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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To describe the cellular source of transforming growth factor β (TGFβ) in the dermis of patients with systemic sclerosis (SSc).

Methods

We performed electron microscopy (EM) with immunogold labeling on skin biopsy specimens from 7 patients with SSc and 3 healthy control subjects. For TGFβ quantification, the numbers of gold particles per square micron were calculated. The origin of mast cells was confirmed and quantified by toluidine blue staining and light microscopy. Degranulation was assessed on toluidine blue–stained sections and on EM images.

Results

In all patients, active TGFβ was observed uniquely in mast cell vesicles, some of which were released into the extracellular space. Patients with progressive SSc and a more recent onset of non–Raynaud's phenomenon symptoms had higher numbers of mast cells and gold particles per mast cell. Mast cells from healthy control subjects also contained active TGFβ but, in contrast to SSc samples, showed a resting character with no or low-level degranulation and uniformly dense osmiophilic vesicles.

Conclusion

Degranulation of skin mast cells can be an important mechanism of TGFβ secretion in SSc.

Systemic sclerosis (SSc) is a heterogeneous connective tissue disease presenting as low-grade inflammation and vasculopathy, which together lead to fibrosis of the skin and internal organs. Transforming growth factor β (TGFβ) is a key player in fibrosis and has shown to be of paramount importance in SSc (1). It has been convincingly demonstrated that TGFβ contributes to fibrosis via the activation of increased levels of TGFβ receptors on fibroblasts (2), leading to activation of the Smad pathway and stimulation of fibroblasts to express collagen or differentiate into myofibroblasts (3). Several cell lines have been reported to express or store TGFβ in SSc. Notably, fibroblasts, endothelial cells, thrombocytes, monocytes, and macrophages are considered to synthesize TGFβ (4). In SSc dermis, the expression of TGFβ is most prominent around vessels. It is associated with infiltrating mononuclear cells, and it colocalizes with type I collagen expression (5–7). However, which of these cells are mainly responsible for TGFβ expression in SSc has not yet been demonstrated.

Mast cells are vesicle-containing secretory cells that reside in connective tissue, notably in skin, the respiratory system, and the gastrointestinal tract. Activation such as that which occurs in the setting of hypersensitivity or anaphylaxis leads to the release of various tissue mediators, including vasoactive amines, proteinases, and TGFβ (8, 9). Their proximity to fibroblasts makes mast cell products available to fibroblasts and stimulates them to produce collagen (4, 9).

In SSc, the number of mast cells is increased in both involved and uninvolved skin, but the number of degranulated mast cells is increased only in involved skin (10). In this study, we used electron microscopy (EM) with immunogold labeling to demonstrate that mast cells are a major source of TGFβ in patients with SSc.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Four-millimeter punch biopsy specimens of involved skin on the forearm were obtained from 7 patients with SSc (4 with diffuse cutaneous SSc and 3 with limited cutaneous SSc), all of whom fulfilled the American College of Rheumatology criteria for SSc (11). The disease course was categorized as improving, stable, or progressive depending on the change in skin thickening and/or organ function in the year preceding skin sampling. Control skin samples were obtained from the forearm of 1 healthy subject and from the breasts of 2 healthy individuals undergoing breast reduction surgery. Ethics approval and written consent were obtained before the intervention.

The skin biopsy specimens were fixed in 1% paraformaldehyde and embedded in LR White resin. For immunostaining, we used a monoclonal mouse antiserum reactive against the active human TGFβ1 and TGFβ2 isoforms (R&D Systems) and a gold-labeled secondary antibody. Staining with immunogold-labeled secondary antibody without primary antibody served as a negative control in each experiment (Figure 1). Sections were examined on a Philips CM100 transmission electron microscope.

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Figure 1. A, Immunoelectron microscopy with anti–transforming growth factor β (anti-TGFβ) antiserum and secondary gold-labeled antibody. B, The same experiment without primary antibody. Staining with immunogold-labeled secondary antibody without primary antibody served as a negative control in each experiment.

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For quantification, we calculated the mean number of gold particles per square micron in all detected mast cells in each sample. Light microscopy on sections stained with toluidine blue (Sigma) was used to confirm the nature of mast cells. To determine the prevalence of mast cells, we counted the number of toluidine blue–positive cells per 100× magnification field. Degranulation was assessed by both toluidine blue staining and EM and was scored in a blinded manner as absent (−), mild (+), moderate (++), or extensive (+++).

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Active TGFβ was uniquely detected in mast cells. Figure 2A shows a mast cell in close proximity to a fibroblast and a proliferating vessel. A higher-magnification view (Figure 1B) showed that TGFβ was abundant in the vesicles of this mast cell. Some TGFβ-containing vesicles were released into the extracellular space (Figure 1C). To confirm that these were mast cells, serial sections were stained with toluidine blue and examined with light microscopy or processed for EM, respectively (Figures 1D and E). No other cells from the patients, notably lymphocytes, fibroblasts, or macrophages, were positive for TGFβ.

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Figure 2. Immunoelectron microscopic and histologic assessment of the dermis of patients with systemic sclerosis. A, Mast cell with typical morphology in close proximity to a fibroblast (arrow) and a proliferating vessel (arrowhead). B, Higher-magnification view of boxed area in A, showing abundant gold particles in the mast cell vesicles. C, Release of transforming growth factor β into the extracellular space by mast cell degranulation. The right panel shows a higher-magnification view of the boxed area in the left panel. D and E, Confirmation of the nature of mast cells (arrows) in serial sections stained by electron microscopy (D) and toluidine blue staining for light microscopy (E).

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Mast cells were more prevalent (≥4 per visual field) in patients with progressive disease and a more recent onset of SSc (duration ≤3 years) (Table 1). The 2 patients with the highest modified Rodnan skin thickness score (12) also had the highest numbers of mast cells, with up to 6 toluidine blue–stained cells in a single field (Figure 3). Patients with stable or improving disease had fewer mast cells (≤3 per visual field).

Table 1. Patient characteristics, mast cell numbers, and TGFβ labeling per mast cell*
Patient/age/sexDisease durationSSc subtypeMRSSImmunosuppressive treatmentDisease courseMaximum no. of mast cells/100× magnification fieldNo. of gold particles/μ2Mast cell degranulation
  • *

    Mast cells were quantified as toluidine blue–positive cells. Gold-labeled transforming growth factor β (TGFβ) particles were counted in all identified mast cells per biopsy specimen. SSc = systemic sclerosis; MRSS = modified Rodnan skin thickness score; RA = rheumatoid arthritis; HSCT = hematopoietic stem cell transplantation.

  • Number of years since the first non–Raynaud's phenomenon symptom.

  • Assessed by toluidine blue staining and electron microscopy and scored as absent (−), mild (+), moderate (++), or extensive (+++).

1/76/M3Diffuse35CyclophosphamideProgressive611.7++
2/58/F2Diffuse30MethotrexateProgressive69.1+++
3/55/F3Limited9Progressive49.8++
4/46/F3Limited8Stable37.8++
5/57/M10Diffuse20CyclophosphamideStable26.0
6/63/F12Limited, RA overlap9Stable46.9+
7/57/F4Diffuse28Autologous HSCTImproving11.5
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Figure 3. Dermal mast cell infiltration in skin sections obtained from systemic sclerosis (SSc) patients 1–7 (see Table 1) and a healthy control (HC), as shown by positive toluidine blue staining (arrows). Patients 1, 2, and 3, who had a progressive disease course, had higher numbers of mast cells in the dermis. Original magnification × 100.

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The number of immunogold-labeled TGFβ molecules per square micron in mast cells was also higher in patients with a progressive disease course compared to that in patients with a stable disease course. One patient, who had a relatively high Rodnan skin thickness score, had successfully been treated with hematopoietic stem cell transplantation prior to skin sampling; this patient had only low numbers of mast cells and gold particles per mast cell. We observed mast cell degranulation in 5 of 7 patients. One patient had diffuse scattering of TGFβ-containing vesicles throughout the dermis, 3 patients had moderate degranulation, and the other 3 patients had mild or no degranulation.

Mast cells in all 3 samples from healthy control subjects also contained TGFβ confined to vesicles, with a mean of 19 immunogold-labeled TGFβ molecules per cell. However, 2 samples from healthy control subjects had 2 dermal mast cells per field with mild degranulation, and 1 healthy individual had a maximum of 5 mast cells per field without degranulation. With standard EM, we observed heterogeneity in mast cell vesicle density in the SSc samples compared to a more homogeneous pattern of osmiophilic structures in samples from healthy control subjects (Figure 4). Some SSc mast cell vesicles were fused in the form of a “tunnel.” These findings reflect an active state of mast cells in SSc skin.

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Figure 4. Standard electron microscopy images showing mast cell vesicle morphology. A, Mast cells from healthy individuals show uniformly dense osmiophilic vesicles, whereas the vesicle density in samples from patients with systemic sclerosis (SSc) is more heterogeneic (asterisks), indicating higher mast cell activity. B, High-magnification view of mast cell vesicles in SSc samples shows fusion of vesicle membranes with each other and with the cell membrane (arrows).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

In this study, we identified degranulating mast cells as a major source of active TGFβ in the skin of patients with SSc. The number of mast cells was higher, and the amount of detectable TGFβ in mast cells was greater in patients with more recent onset of disease and in those with progressive disease, respectively. This suggests that the release of TGFβ by mast cells and trafficking in vesicles could be of importance in the pathogenesis of SSc, e.g., through inflammatory cell attraction or fibroblast stimulation. Functional analyses, however, will be necessary to confirm the profibrotic role of mast cells in SSc. Furthermore, we cannot exclude the possibility that EM is not sensitive enough to detect lower levels of TGFβ when it is expressed in other cells. It is possible that TGFβ anchored in the latent TGFβ complex could not be detected in these experiments because of a masked epitope.

Nonetheless, our analysis demonstrated that degranulating mast cells are a source of TGFβ in SSc and thus might actively contribute to fibrosis by activation of fibroblasts. Our observations are consistent with findings in other fibrotic diseases in which mast cells promote collagen deposition and represent a potential therapeutic target (13, 14). As expected, physiologic storage of TGFβ in the vesicles of resting mast cells from healthy individuals was observed. In contrast, ongoing degranulation, a higher number of mast cells, and mast cell activity reflected by the heterogenic vesicle coloration and fusion of vesicles indicate higher TGFβ turnover by mast cells in patients with SSc.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Hügle had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Hügle, Hogan, van Laar.

Acquisition of data. Hügle, White.

Analysis and interpretation of data. Hügle, Hogan, White, van Laar.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
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