Enhanced expression of SCF in the dermis is a prognostic factor for the regression of urticaria pigmentosa

ARTICLE

Mastocytosis is a heterogeneous disease characterized by increased numbers of mast cells in various organs. Urticaria pigmentosa (UP) is a disorder of mast cell proliferation that occurs in cutaneous tissue [1]. UP in adults usually progresses gradually until the disease becomes systemic, and rarely develops into a hematological disease. Among children with isolated UP, the symptoms are resolved in at least 50% of the cases by adulthood [1-4]. So far, except for the age of onset, there have been few factors identified as being related to the prognosis of UP.

Recent studies have indicated that stem cell factor (SCF) is one of the most important factors affecting the number, phenotype, and function of connective tissue type mast cells in healthy and diseased individuals. SCF is the ligand for the product of the c-Kit proto-oncogene and it stimulates the growth and differentiation of mast cells from CD34-positive cells [5].

In the present study, in order to elucidate prognostic factors of UP, we examined the correlation between the clinical features and the expression of SCF and c-Kit in the skin of patients with UP.

Materials and methods

Patients (Table I)

Skin biopsy specimens that included subcutaneous fat were obtained from 4 adults (3 males and one female; 17-37 years of age) and 4 infants (3 boys and one girl; one day-4 months of age) with UP using a disposable trepan (Dispopunch, Maruho, Japan). The disease duration was up to 16 years, and biopsy specimens were obtained during the early stages of illness (2 weeks-3 years after onset). All patients were diagnosed on the basis of clinical features and histopathological findings. On the most recent visit of patients, 4 months-13 years after biopsy, lesions were counted and compared with those on the day of biopsy. According to the change in the number of eruptions, patients were classified into improved, no-change and aggravated groups. Solitary mastocytoma, systemic mastositosis, and mast cell leukemia were excluded.

Methods

All samples were routinely fixed in formalin. Tissues were embedded in paraffin and sections 4 mm thick were prepared. Toluidine blue staining for the identification of mast cells was performed. Adjacent sections were stained by the LSAB (labelled streptavidin) technique consistent with the manufacturer's instructions. The following primary antibodies were used: anti-SCF polyclonal antibody that recognizes both soluble and membrane-bound SCF (IBL, JAPAN) and anti-c-Kit polyclonal antibody (DAKO JAPAN, JAPAN). The sections were developed with 3,3'-diaminobenzidine solution as the chromogen. The edges of surgical excisions of nevous cell nevous, from the trunks of 8 healthy donors (4 adults and 4 infants) were used for comparison.

Assessment

Mast cells, identified by the presence of metachromatic granules, were counted on every fifth section of each sample under a light microscope at 400 x magnification in 10 fields. The c-Kit positive cells in adjacent sections were also counted in approximately the same field. The ratio of both cell numbers was used for the estimation of c-Kit positive mast cell number. The expression of SCF was observed in terms of distribution and intensity, but did not permit quantitative assessment, and was described using the following categories: (–), negative; (±), partially positive; (+), positive.

Results

1) Toluidine blue staining: In all cases of adult onset UP, toluidine-blue positive cells were found in small numbers around blood vessels in the dermis (Fig. 1a). In infant cases, toluidine blue-positive cells lay closely packed in aggregates (Fig. 2a). The infiltrate extended throughout the entire dermis. The relationship between age and number of toluidine blue-positive cells is shown in Table I. The number of toluidine blue-positive cells infiltrated in adult cases was significantly lower than that in infant cases, regardless of the prognosis. For all control specimens analysed, means of 4.5 ± 0.6 toluidine-blue positive cells in adults and 15.6 ± 4.8 toluidine-blue positive cells in infants were obtained.

2) Expression of c-Kit: The values of the ratio of the number of toluidine blue-positive mast cells to the number of c-Kit positive cells in adjacent sections were not significantly different regardless of prognosis or age of onset (Table I).

3) Expression of SCF:

a) Epidermis: In patients with adult onset UP, basal keratinocytes were weakly stained in an intercellular pattern (Fig. 1b), whereas the epidermis in infants with UP showed intracellular staining throughout (Fig. 2b). In the control tissue sections of adults and infants, the intercellular spaces of the epidermis were stained very faintly and no difference was found between adults and infants (Figs. 1c and 2c).

b) Dermis: In infant cases that showed a tendency to improve, SCF expression was clearly observed in the entire dermis with extracellular patterns (Fig. 3a), while in adult and infant cases that did not show a tendency to improve, SCF was recognized partially or not at all (Fig. 3b). In the control tissue sections of adults and infants, dermal and subcutaneous fibroblasts reacted with a very faint, granular extracellular staining pattern (Fig. 3c).

Since there might be a relationship between maturation of mast cells and the clinical course of UP, we also used anti tryptase antibody (AA1, DAKO JAPAN, JAPAN) to investigate mast cell differentiation in UP. However, we could not find any difference in tryptase expression, in either improving or worsening UP patients (data not shown).

Discussion

SCF, a growth factor known to influence mast cell proliferation and differentiation [7], has been reported to be produced by dermal fibroblasts [8], keratinocytes [9, 10], and dermal endothelial cells [11], whereas its receptor, the c-Kit proto-oncogene product, is expressed only in mast cells and melanocytes [8].

There are few reports regarding SCF expression in UP. Longley et al. [12] showed increased levels of extracellular SCF in the dermis and epidermal keratinocytes, suggesting that overproduction of soluble SCF is responsible for UP. In contrast, Hamman et al. [13] reported that keratinocytes were stained intracellularly throughout the epidermis of normal and mastocytoma patients, whereas epidermal staining was weaker in all UP sections. Their findings might indicate that SCF is important only during the initial development of the disease, and not during the period of maintenance of mastocytosis lesions.

In this study, we found intracellular SCF expression in the epidermis in infant cases that overlayed dense aggregation of mast cells. This finding suggested that SCF in the keratinocyte might have some relation to significant mast cell hyperplasia, as Hamman et al. demonstrated.

Contrary to Longley's description of SCF expression in UP dermis, we found dermal expression of the soluble-form of SCF only in the improving cases. Though we could not find any difference in expression of tryptase, a mast cell differentiation marker, between improving and aggravating cases, soluble SCF in the dermis might be crucial for the differentiation and disappearance of mast cells in UP lesions and for improvement of the clinical features.

Longley et al. also reported one patient with UP and aggressive systemic mastocytosis with massive splenic involvement. They found a mutation that results in constitutive activation and expression of c-KIT in mast cells of both skin and spleen [14]. In the cases of cutaneous limited UP that we have studied, there was no apparent relationship between c-KIT expression by mast cells and the clinical course of UP. Cutaneous and systemic mastocytosis seem to have a different etiology.

All of our patients have received treatment with antihistamines or PUVA therapy, but the medication applied failed to produce an improvement of the symptoms. Recently, Kurosawa et al. reported that combination therapy with ketotifen and ranitidine decreased the elevated blood and urine levels of mediators in systemic mastocytosis [15]. Since the combination of Histamine H1 and H2 receptor antagonists not only affects histamine receptors but also suppresses mast cell degranulation, this might result in therapeutic benefit in patients with UP as well.

CONCLUSION

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