408 Vol. 35, No. 3

© 2012 The Pharmaceutical Society of Japan

Biol. Pharm. Bull. 35(3) 408—412 (2012)

Restriction of Mast Cell Proliferation through Hyaluronan Synthesis by Co-cultured FibroblastsHirotsugu Takano,a Kazuyuki Furuta,a Kazuhito Yamashita,b Mariko Sakanaka,c Naoki Itano,d Eiichi Gohda,b Kazuhisa Nakayama,a Koji Kimata,e Yukihiko Sugimoto,f Atsushi Ichikawa,g and Satoshi Tanaka*,b

a Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University; Kyoto 606–8501, Japan: b Department of Immunobiology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences; Okayama 700–8530, Japan: c Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University; g Institute for Biosciences, Mukogawa Women’s University; Hyogo 663–8179, Japan: d Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University; Kyoto 603–8555, Japan: e Research Complex for the Medicine Frontiers, Aichi Medical University; Aichi 480–1195, Japan: and f Department of Pharmaceutical Biochemistry, Graduate School of Medicine and Pharmaceutical Sciences, Kumamoto University; Kumamoto 862–0973, Japan.Received November 8, 2011; accepted December 1, 2011; published online December 15, 2011

Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the pres-ence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cuta-neous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner.

Key words mast cell; hyaluronan; proliferation; hyaluronan synthase; fibroblast

Accumulating evidence suggests that mast cells play criti-cal roles in regulation of cutaneous inflammatory responses, such as immunoglobulin E (IgE)-mediated type I allergy, urticaria, ultra violet B-induced inflammation, and contact hypersensitivity.1—4) Although these in vivo findings have strongly indicated that mast cells release a variety of pro- and anti-inflammatory mediators, such as histamine, tumor ne-crosis factor-α, interleukin-1β (IL-1β), and IL-10, to regulate the inflammatory processes, it remains unknown how mast cells are activated under these conditions. Appropriate culture models that can reflect the responses of cutaneous mast cells are required to evaluate their functions in vitro, because local mast cells are differentiated under the influence of their im-mediate environment, in which they are ultimately resident.5) Bone marrow-derived cultured mast cells (BMMCs), which can be obtained by prolonged culture of murine bone marrow cells in the presence of IL-3, have been used as a good cul-ture model for mast cells, since they represent several aspects of mast cells, such as surface expression of FcεRI and c-kit, and histamine release upon antigen stimulation.6) However, because BMMCs are incapable of mimicking the specific features of cutaneous mast cells, such as degranulation upon stimulation with substance P or with cationic secretagogues, novel culture models for cutaneous mast cells were required. We established a culture model for cutaneous mast cells, in which BMMCs are co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor (SCF).7) This model reflects several characteristic features of cutaneous mast cells, such as Safranin-positive granules and degranulation upon stimulation

with substance P or with a polycation, compound 48/80. The gene expression profiles revealed that CD44, a primary recep-tor for hyaluronan, was drastically up-regulated during the co-culture period.8) BMMCs were found to be associated with the extracellular matrix containing hyaluronan during the co-culture period. We then explored the functional roles of CD44 during mast cell maturation and found that it positively regu-lates proliferation of mast cells during terminal differentiation and that the number of mast cells is decreased in cutaneous tissues of the CD44-deficient mice.8) CD44-deficient BMMCs transplanted into the cutaneous tissues of mast cell-deficient mice, furthermore, exhibited impaired growth in comparison with the wild-type BMMCs. Although these findings suggest that up-regulation of CD44 in mast cells may be required for their optimal proliferation in the cutaneous tissues, it remains to be clarified how hyaluronan is involved in regulation of cu-taneous mast cell proliferation. In this study, we investigated the effects of exogenously-added hyaluronan on proliferation of cultured mast cells under hyaluronan-deficient conditions.

MATERIALS AND METHODS

Mice Specific-pathogen-free, 8-week-old female Balb/c mice were obtained from Japan SLC (Hamamatsu, Japan). The CD44+/− mice backcrossed for 10—12 generations to C57BL/6 were bred to generate the CD44+/+- and CD44−/−-mice. All animal experiments were performed according to the Guidelines for Animal Experiments of Kyoto University and were approved by the Committee for Animal Experiments.

Regular Article

* To whom correspondence should be addressed. e-mail: tanaka@pharm.okayama-u.ac.jp

Highlighted Paper selected by Editor-in-Chief

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Materials Hyaluronan with various molecular weights (FCH-SU, 5—11×104; FCH-60, 5—7×105; FCH-80, 6—10×105; FCH-120, 1—1.4×106; FCH-150, 1.4—1.8×106; and FCH-200, 1.8—2.2×106) was generously provided by Kikkoman Biochemifa Co. (Tokyo, Japan). The following materials were purchased from the sources indicated: recom-binant mouse IL-3 from R&D Systems (Minneapolis, MN, U.S.A.), mitomycin C, hyaluronidase from bovine testis, compound 48/80, polybrene, an anti-dinitrophenyl (DNP) IgE (clone SPE-7), and DNP-conjugated human serum albumin from Sigma-Aldrich (St. Louis, MO, U.S.A.), 293FT cells, and Lipofectamine 2000 reagent from Invitrogen (Carlsbad, CA, U.S.A.), a retrovirus vector pSINsi-mU6 from TaKaRa Bio (Ohtsu, Japan), biotinylated hyaluronan binding pro-tein (bHABP), and an enzyme-linked immunosorbent assay (ELISA) kit for hyaluronan from Seikagaku Corp. (Tokyo, Japan), A23187 from Calbiochem (San Diego, CA, U.S.A.), S-2586, and S-2288 from Chromogenix (Milano, Italy), and M-2245 from Bachem (Bubendorf, Switzerland). All other chemicals were commercial products of reagent grade.

Preparation of BMMCs and Co-culture with Swiss 3T3 Cells Preparation of BMMCs and the co-culture with a fi-broblastic cell line, Swiss 3T3, were performed as described previously.7) Briefly, BMMCs were co-cultured with mitomy-cin C-treated Swiss 3T3 cells in the presence of the recombi-nant mouse SCF (100 ng/mL). The subculture was performed every 4 d. Greater than 80% of the viable mast cells were con-firmed to be mature mast cells, as assessed by staining with Safranin-O at Day-16.

Measurement of Hyaluronan Conditioned medium col-lected at Day-4 was centrifuged at 800×g for 5 min to remove the cell debris. Concentrations of hyaluronan in the medium were determined using an ELISA kit for hyaluronan with bHABP according to the manufacturer’s instruction.

Degranulation Mast cells were collected and washed in piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (25 mM PIPES–NaOH, pH 7.4 containing 125 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 1 mM CaCl2, and 0.1% bovine serum al-bumin). The cells were incubated for 30 min with 1 μM A23187 or 10 μg/mL compound 48/80 in PIPES buff er. Degranulation of mast cells was evaluated by measuring enzyme activity of a granule enzyme, β-hexosaminidase, as described previously.7)

Measurement of Granule Proteases Mast cells were collected and lysed in phosphate buffered saline (PBS) con-taining 2 M NaCl and 0.5% Triton X-100 at 4°C. The lysate was centrifuged at 10000×g for 30 min at 4°C. The resultant supernatant was subjected to the assays for granule protease activities. The protease activities were measured using the specific chromogenic peptide substrates; chymase, S-2586, tryptase, S-2288, and carboxypeptidase A, M-2245.7)

Retroviral Introduction of Shoet Hairpin RNA (shRNA) for Hyaluronan Synthase 2 We preliminary determined mRNA expression levels of mouse hyaluronan synthase (mHAS) subtypes in Swiss 3T3 cells by the quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The expression level of mHAS1 was below the de-tection limit, and that of mHAS3 was approximately 1.5% of that of mHAS2 in the copy number (data not shown). Target sequences for knock-down of mHAS2 gene were selected by Dharmacon siDESIGN center (http://www.dharmacon.com/sidesign/). The shRNA sequences used in this study were

described as follows (target sequence, oligonucleotides used for the vector construction); Seq. 1 5′-CAA TTG GTC TTG TCT AAC A-3′, 5′-GAT CCA GAA TCA CAT CTG TTT ATA TTT CAA GAG AAT ATA AGC A GC TGT GAT TCT TTT TTT AT-3′, Seq. 2 5′-GAA TCA CAG CTG CTT ATA T-3′, 5′-GAT CCA GAA TCA CAT CTG TTT ATA TTT CAA GAG AAT ATA AGC A GC TGT GAT TCT TTT TTT AT-3′, Seq. 3 5′-ATA TCG TCA TGG TAT TCA T-3′, 5′-GAT CCA ATA TCT TCA TGT TAT TCA TTT CAA GAG A ATG AAT ACC ATG ACG ATA TTT TTT TTA T-3′, Control (a mutated version of Seq. 2, mutated bases indicated by the underlines) 5′-GAT TCA CTG CTG CTA ATA T-3′, 5′-GAT CCA GAT TCA CTG CTT CTA ATA TTT CAA GAG AAT ATT AGC AGC AGT GAA TCT TTT TTT AT-3′. The control shRNA sequence was prepared by introducing three point mutations to the target sequence (indicated by the underlines). The ret-rovirus vector pSINsi-mU6 was used for preparation of the re-combinant retrovirus. The oligonucleotides were annealed and subcloned into BamHI/ClaI site of pSINsi-mU6. 293FT cells were plated in poly-D-lysin coated dishes 1 d before transfec-tion. Each recombinant vectors were co-transfected with both pGP and pE-ampho into 293FT cells using a Lipofectamine 2000 reagent to produce infectious replication-incompetent retrovirus. Swiss3T3 cells seeded 24 h before infection were once washed with PBS, and treated with 0.1% hyaluronidase from bovine testis in PBS containing 0.3% bovine serum al-bumin (BSA) for 1 min at room temperature. The cells were washed with the medium, and the conditioned medium con-taining the recombinant virus was added in the presence of 6 μg/mL polybrene (Day-0). The medium was replaced with the fresh medium at Day 1, and was then replaced again with the fresh medium containing 250 μg/mL G418 at Day-3. G418-resistant clones were then concentrated during the further 2-week culture.

Differential Cell Counting The number of mast cells, which were distributed in different fashions, was determined as follows. The culture supernatant, which contained unbound free mast cells, was collected and centrifuged at 800×g for 3 min to obtain non-adherent cells. The residual culture cells were gently rinsed in PBS and treated with 0.1% hyaluroni-dase from bovine testis for 1 min in PBS containing 0.3% bovine serum albumin. The liberated cells were collected by centrifugation and were designated as hyaluronan-associated cells. The hyaluronidase-resistant adherent cells were treated with trypsin. The trypsinized cells were seeded and fibroblasts were removed as the adherent cells. Non adherent cells were collected and designated as fibroblast-associated cells.

Statistical Analysis Data are presented as means±S.E.M. Statistical significance for comparisons between groups was determined using Student’s t test or analysis of variance (ANOVA). Additional comparisons were made with Dunnet multiple comparison test for comparison with the control groups or Tukey–Kramer multiple comparison test for all pairs of column comparison.

RESULTS

Inhibition of Hyaluronan Synthesis in Swiss 3T3 Cells by Knockdown of HAS2 Swiss 3T3 cells were found to constitutively produce hyaluronan, which formed the extracel-lular matrix in our co-culture system.9) It remains unknown

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whether hyaluronan affects proliferation of the cultured mast cells, although CD44, a primary membrane receptor for hya-luronan, was found to be required for proliferation of cutane-ous mast cells. We planned to suppress hyaluronan synthesis in our co-culture system to investigate the effects of exoge-nous hyaluronan on mast cell proliferation. We prepared mod-ified Swiss 3T3 cells, which constitutively express shRNA for hyaluronan synthase 2 (HAS2) through retroviral infection. All the investigated constructs significantly suppressed hyalu-ronan release from Swiss 3T3 cells (Fig. 1A). Introduction of the point mutations into the most effective construct resulted in failure in suppressing hyaluronan synthesis in Swiss 3T3 cells, excluding the possibility that the expression of shRNA decreased hyaluronan synthesis independently of HAS2 ex-pression (Fig. 1B). A significant decrease in the amount of hyaluronan was also found in the presence of hyaluronidase during the co-culture period (Fig. 1B).

Enhanced Proliferation of the Cultured mast Cells under Hyaluronan-Deficient Conditions Hyaluronan de-ficiency altered distribution of mast cells co-cultured with Swiss 3T3 cells. Greater than 70% of the cultured mast

cells were associated with the extracellular matrix contain-ing hyaluronan during the co-culture period. In contrast, the cultured mast cells was marginally associated with the ex-tracellular matrix and the feeder fibroblasts under both of the hyaluronan-deficient conditions (Fig. 1C). On the other hand, the number of the total cultured mast cells was significantly increased under both of the hyaluronan-deficient conditions (Fig. 1D). Although these findings suggest that hyaluronan has a potential to suppress mast cell proliferation, exogenously-added hyaluronan samples with different molecular weights (10 μg/mL, FCH-SU, 5—11×104; FCH-60, 5—7×105; FCH-80, 6—10×105; FCH-120, 1—1.4×106; FCH-150, 1.4—1.8×106; and FCH-200, 1.8—2.2×106) had no significant effects on pro-liferation of the mast cells co-cultured with HAS2-knockdown Swiss 3T3 cells (data not shown). SCF-induced proliferation of BMMCs was not significantly affected by the presence of FCH-SU, FCH-80, or FCH-200 (data not shown).

Mast Cell Maturation under Hyaluronan-Deficient Conditions We then investigated the several indices related to mast cell maturation, such as granule protease activities, and degranulation in response to secretagogues. No signifi-cant changes were found in the enzymatic activity of granule proteases, such as chymase, tryptase, and carboxypeptidase A, in the cells cultured under both the hyaluronan-sufficient and -deficient conditions (Figs. 2A, B). Hyaluronan deficiency did not affect the degrees of degranulation upon stimulation with compound 48/80, a cationic secretagogue (Fig. 2C) or with a Ca2+ ionophore, A23187. The percentages of Safranin-O-positive mature mast cells were unchanged through the co-culture periods under hyaluronan-deficient conditions (data not shown). These findings suggest that constant release of hyaluronan from the feeder fibroblasts is not required for mast cell maturation.

Roles of the Hyaluronan-CD44 Axis in Mast Cell

Fig. 1. Inhibition of Hyaluronan Synthesis in Swiss 3T3 Fibroblasts Enhanced Proliferation of the Co-cultured Mast Cells

(A) Expression of hyaluronan synthase-2 (HAS2) was suppressed by retroviral shRNA transduction in Swiss 3T3 fibroblasts. Hyaluronan release for 24 h from the parental Swiss 3T3 cells (none) or Swiss 3T3 cells expressing an shRNA construct for HAS-2 (Seq. 1, 2, or 3) was respectively measured. (B) Hyaluronan releases for 24 h from the parental Swiss 3T3 cells (none), Swiss 3T3 cells expressing shRNA (Seq. 2) for HAS2 (HAS2-KD) or its mutated shRNA (control) was measured. The amounts of hyaluronan in the medium in the continuous presence of 0.1% hyaluronidase (+HAase) was also determined. (C) BMMCs were co-cultured with the parental Swiss 3T3 cells (control), or HAS2-KD Swiss 3T3 cells (HAS2-KD), of with Swiss 3T3 cells in the presence of 0.1% hyaluronidase (+HAase). The number of the co-cultured mast cells at Day-4 (control; (1.68±0.0680)×107 cells, HAS2-KD; (2.07±0.186)×107 cells, +HAase; (1.57±0.191)×107 cells) was differentially counted as described in the Materials and Methods. The percent-ages of non-adherent (closed columns), hyaluronan-associated (gray columns), and fibroblast-associated (open columns) mast cells are presented. (D) Growth curves of the co-cultured mast cells are presented (the cell number at Day-0=1, open circles; Control, closed circles; HAS2-KD, open squares; +HAase). The values represent means±S.E.M. (n=3). The values of *p<0.05, and **p<0.01 are regarded as sig-nificant (vs. control). N.D., not detectable.

Fig. 2. Maturation of the Cultured Mast Cells under Hyaluronan-Deficient Conditions

BMMCs were co-cultured with the parental Swiss 3T3 cells (open columns) or HAS2-KD Swiss 3T3 (gray columns), or with Swiss 3T3 cells in the presence of 0.1% hyaluronidase (closed columns). (A, B) Enzymatic activities of chymase and carboxypeptidase A (A), and tryptase (B) of the Day-16 co-cultured mast cells were measured. (C) The Day-16 co-cultured mast cells were mock-stimulated (None) or stimulated with 10 μg/mL compound 48/80, or 1 μM A23187. The level of degranulation was determined by measuring the β-hexosaminidase activity. The values represent means±S.E.M. (n=3).

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Proliferation We then investigated the involvement of CD44 expression in the cultured mast cells. The absence of CD44 impaired proliferation of the mast cells co-cultured with the parental Swiss 3T3 cells, which was consistent with our pre-vious findings. On the other hand, the growth profiles were not different between the wild type and CD44-deficient mast cells when they were co-cultured with HAS2-knockdown Swiss 3T3 cells (Fig. 3A), indicating that CD44 should not be involved in proliferation of the cultured mast cells under hyaluronan-deficient conditions. The absence of CD44 did not affect the distribution patterns of cultured mast cells on HAS2-knockdown Swiss 3T3 cells, whereas it reduced the size and number of mast cell clusters formed on the parental Swiss 3T3 cells (Fig. 3B).

DISCUSSION

Enhanced proliferation of the cultured mast cells under hyaluronan-deficient conditions suggests that hyaluronan syn-thesis by the feeder fibroblasts should limit the size of mast cell population. Tissue mast cell population must be strictly regulated, because terminally differentiated tissue mast cells retain a potential to proliferate in response to hematopoi-etic cytokines, such as SCF and IL-3.10,11) However, it remains largely unknown how proliferative responses of tissue mast cells are regulated. Our results suggest that the extracellular matrix containing hyaluronan should be involved in this regu-lation. Failure of growth inhibition by the exogenously added hyaluronan imply that restriction of mast cell proliferation should not be achieved by hyaluronan alone, but require ongo-ing developments of the extracellular matrix, which is closely associated with HAS2 in fibroblasts. A single hyaluronan chain was found to bind hundreds of the other extracellular matrix proteins and to form a large complex, which is often associated with the surface hyaluronan receptors.12) Because TGF-β, M-CSF, GM-CSF, and IFN-γ were found to suppress the proliferation of BMMCs,13—15) the hyaluronan-containing

large extracellular matrix may negatively regulate the pro-liferation of the cultured mast cells by some other ways, for examples, by capturing these cytokines and growth factors in the vicinity of them.

Accumulating evidence suggests that the biological functions of hyaluronan depend on its molecular mass.16) Hyaluronan molecules with different molecular mass could exhibit even opposite effects on the target cell behavior: high molecular weight hyaluronan (HMW-HA) inhibits the cell cy-cle progression through GTP-loading of Rac whereas low mo-lecular weight hyaluronan (LMW-HA) stimulates it through ERK activation in human vascular smooth muscle cells.17,18) These findings raise a possibility that a decrease in the amount of HMW-HA and a possible increase in the amount of LMW-HA could induce cultured mast cell proliferation in our study. On the other hand, these previous studies also demonstrated that the effects of hyaluronan is exerted by its binding to CD44. Our results using CD44-deficient mast cells suggest that enhanced proliferation under hyaluronan-deficient conditions is somehow independent of CD44 expression in the cultured mast cells and that exogenously-added hyaluronan has no effects on the proliferation of cultured mast cells. The hyaluronan-CD44 axis might not be involved in suppression of cultured mast cells. Hyaluronan may affect mast cell prolifera-tion through the involvement of the other receptors, such as receptor for hyaluronan-mediated motility (RHAMM), Lyve-1, and Toll-like receptor-4, although we have not verified their involvement.

Accumulating evidence suggests that a reduction in the con-centration and average molecular weight of hyaluronan, which are mediated, at least in part, by up-regulated hyaluronidase, are often observed in knee synovial fluids from the patients with osteoarthritis and rheumatoid arthritis.19—22) Since mast cell hyperplasia is a feature of both rheumatoid arthritis and osteoarthritis conditions,23) degradation of the extracellular matrix containing hyaluronan under inflammatory conditions may lead to expansion of local mast cell population. Recently,

Fig. 3. Roles of CD44 in Proliferation of the Cultured Mast CellsBMMCs derived from the wild-type (open columns, +/+) and CD44−/− mice (closed columns, −/−) were co-cultured for 16 d with parental Swiss 3T3 cells (control) or

with HAS2-KD Swiss 3T3 cells. The number of mast cells was compared among these groups (the cell number at Day-0=1). The values represent means±S.E.M. (n=3). The values of **p<0.01 are regarded as significant (vs. wild type BMMCs+control Swiss 3T3 cells). (B) Differential interference contrast microscopy images of the co-cultured mast cells at Day-16 are presented. Bar=100 μm.

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an increase in the number of cutaneous mast cells in response to ultraviolet (UV) irradiation was reported.24) Because colla-gen fragments produced in response to UVB irradiation were found to inhibit hyaluronan synthesis in skin fibroblasts,25) such inhibition of hyaluronan synthesis may result in increase in the number of cutaneous mast cells.

We conclude that hyaluronan synthesis by the co-cultured fibroblasts should restrict proliferation of mast cells in a CD44-independent manner but have no impact on their matu-ration. Our results shed a new light on the mechanism of mast cell hyperplasia under inflammatory conditions with increased degradation and decreased hyaluronan synthesis.

Acknowledgements This study was supported in part by a Grant from the Japan Chemical Industry Association (JCIA) Long-range Research Initiative (LRI), and by KAKENHI (21790100) Grants-in-Aid for Young Scientists (B) from Japan Society for the Promotion of Science (JSPS).

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