Effect of activated human mast cells and mast cell-derived mediators on proliferation, type I collagen production and glycosaminoglycans synthesis by human dermal fibroblasts

ARTICLE

Mast cells are known to play a central role in IgE-mediated allergic reactions, including both immediate and late-phase constituents. Recent studies suggest that mast cells appear in other biological reactions in which IgE is not essential. For example, accumulated mast cells are frequently observed at sites of fibrosis such as keloids, healing wounds or the early stage of systemic sclerosis (SSc) [1-5]. The speculation that mast cells play a role in fibrotic disorders stems primarily from the histological evidence in SSc [2, 4, 5] or fibrotic lung tissue [6]. Furthermore, it is well known that certain drugs used for treatment of allergic disorders improve hypertrophic scars [7]. These facts suggest that mast cells can modulate the various fibrotic processes. It is still unclear, however, whether or not IgE-mediated activation of human mast cells acts as a fibrogenic stimulus for human fibroblasts.

The three major hallmarks in fibrosis are the increased proliferation of fibroblasts, enhanced collagen synthesis by fibroblasts and reconstituted the components of extra-cellular matrix. A number of experimental studies have suggested that mast cell degranulation may induce fibroblast proliferation. The previous experiments, however, have been performed using rodent mast cells because they are easily obtained and managed [8]. The phenotypes of mast cells have been identified in rat, mouse and human models [9, 10]. It is conceivable that there may be functional differences in mast cells of different species. The exact features and functions that characterize rat mast cells cannot always be applied to human mast cells. Human mast cells isolated from the human lung involving complicated purification processes and alterations of membrane receptors have been used for experiments. Recent scientific advances have allowed us to culture human mast cells from umbilical cord blood [11-13]. Both tryptase-positive and tryptase- and chymase-positive cell types were identified by histochemical staining. These cells were functionally matured, histamine-containing, IgE receptor-positive and metachromatic. They can be activated through an IgE-dependent system [11, 12]. Although the HMC-1 mast cell line represents an alternative experimental approach, the general cytoplasmic morphology of these cells reflects their immature nature [14]. Thus, the experimental findings of HMC-1 cells will require further confirmation.

Abnormal metabolism of dermal connective tissue components other than collagen has been investigated in the fibrotic skin disease such as keloids [15] and SSc [16]. It is well known that an increased amount of dermatan sulfate has been demonstrated in skin glycosaminoglycans (GAGs) of patients with SSc or localized scleroderma [17]. In the present study, we investigated whether or not immunologically activated cultured human mast cells and mast cell-derived mediators could affect fibroblast proliferation and the production of type I collagen or glycosaminoglycan by fibroblasts.

Materials and methods

Human fibroblast culture

Normal human dermal fibroblasts were expanded by the explant culture method. In the present experiment, after informed consent was given, we obtained dermal fibroblasts from five young healthy volunteers. Skin specimens were segmented into small tissue samples, and the outgrown fibroblasts were trypsinized and grown in Dulbecco's modified Eagle medium (DMEM; Nihon Seiyaku, Tokyo, Japan) containing 10% fetal calf serum (FCS: Cytosystems, Castle Hill, Australia) at 37° C in 5% CO₂ humidified air. The culture medium was changed every three days. Cells were expanded through two or three passages before the experiments.

Human mast cell culture

Mast cells were generated from human umbilical cord blood. Freshly obtained heparin-treated umbilical cord blood, approximately 20 ml, was layered over Ficoll-Paque (Pharmacia, Uppsala, Sweden), and centrifuged at 400 x g for 20 min. The mononuclear cell layer was collected and suspended at 1 x 10⁶ cells/ml in Media I (IBL Men-eki Seibutsu Kenkyujo, Gunma, Japan) containing 10 mug/ml insulin, 10 mug/ml transferrin, 5 x 10^{- 5} M 2-mercaptoethanol, 25 mM HEPES, 2.6 ng/ml NaSeO, and supplemented by 5% FCS (Cytosystems), 50 ng/ml human recombinant interleukin 6 (IL-6) and 80 ng/ml human recombinant stem cell factor (SCF) (generously provided by Kirin Brewery Co. Ltd., Tokyo, Japan) at 37° C in 5% CO₂ humidified air. The cells were passaged every week and grown in the same media. Cell counting was performed by toluidine blue staining. Mast cells fixed with Carnoy's fluid for 15 min at room temperature were stained using monoclonal murine anti-tryptase (Chemicon, Temecula, Calif.) or anti-chymase (Chemicon, Temecula, Calif.) antibody as originally reported by Irani et al. [18]. In the present study, we used cells aged over 100 days old. At the time of the experiments, 100% of the cells were trypan blue negative and toluidine blue positive. When the cells were immunocytochemically examined for tryptase, nearly 100% of the cells were positively stained. Among tryptase-positive cells, 30% of the cells contained chymase with variable staining intensity.

Activation of human mast cell

Cultured human mast cells were sensitized with 1 mug/ml human myeloma IgE (Chemicon, Temecula, Calif.) for 1 hr at 37° C, washed twice with DMEM without FCS, then resuspended in the same medium prior to the experiments. The cells were either stimulated with anti-IgE or left unstimulated. Mast cells maintained in DMEM without FCS showed no significant change in cell viability during the corresponding period of cell activation.

Co-culture of human fibroblasts with human mast cells

Fibroblasts were seeded into 96-well tissue culture plates at a density of 2.5 x 10³ cells per well and incubated at 37° C in 5% CO₂ humidified air overnight to allow fibroblasts adherence in DMEM containing 10% heat-inactivated FCS. The medium was then replaced with FCS-free DMEM. After 24 hrs of serum deprivation, IgE-sensitized mast cells of varying number (2 x 10~6 x 10⁵ cells/well) were seeded onto the fibroblasts. In order to compare the effects of mast cell activation on fibroblast proliferation, anti-human IgE (Cappel, Durham, NC) or FCS-free DMEM was added. After 24 hrs of co-culture, the medium was aspirated to measure the concentration of type I collagen in supernatant. Measurement of procollagen type I C-peptide in supernatant has been regarded as a reasonable method to quantify type I collagen synthesis [19, 20]. Therefore, type I collagen production by fibroblasts was assessed by measuring the procollagen type I C-peptide concentration using an enzyme-linked immunosorbent assay (ELISA), a procollagen type I C-peptide kit (Takara Shuzo, Kyoto, Japan) [21]. In each experiment, the procollagen type I C-peptide concentration was averaged from triplicate wells. We had confirmed that the type I collagen production by fibroblasts which we used in the present experiments was stimulated by mast cell tryptase [22].

The proliferative response of fibroblasts was measured by [³H]thymidine incorporation. In brief, after 24 hrs of co-culture with human mast cells, the fibroblasts were incubated with [³H]thymidine (specific activity 230 Bq/mmol, American Radiolabeled Chemicals, St. Louis, Mo) for 6 hrs at a final concentration of 10 muCi/ml. The cells were then washed three times with cold phosphate-buffered saline (PBS) and four times with cold 0.1 N perchloric acid solution. After the addition of 0.2 ml of 0.1 NaOH/0.1% sodium dodecyl sulfate, the radioactivity of the cells was measured after being dissolved in 4 ml of Aquasol-2 (New England Nuclear, Boston, Mass.) in a liquid scintillation counter (Beckman LS7000; Beckman Instruments, Fullerton, Calif.). In each experiment, counts per minute (cpm) were averaged from triplicate wells. We had confirmed that the [³H]thymidine incorporation of fibroblasts which we used in the present experiments was stimulated by mast cell tryptase [22].

Disaccharide analysis of glycosaminoglycans

Fibroblasts were seeded onto a 100 mm² tissue culture dish at a density of 5 x 10⁵ cells per dish and cultured in DMEM supplemented with 10% FCS in the presence of 0.1 mM magnesium salt of L-ascorbic acid phosphate (Asc-2p; Wako Pure Chemical Industries, Ltd., Osaka) at 37° C in a 5% CO₂ humidified air. Asc-2p was added to render fibroblasts to the organization of the dermis-like three-dimensional structure in vitro without any pre-treatment with the plastic dish [23, 24]. The culture medium was changed every three days. After 21 days of incubation, the medium was then replaced with FCS-free medium. After 24 hrs of serum deprivation, some human mast cell chemical mediators were added; histamine (Sigma Chemical Company, St. Louis, Mo) at concentrations of 10^{- 3} and 10^{- 9} M, porcine heparin (Sigma Chemical Company, St. Louis, Mo) at concentrations of 0.1 and 10 mug/ml, carboxypeptidase A (Biogenesis, Ltd., ONF, UK) or cathepsin G (Biogenesis, Ltd., ONF, UK) at 1.0 mug/ml. A serum-free medium was used as the control. After 24 hrs of treatment with the chemical mediator, the medium was aspirated and the cell layer was washed twice with PBS. The cell layers were harvested and rinsed with cold PBS three times. The experiments were conducted in triplicate.

Crude GAGs were isolated by a previously described method [25]. In brief, a sample treated with 2% NaOH overnight at 4° C was neutralized with 0.1 N HCl. The sample was then digested with pronase (Sigma Chemical Company, St. Louis, Mo) at a concentration of 0.2 mg/ml, followed by deproteinization with 10% trichloroacetic acid and centrifuged. The supernatant was dialyzed against running water for 2 days and GAGs were precipitated with 0.1% cetylpyridinium chloride in the presence of 0.012 N sodium sulfate. After centrifugation, the precipitate was washed twice with 95% ethanol saturated with NaCl, and then with pure ethanol, and dried. The crude GAGs were dissolved in water at a concentration of 1 mg/100 mul (w/v) and used for further disaccharide analysis.

Fifty mul of the sample solution were evaporated and digested with chondroitinase-ABC or with chondroitinase-AC II (Seikagaku Kogyo, Tokyo, Japan) as described elsewhere [16]. The precolumn labeling with 1-phenyl-3-methyl-5-pyrasolone (PMP) was performed according to Honda et al. [26]. Samples of commercially available chondroitin sulfate (CS)-, dermatan sulfate (DS)- or hyaluronic acid (HA)-derived disaccharides (Seikagaku Kogyo, Tokyo, Japan) were dissolved in 0.3 M NaOH (20 mul). An equal volume of 0.5 M PMP in methanol was added to the solution and the mixture was kept at 70° C for 30 min. Xylose was added as an internal standard. After PMP labeling, 60 mul of 0.1 M HCl was added for neutralization and vortexed. After a double extraction with 50 mul of chloroform, the aqueous layer was evaporated, dissolved in 100 mul of water, and an aliquot was applied to a high-performance liquid chromatography (Model L-6200, Hitachi, Japan) equipped with a Chemco 3C18 column (6 x 100 mm). Elution was performed with a linear gradient of acetonitrile/water (1:3 v/v) in water and 10% of 200 mM phosphate buffer, pH 7.5 containing 5% acetonitrile at a flow rate of 1 ml/min at 50° C. Peaks were detected at 245nm. 4,5-unsaturated hexuronic acid (DELTAUA)-6-sulphated N-acetylgalactosamine [DELTADi-6S], DELTAUA-4-sulphated N-acetylgalactosamine (a subset of CS) [DELTADi-4S (CS)], the main disaccharide unit of chondroitin sulfate A, and DELTAUA-N-acetylglucosamine [DELTADi-HA] were determined by unsaturated disaccharides liberated with chondroitinase AC digestion. DELTAUA-4-sulphated N-acetylgalactosamine (a subset of DS) [DELTADi-4S (DS)] was calculated by deducting the above DELTADi-4S (CS) from the total DELTADi-4S liberated with chondroitinase ABC digestion.

Statistical analysis

Data on [³H]thymidine incorporation and procollagen type I C-peptide concentration were expressed as mean ± SEM. Statistical analysis of these experiments was performed using Statview program (version 4.0; Abacus Concepts, Berkeley, CA, USA) software. The group data were analyzed by variance testing to determine the overall impact of sample treatments within the experiment. Additional post hoc testing by using the Fisher Protected Least Significant Difference (PLSD) test was carried out to determine the statistical significance of individual sample treatments on the parameters in question. The result of analysis of variance is reported as significant only if both the analysis of variance and the Fisher PLSD tests yielded a probability (P) value of 0.05 or lower. Data of the effects of chemical mediators on human fibroblast-derived GAGs were expressed as mean ± SD. Statistical analysis of these experiments was performed with the student's t-test by DA test for Statistics Analysis (Shinkokoueki Pub., Tokyo, Japan).

Results

Effect of IgE-activated mast cells on fibroblast proliferation

Our preliminary studies confirmed that histamine release from IgE-sensitized cultured mast cells was dose-dependent upon added anti-IgE as previously reported [12], and significant histamine release from the cells was observed in response to anti-IgE with an optimal concentration of 10 g/ml (31.5 ± 8.8%, mean ± SEM, n = 3). Therefore, 10 mug/ml of anti-IgE was used in the present experiments. When fibroblast proliferation was measured in the presence or absence of mast cells, peak [³H]thymidine incorporation was seen between 24 and 48 hrs (data not shown). Accordingly, fibroblast proliferation in cultures or co-cultures was measured 24 hrs after the addition of anti-IgE.

The proliferative effect of IgE-sensitized mast cells without anti-IgE activation was assessed (Fig. 1). Anti-IgE alone did not influence the proliferation of fibroblasts in the absence of sensitized mast cells. A low number of mast cells (2 x 10~6 x 10⁴ cells/well) did not alter [³H]thymidine incorporation. However, a high number of mast cells (2 x 10⁵ and 6 x 10⁵ cells/well) significantly increased [³H]thymidine incorporation as compared with the control (p < 0.01 at 2 x 10⁵ and 6 x 10⁵ cells/well).

On the other hand, IgE-sensitized mast cells at 2 x 10⁴, 6 x 10⁴ or 2 x 10⁵ cells/well, when activated by 10 mug/ml anti-IgE, significantly enhanced [³H]thymidine incorporation as compared with the control (p < 0.05 at 2 x 10⁴ cells/ml and p < 0.01 at 6 x 10⁴ and 2 x 10⁵ cells/well). In the presence of mast cells at 6 x 10⁴ cells/well, incorporation without anti-IgE activation was significantly lower than with IgE activation. However, [³H]thymidine incorporation returned to levels similar to control in the presence of IgE-stimulated mast cells at 6 x 10⁵ cells/well (Fig. 1).

Therefore, we observed cells under phase contrast microscopy. It is of note that in the presence of activated mast cells at 6 x 10⁵ cells/well, the fibroblast cell layer was destroyed, in contrast to an intact cell layer in the presence of the same number of mast cells without activation (Fig. 2).

Effect of IgE-activated mast cells on type I collagen production

When type I collagen production by fibroblasts was measured in the presence or absence of mast cells, peak concentration of procollagen type I C-peptide was seen between 24 and 36 hrs (data not shown). Accordingly, type I collagen production by fibroblasts in cultures or co-cultures was measured 24 hrs after the addition of anti-IgE. IgE-sensitized mast cells without anti-IgE activation did not affect the procollagen type I C-peptide concentration in the culture supernatants of fibroblasts as compared with the control. Anti-IgE activated mast cells also failed to affect collagen synthesis (Fig. 3).

Effect of chemical mediators of mast cells on glycosaminoglycan synthesis

The effect of chemical mediators such as histamine, heparin, carboxypeptidase A or cathepsin G on GAG synthesis was examined. The total amount of main disaccharide units in the cell layer was significantly increased by histamine at concentrations of 10 ^-9 and 10 ^-3 mol/l (p < 0.05). The amounts were 14.65 ± 2.72, 21.84 ± 3.88 and 29.73 ± 6.65 nmol/dish at 0, 10 ^-9 and 10 ^-3 mol/l, respectively. In particular, the increased amount of DELTADi-HA was obvious at 10 ^-3 mol/l. The amounts of DELTADi-HA were 8.99 ± 0.69 nmol/dish without histamine and 23.52 ± 5.01 nmol/dish with 10 ^-3 M of histamine, respectively (p < 0.05, mean ± SD), while there was no significant difference in other disaccharide units (Fig. 4). Heparin did not affect the total or individual amounts of main disaccharide units in the cell layer as compared with the control culture. Since carboxypeptidase A and cathepsin G at 1.0 mug/ml destroyed the three-dimensional fibroblast layer, we could not analyze the disaccharide units.

Discussion

The human mast cell contains a wide spectrum of biologically active compounds such as histamine, heparin, carboxypeptidase A, cathepsin G, tryptase, chymase and other mediators [27, 28]. When activated in an IgE-mediated response, mast cells may release these compounds which exhibit both fibrogenic and fibrolytic activities against fibroblasts. For example, histamine and tryptase are mitogenic for fibroblasts [27, 29] and tryptase enhances the expression of type I collagen messenger RNA in human lung fibroblasts [30]. In normal and reconstituted mast cell-deficient mice (W/W^v), it was demonstrated that the enhanced expression of type I collagen messenger RNA was only observed in normal mice after IgE-dependent mast cell activation [31]. Although the most abundant product of the human mast cell is tryptase, its pathophysiological role in connective tissue metabolism is not well defined. Recently, we reported that histamine, carboxypeptidase A, prostaglandin D₂ and tryptase increased proliferation of human fibroblasts and leukotriene D₄ and tryptase increased type I collagen production [22, 32, 33]. However, we reported that cathepsin G cleaved type I procollagen [33]. Although chymase has been reported to increased proliferation of human fibroblasts, it is also reported to cleave type I procollagen [34, 35]. Some mast cell-derived mediators have opposite effects on type I collagen synthesis by human fibroblasts. The present results suggest that although IgE-mediated human mast cell activation may stimulate human fibroblast proliferation, it does not stimulate type I collagen synthesis. Although the mechanism is not unknown, this stimulatory effect was canceled in co-cultures with a higher number of IgE-activated mast cells.

Increased amounts of GAGs in SSc have been reported in previous studies [36, 37]. The increase in DELTADi-4S (DS) and the decrease in DELTADi-HA in sclerotic skin is characteristic of SSc [16]. In the present study, we were not able to perform the same analysis in the three-dimensional fibroblast layer cocultured with IgE-mediated activated mast cells. This is because a large number of activated mast cells destroyed the fibroblast layer. Therefore, we attempted to determine which of the mast cell mediators acts as a cleaving factor in the three-dimensional culture. The three-dimensional culture system is useful in investigating the glycosaminoglycan metabolism of human dermal fibroblasts in vitro [23, 24]. The total amount of main disaccharide units increased significantly in the cell layer in the presence of histamine, and the increased disaccharide unit was mostly DELTADi-HA. No changes were seen in DELTADi-4S (DS). The presence of heparin generated no effects on the total or proportion of each main disaccharide unit in the cell layer. Akimoto et al. [38] investigated the phenotypic distribution and the degranulation of mast cells in the dermis of patients with systemic sclerosis, and concluded that mast cells were involved in the early edematous phase, but not in the sclerotic phase. It is reported that an increased amount of hyaluronic acid has been demonstrated in skin GAGs of patients having edematous lesions such as scleredema [39, 40]. Although the mechanism by which histamine enhances hyaluronic acid synthesis by human fibroblasts is still unknown, the present finding seems to agree with the fact that histamine effects the increased hyaluronic acid levels in bronchoalveolar lavage fluid after histamine inhalation [41]. Taking these findings into consideration, there is a possibility that histamine, the main mediator of human mast cells, may be responsible for the altered disaccharide unit composition in the early stage of SSc. On the other hand, the reasons why carboxypeptidase A and cathepsin G destroyed the three-dimentional fibroblast layer remains unclear.

It is still unclear whether or not an immediate type of mast cell activation is involved in fibrotic conditions. Human mast cells may have not only fibrogenic but also fibrolytic effects on human dermal fibrosis. It was reported that mediators of mast cells affect connective tissue degradation and initiate extracellular matrix catabolic activity [42, 43]. In fact, in rheumatoid arthritic lesions, mast cells stimulate not only synoviocytes and chondrocytes to synthesize metalloproteinases (MMP) but also lymphocytes to modulate the inflammatory cycle [42, 44]. In fibrotic disorders, it is clear that other factors, such as cytokines, matrix proteinases or its inhibitors influence dermal fibrosis and studying their effect on fibroblasts may lead to new therapeutic strategies in the future [45, 46]. Thus, these factors may be one of the determinants in the advance of tissue fibrosis or restoration to normal tissue.

CONCLUSION

In conclusion, it seems that other factors in addition to mast cells are important in development of human tissue fibrosis or sclerosis. Further studies are required to determine whether or not human mast cells promote the development of various fibrosclerotic conditions.

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