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 SCF
expression in the dermis might hold the clue to a better understanding of
the remission of UP. Investigation of the relationship between the maturation
of mast cells and fibroblast-derived factors may be a useful approach to
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