Expression of basic fibroblast growth factor and its receptor by fibroblast, macrophages and mast cells in hypertrophic scar

ARTICLE

Basic fibroblast growth factor (bFGF) is a multifunctional polypeptide that promotes growth and differentiation of a broad spectrum of cell types [1, 2]. Since bFGF is a potent mitogenic and chemotactic factor for endothelial cells and fibroblasts, it has been implicated in the wound healing process [3-6]. Recent studies have directly focused on fibroproliferative disorders such as Dupuytren's contracture, pulmonary fibrosis and carbon tetrachloride-induced hepatic fibrosis [7-9].

Hypertrophic scar (HS) and keloid can be defined as an abnormal wound healing process characterized by excessive accumulation of collagen and proliferation of fibroblasts [10]. Tan et al. [11] demonstrated that keloid-derived fibroblasts responded to bFGF similarly to those from normal skin in the production of proteoglycan and collagen.

bFGF is reported to be stored in basement membrane in vivo [12]. However, the mechanism of bFGF secretion is not fully understood because bFGF lacks signal peptide sequence [13-16]. The cellular source of bFGF is also a matter of controversy. Many investigators believe that major cellular sources of tissue bFGF are monocytes/macrophages [17-20]. Some authors [21-23], however, nominated mast cells (MCs), indicating the bFGF release by degranulation of MCs [21]. The local proliferation of MCs in HS or keloid has been reported [24]. To our knowledge, there has been no report on the expression of bFGF and bFGF-R in HS or keloid.

To investigate the role of bFGF in HS or keloid formation in vivo, we examined the localization of bFGF and bFGF receptor (bFGF-R) expressed by macrophages and MCs as well as fibroblasts, with a double immunostaining method. As we observed extraordinary immunoreactivity to bFGF and bFGF-R by the fibroblasts in HS or keloid, we also applied the same staining to cultured fibroblasts.

Materials and methods

Selection of patients

After informed consent was obtained, skin biopsy was performed from the lesional skin of 10 patients with scars (40.7 ± 23.7 years old, mean age ± SD). The distinction between keloid and hypertrophic scar is not universally accepted [10]. We classified the scar tissues into two categories based on the histopathological features according to Linares et al. [25]; immature hypertrophic scar in which nodules or whorl-like collagen bundles were observed as pathological findings (hypertrophic scar, HS, n = 5), and semimature or mature scar from hypertrophic or non-hypertrophic healing in which collagens showed parallel banding (non-HS, n = 5). Disease duration ranged from 4 months to 18 years (6.5 ± 7.5 years) in HS and from 3 years to 20 years (10.6 ± 7.0 years) in non-HS.

No patient took any medication at the time of biopsy. The location of the scars was variable. For comparison, skin specimens were obtained from 5 healthy volunteers (45.2 ± 20 years old).

Sample processing

Skin samples were cut into a few pieces. One piece was fixed in 3.7% neutral formaldehyde solution and processed for staining with hematoxylin-eosin. Another one was snap-frozen in liquid nitrogen and stored at ­ 80° C until use. Six-µm thick frozen sections were prepared with a microtome (Tissue Teck II Cryostat, Miles Inc., USA) and fixed in cold acetone for 7 min.

Fibroblast culture

Fibroblasts were obtained from HS (n = 3), non-HS (n = 2) and normal skin (n = 2) by explant culture method. Fibroblasts were grown in Dulbecco's modified Eagles medium supplemented with 10% fetal bovine serum in a humidified atmosphere in 5% CO₂ at 37° C. For immunohistochemistry, cells were cultured on multispot glass slides (Toyobo Engineering, Japan) for several days. Subconfluent cells were washed with phosphate buffered saline (PBS), allowed to air dry, fixed in ice-cold acetone, and subjected to immunostaining. Fibroblasts at 3rd or 4th passage were used in the present study.

Antibodies

The rabbit polyclonal anti-human bFGF antibody (BT-583) was purchased from Biomedical Technologies Inc. (MA, USA). Mouse IgM type anti-human bFGF-R monoclonal antibody (MAB 125) was purchased from Chemicon International Inc. (USA). Mouse monoclonal anti-CD68 antibody (EBM11, Dako, Denmark) or mouse monoclonal anti-tryptase antibody (Chemicon International Inc., USA) was used as a marker for macrophages or MCs respectively. Primary antibody to bFGF or bFGF-R was diluted at 1:50 or 1:150, respectively. Anti-CD68 antibody or anti-tryptase antibody was diluted at 1:100 or 1:200, respectively.

Immunohistochemical studies

Immunostaining was performed with three-step avidin-biotin complex (ABC) immunoperoxidase method (LSAB kit, Dako, CA, USA) according to the manufacturer's instruction, except that the second antibody was replaced with biotinylated anti-rabbit or anti-mouse IgM antibody. Color was developed with 3-amino-9-ethylcarbazole (AEC) as chromogen. Tissue sections were lightly counterstained with methyl green (Muto Pure Chemicals Ltd., Japan) and subsequently mounted in 90% glycerol.

To investigate the association of bFGF/bFGF-R with macrophages and MCs, double immunohistochemical staining was performed. For double immmunostaining, the sections were first incubated with anti-bFGF or anti-bFGF-R overnight at 4° C followed by the ABC immunoperoxidase technique as described above. The sections were then incubated with anti-CD68 or anti-tryptase antibody overnight at 4° C followed by incubation with alkaline phosphatase-conjugated anti-mouse IgG. Alkaline phosphatase activity was visualized by incubation with Fast blue RR and naphthol AS-TR solution (Vector Blue, Vector Lab Inc., CA, USA) containing levamisole (0.24 mg/ml) to inhibit endogenous alkaline phosphatase activity.

At least three tissue specimens from each skin sample were examined for each antibody.

Cell counting

The double immunostained specimens were serially photographed with Nikon photomicroscope EIM and EFM (x 264 magnification) to count positive cells. Blue colored cells were considered to be macrophages or MCs. Among the blue colored cells, the cells which also had a reddish brown color were considered to be bFGF or bFGF-R positive macrophages or MCs. We calculated the percentages of (bFGF⁺ and CD68⁺ cells)/CD68⁺ cells, (bFGF-R⁺ and CD68⁺ cells)/CD68⁺ cells, (bFGF⁺ and tryptase⁺ cells)/tryptase⁺ cells, and (bFGF-R⁺ and tryptase⁺ cells)/tryptase⁺ cells. For example, the number of macrophages (CD68⁺ cells) expressing bFGF (bFGF⁺ cells) were divided by the total number of macrophages(CD68⁺ cells) and multiplied by 100.

Statistical analysis

Statistical analysis was performed with unpaired Student's t-test.

Results

Immunolocalization of bFGF/bFGF-R in normal skin

In normal skin samples, bFGF and bFGF-R were expressed by the cytoplasms of keratinocytes, some of the sweat and sebaceous gland cells, hair follicle cells, endothelial cells and a few unidentified cells around the vessels in the upper dermis (Fig. 1a, b).

Immunolocalization of bFGF/bFGF-R in scar tissue

Beside the similar staining found in normal skin, cytoplasmic bFGF immunoreactivity was found in many spindle cells and some round cells between collagen bundles in HS (Fig. 2a). Although bFGF-R exhibited a similar distribution to bFGF, bFGF-R positive cells were distributed much more widely than bFGF positive cells (Fig. 2b). Although intensively positive cells for bFGF or bFGF-R were occasionally found in some specimens of non-HS, their number was obviously smaller than that in HS (data not shown).

Double immunohistochemical staining of anti-bFGF/bFGF-R
antibody with anti-CD68/tryptase antibody

In HS, most of the positive cells for bFGF or bFGF-R between collagen bundles were regarded fibroblasts because they were negative for both CD68 and tryptase (Fig. 2c, d, e).

In HS, the percentage of bFGF positive macrophages was 54.8 ± 25.3 (mean ± SD)%, which was significantly higher in than normal skin (7.6 ± 10.8%, p < 0.005). The percentage of bFGF positive MCs was not different from normal skin (15.5 ± 8.8% vs 9.8 ± 3.8%). With regard to bFGF-R, positive rates both of macrophages and MCs were not significantly different from normal skin (51.7 ± 11.6% vs 37.3 ± 20.4% and 18.7 ± 19.3% vs 9.2 ± 9.1%, respectively). In non-HS, the percentages of the labeled cells with bFGF and bFGF-R in macrophages and MCs did not significantly differ from those of normal skin. The percentage of bFGF-R positive macrophages in non-HS was lower than in normal skin because of the increase of bFGF negative macrophages in non-HS. There was a significant statistical difference in the positive rate to bFGF-R in macrophages between HS and non-HS (Table).

Immunoreactivity to bFGF and bFGF-R in cultured fibroblasts from scar and normal skin

The nuclei of fibroblasts from HS were strongly labeled with bFGF (Fig. 3a). In contrast, fibroblasts from non-HS and normal skin showed faint immunoreactivity to bFGF (Fig. 3b). The cytoplasm of fibroblasts from HS, non-HS and normal skin was similarly labeled with bFGF-R (data not shown).

Discussion

Our results of bFGF and bFGF-R immunolocalization in normal skin were in accordance with the previous observation that they are located in endothelial, epithelial and sebaceous cells [16, 26]. The important findings in the present study are that bFGF and bFGF-R were abnormally expressed by fibroblasts and that bFGF was also highly expressed by macrophages in HS but not in non-HS or normal skin.

Many investigators support the hypothesis that monocytes/macrophages are the major source of tissue bFGF during inflammation and neovascularization. On the other hand, there are some reports that MCs are a main source of tissue bFGF in cutaneous hemangioma, chronic lung disease and pulmonary fibrotic disorders [21-23]. In our study, the expression of bFGF in macrophages was significantly increased but not in MCs, suggesting that macrophages, but not MCs, may be a main cellular source of bFGF in HS. The positive rate for bFGF in macrophages had a wide range in HS. We could find no correlation with disease duration (data not shown).

Morita et al. [27] demonstrated a great number of macrophages expressing bFGF-R in renal tubulointerstitial fibrosis, suggesting two possibilities; macrophages themselves synthesized bFGF-R to be activated, or they phagocytosed bFGF-R. Logan et al. [28] demonstrated the expression of bFGF-R by tissue macrophages after brain injury. In our study, however, macrophages and MCs did not show increased expression of bFGF-R in comparion with normal skin.

It is of note that fibroblasts from HS showed intensive immunoreactivity to bFGF and bFGF-R. Gonzalez et al. [7] demonstrated that bFGF was abnormally expressed by the fibroblasts of Dupuytren's contracture, especially in the fibrous nodular area that is the organizing center of the lesion. Hasebe et al. [29] showed an increased expression of bFGF-R but not of bFGF by fibroblasts in the fibrotic focus of invasive carcinoma. The density of dermal fibroblasts in HS and keloid is higher than in normal skin [10, 30]. Taken together with these reports and our results, it is speculated that fibroblasts in HS are actively proliferating by virtue of the abnormal expression of both bFGF and bFGF-R. The autocrine and paracrine mechanism(s) may work towards their proliferation.

The increased immunoreactivity to bFGF was observed in the nuclei of cultured fibroblasts from HS tissue. Although the intracellular localization of bFGF is a matter of controversy [31], the strong nuclear staining of cultured fibroblasts from HS may be of great interest. This finding can be explained by the concept of the intracrine mechanism [32], in which a growth factor can exert its bioactivity within a cell with no need to be secreted or to bind its receptors on the cell surface. Bouche et al. [33] reported that bFGF would act directly on the nuclei of endothelial cells to regulate the transcription of ribosomal genes.

bFGF-R was equally expressed by cultured fibroblasts from non-HS and normal skin, which contrasts with our in vivo finding. This result suggests that the expression of bFGF-R by cultured fibroblasts may not differ irrespective of their origin. Tan et al. [11] reported that both keloidal and normal fibroblasts equally synthesized type I collagen after bFGF stimulation. Their results may support our speculation. With respect to the discrepancy in bFGF-R expression between in vivo and in vitro, alteration in fibroblast phenotype during the culture process seems to be most likely.

The previous study demonstrated that the expression of bFGF is regulated in the early phase of wound healing [6]. Our observations suggest that the persistent expression of bFGF and bFGF-R may play an important role in the development of HS, in which macrophages may act as cellular sources of bFGF as well as fibroblasts.

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