JP5366077B2 - A skin disease model with inflammation and hyperproliferation - Google Patents

Description

  The present invention provides a skin disease model that can be used for the investigation of the etiology / onset mechanism of skin diseases associated with inflammation and hyperproliferation, for example, psoriasis, methods effective for the treatment, and screening for effective drugs.

  Psoriasis is one of the most common skin diseases. The morbidity rate in Western countries is about 2%, and the morbidity rate is increasing in Japan, although the frequency is low (Non-patent Document 1: Menter A et al., Lancet (1991) 338: 231-234). The disease is characterized by a red thickened scaly epidermis resulting from hyperproliferation and poor differentiation of epidermal keratinocytes, as well as marked infiltration of immune cells such as T cells, macrophages and neutrophils. Psoriasis is rarely fatal and the lesion is easy to treat. However, the symptoms often recur throughout their lives and remain a serious challenge for the patient's quality of life.

  Despite extensive research on the pathogenesis in recent years, it is still necessary to elucidate the molecular and cytological mechanisms that cause skin lesions with inflammation and hyperproliferation, including psoriasis (Non-patent document 2: Chamian F et al., Curr Opin Rheumatol (2004) 16: 331-337). In order to elucidate the pathogenesis of skin diseases associated with inflammation and hyperproliferation such as psoriasis, great efforts have been expended in developing appropriate animal models. Injection of activated T cells isolated from a patient with psoriasis into normal or non-lesional psoriatic skin tissue transplanted into SCID mice induces psoriatic lesions in that tissue, thereby causing T cells in the onset of psoriasis Has been demonstrated (Non-Patent Document 3: Wrone-Smith T et al., J Clin Invest (1996) 98: 1878-1887).

  The classic finding of this disease that keratinocytes play an important role in the development of psoriasis is the number that constitutively active STAT3 expression in the mouse epidermis induces psoriatic-like skin lesions and leads to psoriasis pathogenesis It was re-recognized by showing that the expression of species molecules was simultaneously enhanced (Non-Patent Document 4: Sano S et al., Nat Med (2005) 11: 43-49). Recently, it has been found that epidermis-specific deletion of JunB and cJun in adult mice results in a psoriatic-like skin phenotype (Non-Patent Document 5: Zenz R et al., Nature (2005) 437: 369- 375). In these mice, arthritis was also observed, which is often observed in human psoriasis cases but rarely occurs in animal models. Therefore, the JunB / cJun-deficient mouse model is considered to be the most highly mimicking human psoriasis. Such mouse models require laborious work such as laboratory animal breeding, administration of test substances to laboratory animals, skin observation, blood tests, etc. It has been reviewed from the viewpoint.

Lancet (1991) 338: 231-234 Curr Opin Rheumatol (2004) 16: 331-337 J Clin Invest (1996) 98: 1878-1887 Nat Med (2005) 11: 43-49 Nature (2005) 437: 369-375 Biochem Biophys Res Commun (2004) 322: 1111-1122 Trends Immunol (2003) 24: 155-158 Nature (1987) 330: 80-82 Arthritis Rheum (2004) 50: 3792-3803 Neurosci Lett (1998) 247: 195-197 Digestion (1995) 56: 406-414 J Biochem (Tokyo) (1990) 108: 650-653 Am J Clin Pathol (1987) 87: 681-699 J Biol Chem (2001) 276: 35818-35825 J Invest Dermatol (1992) .99: 299-305 Hum Genet (2002) 111: 310-313 J Biol Chem (1992) .267: 7499-7504 J Immunol (1993) 150: 2981-2991 Biotechniques (1998) 24: 954-958, 960, 962 Hum Mol Genet (2001) 10: 1793-1805 Br J Dermatol (2003) 149: 484-491

  The present invention provides a skin disease model with inflammation and hyperproliferation including psoriasis, which can replace an animal model.

  One of the earliest events after the deletion of JunB / cJun protein is the induction of S100A8 and S100A9. S100A8 and S100A9 belong to the EF-hand calcium-binding S100 protein family composed of more than 20 members (Non-Patent Document 6: Marenholz I et al., Biochem Biophys Res Commun (2004) 322: 1111-1122). Both proteins are secreted by neutrophils, activated monocytes, and macrophages, function as chemotactic molecules in those cells, and participate in a positive feedback loop for the recruitment of inflammatory cells (Non-Patent Document 7: Roth J et al., Trends Immunol (2003) 24: 155-158). S100A8 and S100A9 positive bone marrow cells are the first cells that infiltrate in the inflammatory region (Non-patent Document 8: Odink K et al., Nature (1987) 330: 80-82). Rheumatoid arthritis (non-patent document 9: Liao H et al., Arthritis Rheum (2004) 50: 3792-3803), multiple sclerosis (non-patent document 10: Bogumil T et al., Neurosci Lett (1998) 247: 195-197), Crohn's disease (Non-patent document 11: Lugering N, et al., Digestion (1995) 56: 406-414), and connective tissue disease (non-patent document 12: Kuruto R, et al., J Biochem). High S100A8 and S100A9 serum levels have been observed in a number of human inflammatory diseases, including (Tokyo) (1990) 108: 650-653). Therefore, S100A8 and S100A9 are thought to play an important role in the induction and propagation of inflammation.

  Production of S100A8 and S100A9 is not limited to inflammatory cells, but is observed in some epithelial cells, for example, activated or transformed (Non-patent Document 13: Brandtzaeg P et al., Am J Clin Pathol (1987). 87: 681-699). In the epidermis, S100A8 and S100A9 are wound healing (Non-Patent Document 14: Thorey IS et al., J Biol Chem (2001) 276: 35818-35825) and psoriatic lesions (Non-Patent Document 15: Madsen P et al. , J Invest Dermatol (1992) 99: 299-305), which is upregulated in hyperproliferative keratinocytes. The gene is encoded within the psoriasis sensitive region PSORS4 (Non-patent Document 16: Semprini S et al., Hum Genet (2002) 111: 310-313).

  Despite these findings, little is known about the biological functions performed by S100A8 and S100A9 in epithelial cells, except for the possibility of chemotactic effects on inflammatory cells (Non-patent Document 17: Lackmann M et al.). al., J Biol Chem (1992) 267: 7499-7504; Non-Patent Document 18: Lackmann M et al J Immunol (1993) 150: 2981-2991). Therefore, the present inventor aimed to elucidate whether S100A8 / A9 acts directly on epidermal keratinocytes. As a result, exogenous S100A8 / A9 stimulates normal human epidermal keratinocytes (NHEK) to produce inflammatory cytokines that are up-regulated in psoriatic lesions, and S100A8 / A9-inducible cytokines are S100A8 in NHK. And have been found to stimulate the production and secretion of S100A9. Furthermore, it was also found that S100A8 / A9 itself enhances NHEK proliferation. These results suggested the existence of a positive feedback mechanism of NHEK proliferation and inflammation involving S100A8 / A9 as a major mediator, and the possibility that S100A8 / A9 may play an important role in the development of psoriasis.

Accordingly, this application includes the following inventions:
(1) An in vitro model system for skin diseases accompanied by inflammation induction and hyperproliferation, characterized in that inflammatory cytokines are induced by causing at least one of S100A8 and S100A9 to act on cultured keratinocytes, and proliferation is enhanced.
(2) The in vitro model system for skin diseases according to (1), wherein both S100A8 and S100A9 are allowed to act.
(3) The in vitro model system for skin diseases according to (1) or (2), wherein the hyperproliferative skin disease is psoriasis.
(4) The in vitro model system of the skin disease of any one of (1) to (3), wherein the S100A8 and S100A9 are recombinant proteins.
(5) A method for creating an in vitro model system for skin diseases accompanied by inflammation and hyperproliferation, wherein at least one of S100A8 and S100A9 is allowed to act on cultured keratinocytes to enhance the expression and proliferation of inflammatory cytokines Features, a method.
(6) The method of (5), wherein both S100A8 and S100A9 are allowed to act.
(7) The method according to (5) or (6), wherein the hyperproliferative skin disease is psoriasis.
(8) The method according to any one of (5) to (7), wherein S100A8 and S100A9 are recombinant proteins.

  An object of the present invention is to provide a skin disease model with inflammation and hyperproliferation that can replace an animal model. This skin disease model can be used to investigate the etiology / onset mechanism of skin diseases involving inflammation and / or hyperproliferation, for example, psoriasis, methods effective for the treatment, and screening for effective drugs.

The production and secretion of S100A8 and S100A9 by NHEK is shown. The preparation of recombinant S100A8 and S100A9 proteins and the morphological changes when they are added to NHEK are shown. (A) The purity of recombinant S100A8 and S100A9 is shown. (B) shows morphological changes of NHEK by S100A8 / A9 (each 5 μg / mL). GST was used as a control. The gene whose expression was enhanced by S100A8 / S9 in NHEK assayed by RT-PCR is shown. The amount of transcript is displayed as a magnification relative to the value of the untreated control. FIG. 3 shows stimulation of production and secretion of S100A8 and S100A9 in NHEK by cytokines. The expression level of tubulin was shown as a control of the amount of cell extract. The growth stimulation of NHEK by S100A8 and S100A9 is shown.

Cultured Keratinocytes Cultured keratinocytes can be obtained by culturing keratinocytes derived from the epidermis of an animal in an appropriate medium, such as EpiLife (Cascade Biologies, USA, Portland, Oreg.). The cells are not limited to humans but may be cells derived from the epidermis of mice, rats, pigs and the like, but preferably human-derived cells are used.

S100A8 and A9
The amino acid sequences of S100A8 and A9 and the DNA sequence encoding them are disclosed in Non-Patent Document 16, for example. S100A8 and A9 that can be used in the present invention are usually human-derived natural types or recombinant proteins, but modified, heterologous, or non-purified products can be used as long as they have activity. Recombinant proteins of S100A8 and A9 are prepared according to methods well known in the art, for example, by inserting an isolated or PCR-synthesized S100A8 or A9 gene (cDNA) into, for example, a plasmid, virus or the like to prepare an expression vector. Large-scale preparation is possible by introducing and expressing in a host cell, for example, a cultured cell such as a microorganism, animal cell or plant cell.
S100A8 and A9 are preferably the same type as keratinocytes, but even if they are different, they can be used if they can induce inflammatory cytokines and can proliferate keratinocytes. S100A8 and / or A9 is dissolved in water or a medium, for example, a medium suitable for culturing keratinocytes, such as the EpiLife medium, and added to the culture system. Although the amount added cannot be generally specified, the concentration is about 1 ng / ml to 1 mg / ml, preferably about 10 ng / ml to about 100 μg / ml, more preferably about 100 ng / ml to about 10 μg / ml. The addition of S100A8 and / or A9 is preferably performed in the presence of calcium chloride. The culture conditions such as the culture time and the culture temperature in the presence of S100A8 and / or A9 are not particularly limited, and the time is sufficient for the excessive growth of keratinocytes. The culture is preferably performed at 30 to 37 ° C. for 1 to 14 hours, more preferably at 34 to 37 ° C. for 2 to 7 hours, preferably under 5% CO ₂ .
Examples of inflammatory cytokines include, but are not limited to, TNF, CXCL1, CXCL2, CXCL3, IL8, IL6, IL1F9, IL1RN, and the like.

  The skin disease model according to the present invention can be used for elucidation of mechanisms such as the onset, progression, and cure of skin diseases accompanied by inflammation and hyperproliferation, search for effective disease treatment methods, and screening for effective therapeutic agents. A skin disease accompanied by inflammation and hyperproliferation refers to a condition in which, in addition to skin inflammation, keratinocytes on the epidermis abnormally overgrow and skin thickening is observed. Psoriasis is a typical symptom including all diseases that show epidermis thickening with various eczema symptoms such as erythema, desquamation, lichenification, such as psoriasis, atopic skin disease, seborrheic dermatitis.

  Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this.

Materials and Methods Cells and Materials Neonatal human epidermal keratinocytes (NHEK; KURABO, Osaka, Japan) were prepared using EpiLife ™ medium containing 0.03 mM low concentration of calcium and HKGS growth additive (Cascade Biologics) or Cultured in EpiLife ™ -KG2 medium (Cascade Biologies, Portland, Oreg., USA). HKGS contains 0.2 ng / ml EGF, 5 μg / ml insulin, 0.18 μg / ml hydrocortisone, 5 μg / ml transferrin, and 0.2% (v / v) bovine pituitary extract. Contains. Keratinocytes were subcultured, and those in the second to fourth passages were used for the experiment. To measure DNA synthesis, tritium thymidine (1 μCi / mL; ARC, St. Louis, MO, USA) was added to the cultures 1 hour prior to cell recovery. Recombinant human EGF, TNF-α and IL-6 were purchased from PEPROTECH EC (London, UK). Recombinant human IL1F9, IL-8 / CXCL8, and CXCL1 were purchased from R & D Systems (Minneapolis, Minnesota, USA).

Preparation of recombinant proteins S100A8 and S100A9 cDNAs were amplified by PCR and cloned into pGEX6P1 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA: BamH1-Xho1 site). The nucleotide sequence of the vector was confirmed by DNA sequencing. Escherichia coli (BL21-Codon Plus-(DE3) -RIL; STRATAGENE, La Jolla, CA, USA) was transformed with this vector (pGEX6P1, pGEX6P1-S100A8 and pGEX6P1-S100A9). The recombinant GST fusion protein was purified by glutathione-agarose affinity chromatography using a Sephadex 4B column (GE Healthcare Bio-Sciences). GST was released by cutting with PreScission protease (GE Healthcare Bio-Sciences) and removed from the final preparation using a Sephadex 4B column.

Western blot analysis Western blot analysis was performed under general conditions. The antibodies used were as follows: anti-human tubulin antibody (Sigma, St. Louis, MO, USA), rabbit anti-human S100A8 (Calgranulin A) antibody (FL-83; Santa Cruz Biotechnology, USA, CA) , Santa Cruz) and rabbit anti-human S100A9 (Calgranulin B) antibody (H-90; Santa Cruz Biotechnology). Signals were obtained by HRP (horseradish peroxidase) -labeled anti-mouse antibody and anti-rabbit IgG antibody (Cell Signaling Technology, Beverly, Mass., USA), then chemiluminescence system (ECL plus, GE Healthcare Bio-Sciences) Visualized. In order to confirm S100A8 and S100A9 secreted into the culture medium, the protein in the medium was precipitated with methanol and dissolved in the same amount of buffer as the cell extract. Cell extract and culture medium-derived preparations were subjected to equal Western blot analysis so that relative amounts could be directly compared.

DNA microarray
Growth phase NHEK cultured in EpiLife ™ -KG2 (Cascade Biologies) was replaced with the same medium with or without 2 mM calcium chloride, S100A8 and S100A9 (each 10 μg / ml) for 3 hours Exposed. Total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) and purified using RNeasy spin columns (Qiagen, Valencia, CA, USA). The 260 nm / 280 nm luminous intensity ratio of the extracted RNA samples was 2.0 or more. Cy3- and Cy5-labeled RNA was prepared from these RNA samples and hybridized to a human whole genome oligo microarray (Agilent Technologies) according to the manufacturer's recommended protocol. The fluorescence image of each spot on this array was taken in by Microarray Scanner Bundle G2565BA (Agilent Technologies), and the obtained TIFF image was digitized by Feature Extraction software (Agilent Technologies). Spots flagged with gIsFeatNonUnifOL, rIsFeatNonUnifOL, gIsBGNonUnifOL, and rIsBGNonUnifOL were excluded from further analysis.

Real-time quantitative PCR
NHEK was exposed to S100A8 / A9 under conditions similar to those described for the DNA microarray and using the MagNA ™ Pure mRNA extraction kit and MagNA Pure ™ instrument (Roche Diagnostics, Tokyo, Japan). mRNA was extracted. The resulting mRNA was reverse transcribed using SuperScript ™ II (Invitrogen Corporation, Carlsbad, Calif., USA). Real-time quantitative PCR was performed on a LightCycler fast thermal cycler system using the LightCycler FastStart DNA master SYBR green I kit (Roche Diagnostics) according to the manufacturer's instructions. Typical reaction conditions were 40 cycles consisting of a 10 minute activation step followed by denaturation at 95 ° C. for 15 seconds, annealing at 60 ° C. for 10 seconds, and extension at 72 ° C. for 10 seconds. The primers used are shown in Table 1 below. The final concentration of each primer was 0.2-0.25 μM in a total reaction volume of 20 μl. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a control gene. The specificity of the amplified fragment was confirmed by melting curve analysis. The expression level of each gene was quantitatively analyzed using LightCycler analysis software (Non-patent Document 19: Morrison TB et al., Biotechniques (1998) 24: 954-958, 960, 962). The amount of target mRNA was normalized by the amount of GAPDH, and finally expressed as a ratio to the amount of untreated control mRNA.

Results Secretion of endogenous S100A8 and S100A9 from NHEK The inventors first tested whether S100A8 and S100A9 were produced and secreted by normal human keratinocytes (NHEK). NHEK is generally cultured in a basic medium EpiLife supplemented with 1% HKGS (growth additive; manufactured by Cascade Biologics). Under this condition, NHEK produced large amounts of S100A8 and S100A9, which were easily secreted into the medium (FIG. 1). When analyzed after 24 hours of incubation, most of the produced S100A8 and S100A9 was recovered from the medium (approximately 20 ng S100A9 in 10 ml medium). HKGS induced and secreted the production of these proteins in a dose-dependent manner. HKGS contains 0.2 ng / ml EGF, 5 μg / ml insulin, 0.18 μg / ml hydrocortisone, 5 μg / ml transferrin, and 0.2% (v / v) bovine pituitary extract. ing. Even EGF alone could enhance the production / secretion of S100A8 and S100A9 (FIG. 1). Tubulin was tested as a control.

Effects of exogenous S100A8 / A9 on NHEK gene expression S100A8 and S100A9 are generally considered to act as activators and chemotactic attractants for neutrophils and monocytes / macrophages (Non-patent literature) 17 and 18). However, when human epidermal keratinocytes secrete S100A8 and S100A9, the keratinocytes themselves are necessarily exposed to these proteins. Therefore, in order to examine the direct effects that S100A8 and S100A9 may have on epidermal keratinocytes, the present inventors added a mixture of purified recombinant S100A8 and S100A9 proteins (S100A8 / A9) onto NHEK. The protein preparation was fully purified as shown in FIG. 2A. These proteins were produced and purified as GST fusion proteins as described in the section of materials and methods. The gel was stained with Coomassie Brilliant Blue. The arrow indicates the position of GST.
The inventor has also found that S100A8 / A9 results in morphological changes in cells. That is, the edges of the cells became smoother and the adhesion between cells disappeared (FIG. 2B). This effect was considered to be induced by S100A8 / A9, not the contaminants in the preparation, because GST prepared under the same conditions did not show any such action. With this observation, the inventor analyzed for possible changes in the gene expression profile of NHK by a DNA microarray covering 41,765 genes, resulting in 19 cells exposed to S100A8 and S100A9 over 3 hours. Was found to be enhanced (> 2.0 times) (Table 2). The gene group whose expression is enhanced includes IL-8 / CXCL8, CXCL1, CXCL2, CXCL3, CCL20, IL-6, SPRR and TNFα and related proteins. Of particular note is that the majority of the up-regulated genes are in common with genes known to be expressed in the psoriatic epidermis, most of which are inflammatory cytokines (non- Patent Document 20: Bowcock AM et al., Hum Mol Genet (2001) 10: 1793-1805). In order to confirm the enhanced expression of these genes, the present inventor performed RT-PCR of NHEK exposed to S100A8 / A9 for 3 hours (FIG. 3). The mRNA level of each gene increased depending on the dose of S100A8 / A9 and the type of gene. The degree of upregulation of the tested genes was about 2 times (TNFα) to about 9 times (CXCL8 / IL-8), which almost coincided with the results of the DNA microarray.

Production / secretion of S100A8 and S100A9 enhanced by S100A8 / A9-inducible cytokines in NHEK Next, we will examine whether S100A8 / A9-inducible cytokines affect the production of S100A8 and / or S100A9 in NHK Tested. NHK was incubated for 24 hours in EpiLife basal medium without HKGS but supplemented with cytokines. The same amount of total protein preparation from cell extract and culture medium was subjected to Western blot analysis so that the fraction of total secreted protein could be roughly calculated. As shown in FIG. 4, all S100A8 / A9 inducible cytokines tested, ie CXCL1, CXCL8 / IL-8, IL-1 F9, IL-6, and TNFα, produced and rapidly produced S100A8 and S100A9 in a dose-dependent manner. Urged secretion. Interferon γ also showed a similar effect. As a control, a test using tubulin instead of S100A8 or S100A9 was also tested. These test results suggest that the cytokines induced by S100A8 and S100A9 and S100A8 / A9 are members of a positive feedback loop for production and secretion in NHK. It has been reported that the combination of TNFα and interferon γ efficiently enhances mRNA levels of S100A8 and S100A9 in NHK (Non-patent Document 21: Mork G et al., Br J Dermatol (2003) 149: 484-). 491).

Stimulation of NHEK proliferation by S100A8 and S100A8 / A9 To test the effect of S100A8 and S100A9 on NHK proliferation, NHK cells were separated from EpiK but without HKGS but with S100A8, S100A9 or a mixture of both proteins. Incubated in medium for 24 hours. One hour before cell recovery, ³ H-thymidine (1 μCi / mL) was added to the medium, and the radioactivity in the insoluble fraction was measured. GST was used as a control. As shown in FIG. 5, S100A8 significantly enhanced NHK proliferation, but S100A9 did not. The mixture of both proteins enhanced NHEK growth more strongly than the effect of S100A8 alone. The optimum concentration of S100A8 / A9 was about 100 ng / ml, and the growth enhancement decreased with increasing concentration. IL-1 F9, one of S100A8 / A9-inducible cytokines whose functions are not clearly elucidated, also stimulated NHK proliferation.

Claims (7)

In the culture of cells composed of keratinocytes, inflammatory cytokines are induced in the keratinocytes by causing S100A8 and S100A9 to act on the keratinocytes, and the proliferation thereof is promoted. In vitro model system.   2. The in vitro model system of skin disease according to claim 1, wherein the hyperproliferative skin disease is psoriasis. The skin disease model according to claim 1 or 2 , wherein the S100A8 and S100A9 are recombinant proteins. A method for creating an in vitro model system for skin diseases involving inflammation and hyperproliferation, wherein S100A8 and S100A9 are allowed to act on a culture of cells composed of keratinocytes to enhance the expression and proliferation of inflammatory cytokines in the keratinocytes A method characterized by that.   The method according to claim 4, wherein both S100A8 and S100A9 are allowed to act.   6. The method of claim 4 or 5, wherein the inflammatory hyperproliferative skin disease is psoriasis. The method according to claim 5 or 6 , wherein the S100A8 and S100A9 are recombinant proteins.