RESULTS
Digital dermal accumulation of mast cell in SSc patients
We compared the density of dermal mast cells in the fingers and forearms in 46 SSc patients, 29 non-SSc patients, and 10 normal controls by digital tissue pathology using NanoZoomer Digital Pathology. The density of dermal mast cells, detected by toluidine blue staining, were significantly higher in SSc patients [both forearms (24.0±9.0/mm2) and fingers (22.4±8.2 /mm2)] compared with non-SSc patients (12.9±6.9 /mm2) and normal controls (5.5±3.1 /mm2) (p＜0.001, each, Figure 1A). The digital and forearm dermal mast cell densities in the SSc group were comparable. The results showed accumulation of dermal mast cells in fingers, and that digital toluidine blue-positive mast cell density is comparable to that in the forearm in SSc patients.
The c-Kit detects a receptor for stem cell factor expressed in mature mast cells, hematopoietic stem cells and germ cells (cite a reference here). The use of the same anti-c-Kit antibody confirmed the findings of the toluidine blue analysis, i.e., accumulation of dermal mast cells in the fingers and forearms of the SSc group, relative to those detected in the control (18.0±11.9/mm2) and non-SSc groups (6.5±3.0/mm2), and similar to the density of these cells in the fingers (29.2±11.3 /mm2) and forearms (27.0±10.1/mm2) (p＜0.001, each, Figure 1B). The density of mast cells identified by toluidine blue staining correlated strongly with that detected by c-Kit-staining (Figure 1C) (total SSc group: R2=0.708, p＜0.001; SSc group (fingers): R2=0.615, p＜0.001; SSc group (forearms): R2=0.823, p＜0.001). These results suggest that c-Kit immunostaining is comparable to toluidine blue staining in identifying dermal mast cells.
Figure 2 shows serial sections of the dermis in a representative patient with lcSSc (mRSS 8) (Figure 2A-F), patient with non-SSc (Figure 2G-I), and a healthy individual (Figure 2J-L). Hematoxylin-eosin (H&E) staining showed deposition of collagen fibers in the dermis in the fingers of lcSSc, compared with the healthy individual (Figure 2A, J). The forearms of the lcSSc patient also showed homogeneous collagen fibers and perivascular and interstitial edema (Figure 2D). No morphological difference was noted in dermal mast cells between the fingers and forearms. In the skin tissues of the normal control and non-SSc patient (rheumatoid arthritis complicated with cutaneous vasculitis), no collagen fiber hyperplasia was observed (Figure 2G, L). The density of mast cells was 37.0/mm2 in toluidine blue staining (Figure 2E) and 40.3/mm2 in c-Kit immunostaining in the forearms of the lcSSc patient (Figure 2F), which were comparable to those in the fingers of the same patient (Figure 2B, C) but more than those in the non-SSc patient (Figure 2H and I) and normal individual (Figure 2K and L).
It is noteworthy that more than half of the digital mast cells of lcSSc were degranulated (data not shown). Comparable findings were noted in staining for tryptase. In skin tissues of the normal control and non-SSc patient, only a marginal number of mast cells were observed (Figure 2I, L). Similar results were obtained in immunostaining for tryptase and chymase, specific serine proteases of mast cells (data not shown).
Digital dermal mast cell accumulation in dcSSc group and anti-topoisomerase I antibody-positive group
In the next step, we assessed the density of mast cells in fingers and forearms in several lcSSc and dcSSc patients and analyzed the relevance of mast cell skin accumulation to SSc, SSc disease type and serum autoantibodies. The digital dermal mast cell density of the dcSSc group (28.3±9.0/mm2) was significantly higher than that of the lcSSc group (20.8±7.7/mm2, p＜0.05, Figure 3A). However, no significant difference was noted in the density of these cells found in the forearm dermis between the two groups.
Digital dermal mast cell density was significantly higher in the anti-topoisomerase I antibody-positive group (32.4±7.5/mm2) compared with that of the negative group (21.1 ±7.6/mm2, p＜0.05, Figure 3B). However, there was no significant difference in the forearm dermis between the two groups. On the other hand, no significant difference was noted between digital and forearm dermal mast cell densities, irrespective of anti-centromere antibody (Figure 3C) (fingers: 21.0±6.9/mm2 vs. 24.7±9.5/mm2, p=0.124, forearms: 23.4±4.4/mm2 vs. 21.5±9.5/mm2, p=0.399). These results indicated that density of mast cells in fingers, but not in forearm, correlates with the incidence of dcSSc as well as anti-topoisomerase I antibody.
Dermal mast cell density in fingers correlates with mRSS
Because digital dermal mast cell density was higher in patients positive for anti-topoisomerase I antibody, we considered that digital dermal mast cell density reflects the severity of SSc. In fact, digital dermal mast cell density correlated significantly with mRSS, which is used for clinical evaluation of skin thickness, in 17 body surface areas, whereas that in the forearm was not (Figure 4A). Although digital and forearm dermal mast cell densities did not correlate significantly with the local mRSS, the skin score for the fingers and forearms, that of fingers, but not forearms, tended to increase with the local mRSS score of the fingers (Figure 4B).
Furthermore, digital dermal mast cell density tended to correlate negatively but significantly with disease duration (r=-0.335, p=0.031, Figure 4C), whereas mast cell density in the forearms did not (r=0.070, p=0.661, data not shown), suggesting the possible involvement of digital dermal mast cell density in the pathological process of SSc at a relatively early stage of the disease process. Interestingly, accumulation of mast cells was specially observed in fingers of patients with mRSS score of ＜5 (n=10), indicating that digital dermal mast cells could be involved in the initial stage of the pathological processes in SSc.
The above results suggest the involvement of mast cells in the pathological process in SSc and that the density of dermal mast cells in the fingers, the site of appearance of the initial symptoms, may serve as an indicator of the severity of systemic skin sclerosis in SSc.