand activity enhancer) A method of increasing the expression of a substance is provided. Also provided is a method of inhibiting Fas ligand (e.g., treating a subject with a Fas ligand activity inhibitor) to reduce the expression of one or more chemokines selected from the group consisting of Mip-1 alpha, RANTES, and IP-10 .

In another aspect, a chemokine modulator comprising an sFas ligand as an active ingredient is provided. More specifically, there is provided an agent for promoting expression of at least one chemokine substance selected from the group consisting of Mip-1 alpha, RANTES, and IP-10, which comprises an sFas ligand or an sFas ligand activity enhancer as an active ingredient. Also provided is an agent for inhibiting the expression of one or more chemokines selected from the group consisting of Mip-1 alpha, RANTES, and IP-10 comprising an sFas ligand inhibitor as an active ingredient.

In another aspect, there is provided an application for modulating chemokine materials of sFas ligands. More specifically, there is provided an application for use in promoting expression or promoting expression of one or more chemokines selected from the group consisting of sFas ligands or sFas ligand activators Mip-1 ?, RANTES, and IP-10. Also provided is an agent for use in inhibiting the expression of, or inhibiting the expression of, one or more chemokines selected from the group consisting of MIP-1 alpha, RANTES, and IP-10 of sFas ligand inhibitors.

In an embodiment according to the present invention, expression of chemokine may be increased by administering sFas ligand or its activity enhancer to the subject, or culturing sFas ligand together to increase the expression of chemokine, or administering sFas ligand inhibitor to the subject have.

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

Since the above-mentioned chemokine materials are known to be involved in the inflammatory reaction, another aspect of the present invention provides a technique for inhibiting the inflammatory reaction by inhibiting the sFas ligand. In particular, there is provided a technique for preventing, treating, alleviating, and / or alleviating inflammation associated with one or more chemokines selected from the group consisting of Mip-1 alpha, RANTES, and IP-10, such as arthritis, especially rheumatoid arthritis .

Accordingly, one example of the present invention provides a method for preventing and / or treating arthritis, which comprises administering an sFas ligand inhibitor to a patient suffering from arthritis, preferably rheumatoid arthritis. The method of preventing and / or treating arthritis may further comprise the step of identifying patients with arthritis, preferably rheumatoid arthritis, prior to administering the sFas ligand inhibitor.

In another example, there is provided a composition for the prevention and / or treatment of arthritis, preferably rheumatoid arthritis, comprising an sFas ligand inhibitor.

In another example, there is provided an application for use in the prevention and / or treatment, or prevention and / or treatment of osteoarthritis, preferably rheumatoid arthritis, of an sFas ligand inhibitor.

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

In particular, the sFas ligand according to the present invention is characterized in that the regulation of chemokine expression and the inflammatory reaction regulating activity such as arthritis are independent of binding with Fas and apoptosis.

In addition, the present inventors have also found that the sFas ligand binds to NF-kB related proteins 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_06708) It was further confirmed that the protein expression was increased.

As used herein, the term "Fas ligand" means a soluble Fas (sFas) ligand unless otherwise specified. In addition, the sFas ligand in the present invention may be of mammalian origin, preferably of rodent or human origin, and may be, for example, an accession number AAA19778 (SEQ ID NO: 1), or AAC50071 (SEQ ID NO: 2) .

As used herein, 'enhancing the activity of sFas ligand' means all actions that enhance the activity of sFas ligand, as well as increase the expression of sFas ligand. The sFas ligand activity enhancer is a substance that promotes the activity of sFas ligand by inducing activation of cells expressing or secreting sFas ligand. For example, GPI (Glucose-6 -Phosphate Isomerase) protein, an anti-GPI antibody, or a GPI-anti-GPI antibody complex, but the present invention is not limited thereto.

As used herein, the term " inhibition of sFas ligand " means reduction or elimination of the action of sFas ligand, including suppression, elimination and inactivation of sFas ligand, as well as reduction or elimination of activity of Fas ligand / ≪ / RTI > The Fas ligand inhibitor refers to any substance that reduces the expression of, reduces, and / or blocks the action of Fas ligand. For example, Fas ligand blocking antibody, FAS ligand solubilization inhibitor 3 inhibitor (eg, Stromelysin-1 inhibitor, eg, C 1 -hydroxy-3-isobutyl-2-methyl-N 4 2- (methylamino) -2-oxo-1-phenylethyl succinamide ₂₇ H ₄₆ N ₁₀ O ₉ S, etc.), and the like.

The subject may be a mammal, preferably a human or rodent, or a cell or tissue isolated therefrom. Preferably, the subject is a patient suffering from a pathological condition such as inflammation due to at least one expression selected from the group consisting of chemokines such as Mip-1 alpha, RANTES, and IP-10, or a patient suffering from such pathological conditions, Derived cells, tissues, or body fluids. In one embodiment of the present invention, the inflammation may be arthritis, and the inflammation-induced cell may be an arthritis-inducing cell, more specifically, an immune cell and / or synovial cell infiltrated into the arthritis-induced joint, Cells, and the like.

The dose of the Fas ligand inhibitor may be appropriately adjusted depending on the condition of the patient, degree of progress of inflammation and the like. For example, the daily dose may be 0.1 to 100 ug / kg (body weight) 50 ug / kg (body weight), but is not limited thereto.

As shown in the following Examples, the method for preventing and / or treating inflammation, particularly arthritis according to the present invention is effective not only for prevention of onset and therapeutic effect in the early stage of onset, And has an advantage that the effect is excellent.

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

Reacting the candidate material with the sample; And

Measuring the Fas ligand activity of the sample

Lt; / RTI >

The candidate substance may be determined as a chemokine expression inhibitor when the activity of the Fas ligand in the sample treated with the candidate substance is removed or the Fas ligand activity in the sample not treated with the candidate substance is decreased .

The chemokine may be at least one selected from the group consisting of Mip-1 alpha, RANTES, and IP-10. Since the chemokine-related substances are known to be involved in the inflammatory reaction, the chemokine expression inhibitor selected by the screening method can be used as an agent for preventing and / or treating arthritis such as inflammation, especially rheumatoid arthritis. Thus, the screening method may be a method of screening an inflammation inhibitor, and / or a therapeutic agent for arthritis (e.g., 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 Cells or eyeballs.

The Fas ligand activity can be measured by a conventional method, and it can be easily understood by those skilled in the art. For example, by measuring the change in the thickness of the joints for about 1 to 20 days, preferably about 8 to 12 days, for example, 10 days compared to the non-injected individuals when the animal is infused with soluble FasL to induce arthritis The degree of activation can be assessed. Alternatively, for example, immunohistocytes infiltrated into the joints after induction of arthritis are obtained, and the chemokines expressed or secreted in the group incubated with soluble sFasL and the group not containing them are subjected to realtime PCR, Cytoflowmetry, FACS (fluorescence activated cell sorter) or ELISA , The degree of activity can be evaluated. For example, if the expression of the chemokine is greater than that of the untreated group by comparing the expression of the group treated with sFasL and the group treated with sFasL, the chemokine expression of the chemokine Can be judged to be increased. Realtime PCR is useful for comparing relative amounts between groups, ELISA for absolute amounts, and FACS (fluorescence activated cell sorter) for measuring cell activation.

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

The screening chemokine expression inhibitor has the advantage of not only preventing inflammation, especially preventing the onset of arthritis and treating the onset of onset but also having an excellent disease relief and therapeutic effect even in the middle and late stages of the onset.

The technology for controlling chemokine expression using the Fas ligand proposed in the present invention and the technology for preventing and / or treating arthritis such as inflammation, especially rheumatoid arthritis and the like, and a novel technology for detecting chemokine expression inhibitors using the same, effectively prevent arthritis from occurring It is also a very useful technique for treating many patients suffering from arthritis by allowing them to obtain excellent therapeutic effects even after a certain degree of disease has progressed.

.

FIGS. 1A to 1C show the degree of expression of chemokine according to the Fas ligand concentration, wherein 1a shows the expression level of MIP-1α, 1b shows the expression level of RANTES, and 1c shows the expression level of IP-10.
FIG. 2A shows the thicknesses and clinical indices of joints in three kinds of mouse models in which arthritis was induced by K / BxN serum transfer according to Example 2,
2b is a joint photograph of the three kinds of mice,
2c show the expression levels of IFNγ, IL-4 and TGFβ in the three kinds of mice.
Fig. 3 shows the joint thickness and clinical index in z-VAD treated mice according to Example 3. Fig.
FIG. 4A shows the joint thickness and clinical index of the mouse of Example 4,
4b shows the result of measuring the joint thickness and clinical index of the mouse of Example 5,
4c show the degree of expression of IFNγ, IL-4 and TGFβ in the mouse joint of Example 5.
FIG. 5A shows the joint thickness and clinical index of the arthritic mouse of Example 6,
5b shows the result of measuring the joint thickness and clinical index of the arthritis middle-stage mouse of Example 6,
5c shows the result of measuring the joint thickness and clinical index of the mouse at the end of arthritis of Example 6,
5d to 5f show the degree of expression of IFNγ, IL-4 and TGFβ in the early, middle and late mouse joints of Example 6, respectively.
Figure 6 shows the joint thickness and clinical indices when inhibiting the in vivo production of soluble Fas ligand.
Figure 7a shows the number of inflammatory cells in the upper chamber and the number of inflammatory cells in the bottom chamber after 24 hours, and 7b shows the number of cells in the upper and bottom chambers after 24 hours, respectively. will be.

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  One: Morocco Ligand Chemokine  Expression increase effect test

Serum of K / BxN mice (obtained by cross-breeding KRN TCR transgenic mice and NOD mice from Drs D. Mathis of Harvard medical school, Boston, MA and C. Benoist) to normal B6 mice (Orient Bio) To induce arthritis. After 10 days of observation, the infiltrated cells and the synovial cells in the joints were separated and cultured in vitro with a soluble Fas ligand (accession number AAA19778 (SEQ ID NO: 1), 1 μg, 5 μg, 10 μg (R & D system) Respectively. The culture medium was prepared by adding 10% fetal bovine serum (Gibco), 1% penicillin streptomycin (Gibco) and DMEM (Dulbeccos's Modified Eagle's Medium) (WelGene) well / 10 < ⁶ > cells.

After culturing for 24 hours at 37 ° C in a 5% CO2 incubator, the cells and culture medium were separated by centrifugation, and RNA was extracted from the cells. RNA extraction was carried out using the RNasy mini prep kit from Qiagen, and 3 μg of the RNA was synthesized in cDNA using the MMLV reverse transcription kit, which was again produced by promega. Real-time PCR ABI 7500, manufactured by Applied Biosystem, was used to measure the expression of chemokines Mip-1 alpha, RANTES, and IP-10. Chemokine primers were also prepared by Applied Biosystem and the expression rates of chemokines were standardized by GAPDH, a type of house keeping gene, which does not change well depending on the external conditions.

The results thus obtained are shown in Figs. 1A to 1C. As can be seen in Figs. 1a and 1c, the expression of all three types of chemokines was increased in the case of treatment with soluble Fas ligand, and it was found that the expression was increased in proportion to the treatment concentration.

Example  2: in animal models Rheumatism  Arthritis induction test 1

In order to investigate the role of Fas ligand in the antibody-induced arthritis model, we examined the effects of Fas ligand-deficient gld mice, Fpr-deficient lpr mice, and control (normal) B6 mice with both Fas and Fas ligands RA was induced. First, blood was obtained from a naturally induced K / BxN mouse (obtained from Drs.D. Mathis of Harvard medical school, Boston, MA, and obtained by crossing with KRN TCR transgenic mice and NOD mice from C. Benenoist) Were collected. 150 [mu] l of the K / BxN serum obtained above was injected intraperitoneally (i.p.) into the B6 (Orient Bio), lpr (central experimental animal) and gld mouse The thickness of the joints was measured with a caliper (Manostat, Switzerland).

Clinical index refers to:

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. As can be seen in FIG. 2a, B6 mice and lpr mice were injected with serum, and the joints began swelling from day 4, and the highest index was observed at 8 to 9 days. However, in the gd mice lacking the Fas ligand, the joints were hardly swollen after 10 days of serum injection, and the clinical index remained at 3 to 4 points. In addition, a joint photograph of the test mouse is shown in Fig. 2B. As can be seen in Fig. 2b, it was observed that the joints were hardly swollen in the gld mice.

In the K / BxN serum transfer model, the expression of cytokines, which are important in controlling inflammation of the joints, was confirmed in the joint mRNA. Serum transfers were performed and after 10 days of observation, the mice were euthanized and the joints were excised. RNA was isolated from the joint tissues using the RNeasy kit (Qiagen), and all procedures were carried out according to the manual provided by the manufacturer of the RNeasy kit (Qiagen). CDNA was synthesized from 3 μg of the separated RNA using Reverse Transcription System (Promega Corporation, Madison, Wisconsin). Expression of each cytokine (IL-4, IFNγ, and TGFβ) was measured using an Applied Biosystems 7500 sequence detection system (Perkin-Elmer Biosystems) and all results were averaged compared to GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) . Each primer used was synthesized in Applied Biosystem (Foster City, Calif.) As follows.

TGF-β1 (forward): 5'-GCAACATGTGGAACTCTACCAGAA-3 '

TGF-β1 (reverse): 5'-GACGTCAAAAGACAGCCACTCA-3 '

TGF-β1 specific probe: FAM-ACCTTGGTAACCGGCTGCTGACCC-TAMRA

The results obtained above are shown in Fig. 2C. As shown in Figure 2c, expression of IL-4 and IFN gamma in B6 and lpr mice was increased compared to gld mice whereas expression of TGF beta was increased in gld mice. These data indicate that the Fas ligand plays an important role in inducing joint inflammation in the K / BxN serum transfer model, and this role is independent of Fas.

A typical function of the Fas ligand known so far is to bind to its receptor, Fas, to induce apoptosis of the target cell. If Fas ligand acts on joint inflammation through apoptosis by interaction with Fas, lpr mice deficient in Fas and gld mice deficient in Fas ligand should show the same arthritis inducing pattern. However, as shown in Figs. 1a-1c, the lpr mice deficient in Fas and the gld mice deficient in Fas ligand exhibited different patterns in the induction of arthritis. This suggests that Fas ligand does not contribute to the pathogenesis of arthritis by inducing apoptosis through binding to Fas but may be involved as another mechanism.

Example  3: In animal models Rheumatism  Arthritis induction test 2

To further demonstrate that the Fas ligand is involved in arthritis induction by a separate mechanism independent of apoptosis by binding to Fas, the following test is additionally performed.

B6 mice, as used in Example 2, were treated with a 50 ug or 100 ug of a caspase inhibitor capable of inhibiting apoptosis (z-VAD, Calbiochem) Serum was injected to induce arthritis. The thickness and clinical indices of the joints were measured in the same manner as in Example 2, and the results are shown in FIG.

As can be seen in FIG. 3, the arthritic score (joint thickness and clinical index) was increased in the same pattern in both z-VAD treated and untreated cases. Through the above data, it was confirmed that apoptosis was not involved at all in inducing arthritis through K / BxN serum transfer.

Example  4: In animal models Rheumatism  Arthritis induction test 3

To further confirm the role of the Fas ligand, the test was conducted by inducing arthritis by adoptive transfer of splenocytes of B6, lpr, and gld mice to gld mice, respectively.

First, B6, lpr, and gld mice were euthanized to collect spleens, then homogenized with 1 ml of red blood cell (RBC) lysis buffer (Qiagen), and RBCs were removed to obtain lymphocytes. After filtration through a sterilized 40 μm cell strainer (BD bioscience), the day before the transfer of the serum, 1 × 107 spleen cells per gld mouse were injected intravenously (iv) with sterilized PBS together with a total volume of 300 μl.

The joint thickness and the clinical indices of the mice transplanted with splenocytes and the mice not transplanted with splenocytes 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 FIG. 4a, the arthritis score, which is expressed as joint thickness and clinical index, was increased compared to gld mice not transplanted in gld mice transplanted with splenocytes from B6 and lpr mice. On the other hand, gld mice that transferred splenocytes from gld mice showed almost no arthritis induced, similar to untransferred gld mice. The data show that even if Fas is deficient, arthritis can be induced if Fas ligand is present.

Example  5: In animal models Rheumatism  Arthritis induction test 4

Next, sFas ligand was injected into gld mice, and K / BxN serum was transferred to test for arthritis induction.

2 μg or 10 μg of mouse sFas ligand (R & D) was injected into the abdominal cavity of gld mice on the day before serum transfer, and the joint thickness and clinical index were measured in the same manner as in Example 2, and the results are shown in FIG. As shown in FIG. 4B, when 2 μg of sFasL was injected, there was no significant change in joint thickness and clinical index. However, when 10 μg of sFasL was injected, the joint thickness and clinical index increased to the extent similar to wild-type B6 mice . The degree of expression of IFNγ, IL-4 and TGFβ on the joint mRNA in the gld mice injected with sFasL 10 μg was measured and shown in FIG. 4c. As shown in Fig. 4C, the expression of IFNγ and IL-4 on the joint mRNA was increased while the expression of TGFβ was decreased in the gld mice injected with 10 μg of sFasL.

Example  6: Morocco Ligand  Arthritis treatment test through inhibition

These results indicate that Fas ligand is one of the important pathogenesis of arthritis in K / BxN serum transfer model. These results, on the contrary, suggest that the blocking of Fas ligand activation plays an effective role in the prevention and treatment of arthritis. To confirm this hypothesis, the onset stage of arthritis was divided into early, mediated, and late phases to block the activation of Fas ligand and to observe the condition of arthritis.

Fas ligand blocking antibody (BD bioscience) was mixed with PBS in an amount of 10 μg, and the whole volume was adjusted to 300 μl and injected intravenously (iv) of B6 mouse. The Fas ligand blocking antibody was treated from the day before to the day of serum transfer to the day after the transfer of serum to block the role of Fas ligand at the early stage of arthritis development. The animal model was measured for joint thickness and clinical indices in the same manner as in Example 2, and is shown in FIG. 5A. As shown in FIG. 5A, it was confirmed that the onset of arthritis was remarkably inhibited in the mouse treated with the Fas ligand blocking antibody.

Between 5 and 9 days after transfection, which was considered to be the middle stage of joint inflammation, Fas ligand blocking antibody was mixed with PBS in an amount of 10 μg or 20 μg, and the total volume was adjusted to 300 μl and injected intravascularly into B6 mouse. The clinical indices were measured and are shown in Figure 5b. As shown in FIG. 5B, the Fas ligand blocking antibody was also inhibited in the middle stage of arthritis, and the inhibitory effect was more significant when the Fas ligand blocking antibody concentration was higher Respectively.

Finally, the Fas ligand blocking antibody was mixed with PBS in an amount of 20 μg from the 8th day of serum transfer, which is the highest stage of arthritis score (joint thickness and clinical index) to 11 days, and the total volume was adjusted to 300 μl, (Iv). The joint thickness and clinical indices of the mice 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 inhibited at the late stage of arthritis development, at the end of the term, as in the middle stage when Fas ligand blocking antibody was treated.

In addition, the degree of expression of cytokines (IL-4, IFN gamma and TGF beta) on the joint mRNA of the serum-transferred B6 mouse model was measured at the early stage, middle stage and late stage of arthritis, respectively, and the results are shown in FIGS. 5d to 5f . As shown in FIGS. 5d to 5f, expression of IL-4 and IFNγ was decreased and expression of TGFβ was increased in mice injected with Fas ligand blocking antibody at early stage, middle stage and late stage of arthritis development.

Example  7: soluble Morocco Ligand  Create Suppressed  Arthritis induction experiment

Indeed, the inhibition of arthritis induction was tested when inhibiting the in vivo production of soluble Fas ligand, which promotes arthritis induction. The wild-type B6 mouse, the Fas-deficient lpr mouse, and the Fas ligand-deficient gld mouse were injected with the Metrix metalloproteinase-3 inhibitor (MMP-3 inhibitor, Calbiochem, Ac- Arg-Cys- Pro-Asp-NH ₂ ) was injected into the peritoneal cavity and the joint thickness and clinical index were measured in the same manner as in Example 2. The results are shown in FIG. As shown in FIG. 6, when the production of sFas ligand was inhibited in wild-type B6 mice, joint thickness and clinical indices were observed to be decreased, but these effects could not be observed in lpr mice and gld mice. Through the above data, it was confirmed that the therapeutic effect of arthritis of MMP inhibitor is obtained through inhibition of sFas ligand formation, and generation of sFas ligand is an important factor promoting arthritis induction.

Example  8: soluble Morocco Ligand  Direct Chemokine  Examination of roles

The above data show that the production of sFas ligand promotes the expression of chemokines, which plays a role in inducing arthritis. Next, we examined whether the sFas ligand itself could act as a chemokine to stimulate the infiltration of inflammatory cells, not simply as a chemokine expression regulator. After induction of arthritis in wild-type B6 mice, inflammatory cells infiltrated into the joints on day 8, which had the highest joint thickness and clinical index, were obtained in the same manner as in Example 2. The upper chamber of an 80 μm transwell plate (BD Falcon) (RPMI 1640, 10% fetal bovine serum, 1% antibiotics), and the bottom chamber was loaded with 0 ng, 50 ng and 100 ng of sFas ligand in 700 μl culture, respectively. After 24 hours, the number of cells migrated from the upper chamber to the bottom chamber according to the sFas ligand is shown in FIG. 7a. The number of cells in the upper chamber and the bottom chamber after 24 hours is shown in FIG. 7B.

As can be seen from FIGS. 7A and 7B, the number of cells migrating to the bottom chamber among the inflammatory cells in the upper chamber increased in proportion to the sFas ligand concentration. Based on the above results, it was confirmed that sFas ligand not only regulates chemokine expression but also sFasL itself acts as a chemokine.

Taken together, these results suggest that Fas ligand blockade inhibits chemokine expression and inhibits the inflammatory response, and is effective in suppressing arthritic symptoms not only in the early stages of inflammation, especially arthritis but also after onset. These results could also provide a breakthrough in the development of prevention and treatment of inflammation, including arthritis. Further, sFasL itself provides a new use as a chemokine.

Claims (24)

An inhibitor which inhibits soluble Fas (sFas) ligand as an active ingredient,
Wherein the inhibitor is at least one selected from the group consisting of a Fas ligand blocking antibody, a Fas ligand solubilization inhibitor, and an MMP-3 inhibitor.
MIP-1 ?, RANTES and IP-10. delete delete An inhibitor which inhibits soluble Fas (sFas) ligand as an active ingredient,
Wherein the inhibitor is at least one selected from the group consisting of a Fas ligand blocking antibody, a Fas ligand solubilization inhibitor, and an MMP-3 inhibitor.
A composition for preventing or treating inflammation caused by at least one chemokine selected from the group consisting of MIP-1 ?, RANTES and IP-10. delete 5. The composition of claim 4, wherein the inflammation is arthritis by at least one chemokine selected from the group consisting of MIP-1 alpha, RANTES, and IP-10. 7. The composition of claim 6, wherein the arthritis is rheumatoid arthritis. delete delete delete delete delete delete delete delete delete 1. A method of inducing inflammation by at least one chemokine selected from the group consisting of MIP-1 alpha, RANTES and IP-10, comprising administering a soluble Fas ligand to the tissue or cells separated from the mammal. delete delete delete delete Reacting the candidate material with the sample; And
Measuring the Fas ligand activity of the sample,
When the activity of the Fas ligand in the sample treated with the candidate substance is removed or the Fas ligand activity in the sample not treated with the candidate substance is reduced, the candidate substance is selected from the group consisting of MIP-1α, RANTES and IP-10 Lt; RTI ID = 0.0 > chemokine < / RTI > of the selected one or more chemokines.
A method for screening a chemokine expression inhibitor. Reacting the candidate material with the sample; And
Measuring the Fas ligand activity of the sample,
When the activity of the Fas ligand in the sample treated with the candidate substance is removed or the Fas ligand activity in the sample not treated with the candidate substance is reduced, the candidate substance is selected from the group consisting of MIP-1α, RANTES and IP-10 ≪ RTI ID = 0.0 > chemokine, < / RTI >
MIP-1 alpha, RANTES, and IP-10. 24. The method according to claim 23, wherein the inflammation is arthritis caused by at least one chemokine selected from the group consisting of MIP-1 alpha, RANTES and IP-10.

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