ARTICLE
Auteur(s) :, Achiléa
L Bittencourt*, Maria de Fátima Oliveira, Carlos
Brites, Johan Van Weyenbergh, Maria das Graças da Silva Vieira,
Iguaracyra Araújo
Departments of Pathology and Dermatology and Laboratory of
Retrovirology, Hospital Universitário Prof. Edgard Santos, Federal
University of Bahia
Laboratory of Immunoregulation and Microbiology (LIMI),
CPqGM-FIOCRUZ Salvador, Bahia, Brazil
Department of Pathology, Hospital Universitário Prof. Edgard
Santos, Federal University of Bahia, Rua João das Botas s/n, Canela
CEP 40110-060. Salvador, Bahia, Brazil Fax: (+55) 71 3396 335.
accepté le 9 Novembre 2004
Carriers of human T-cell lymphotropic virus type I (HTLV-I) may
develop many diseases such as adult-T cell leukemia/lymphoma (ATL),
an aggressive form of T-cell lymphoma, HTLV-I-associated
myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic
neurological disease and infective dermatitis associated with
HTLV-I (IDH), a severe form of childhood infected eczema [1].IDH
was described in Jamaica in 1966 by Sweet [2] but only in 1990 was
it related to HTLV-I [3]. Most cases have been reported in Jamaica
[3, 4] but some have been reported in Colombia, the Dominican
Republic, Trinidad-Tobago, and among Haitians resident in Miami
[5-8]. In Brazil, one case of IDH was reported in Rio de Janeiro
[9]. In Japan, where the frequency of HTLV-I carriers is high, only
two cases of IDH have been reported [10].IDH is associated with a
non-virulent Staphyloccus aureus or beta-hemolytic Streptococcus
infection of the skin and nasal vestibules, and involves mainly the
scalp, external ear and neck. Conjunctivitis has been observed in
most cases [5]. This disease is considered to be a risk factor for
the development of ATL and HAM/TSP [11-14].Since some infected
children may eventually present other kinds of eczema, it is
important to discriminate between IDH and atopic or seborrheic
dermatitis, with the differential diagnosis being based on clinical
features [5]. The differential diagnosis with seborrheic dermatitis
is a source of concern mainly in puberty [15] but in the majority
of IDH cases the lesions begin earlier.In the present
investigation, we studied the histopathological and
immunohistochemical patterns of 19 patients with IDH in order to
determine if they differ from atopic and seborrheic dermatitis and
if they present histological aspects of cutaneous lymphoma.
Material and methods
Patients
The study group consisted of 19 children, 10 girls and 9 boys, 10
of them mulattos and 9 blacks, all of very low social status. The
age at diagnosis ranged from 2 to 14 years (mean: 8.00 ± 3.63
years) in 18 children and was undefined in one child who had been
abandoned. According to mothers’ information, age at the onset of
symptoms ranged from 2 months to 6 years (1.94 ± 1.74 years) in 18
children. All patients fulfilled the major criteria for the
diagnosis of IDH [5]. Dermatological examination revealed extensive
crusty and erythematous lesions, miliary follicular papules, and
retroauricular fissures. Disseminated scaly and erythematous
papules or plaques were also observed in seven cases. The
preferential sites of involvement were: scalp (100%),
retroauricular areas (100%), neck (87%) and external ears (83%). In
10 cases the disease was more severe, involving all the segments of
the body. Blepharoconjunctivitis was observed in 10/19 cases. The
patients presented mild to moderate pruritus. All were treated with
sulfamethoxazole/trimethoprim, with disappearance or marked
improvement of the lesions but with relapses occurring when the
medication was discontinued. The recurrent lesions were less severe
and more localized. Patient follow-up ranged from 0.5 to 7 years
(median: 3.35 years). In the four cases histologically mimicking
mycosis fungoides (MF), follow-up ranged from 1 to 7 years (median:
3.7 years). No patient presented clinical evidence of lymphoma
during follow-up (Oliveira, personal communication) but five of the
children developed HAM/TSP during follow-up (Chagas, personal
communication) [14].
Serological diagnosis
Antibodies to HTLV-I/II were investigated by diagnostic
enzyme-linked immunosorbent assay (ELISA - Cambridge Biotech,
Worcester, MA, USA) and confirmed with a Western blot capable of
discriminating between HTLV-I and HTLV-II (HTLV Blot 2.4, Genelab,
Singapore). Serologic assays for HIV were also performed. All
patients were serologically positive for HTLV-I and negative to
HTLV-II and HIV.
Histopathological and immunohistochemical studies
Punch skin biopsies were performed in the scalp lesion of all
patients and in one case a biopsy was simultaneously obtained from
a papular lesion of the abdomen. The biopsies were fixed in 10%
buffered formalin, the blocks were embedded in paraffin, and
histological sections were stained with hematoxylin and eosin (HE).
The immunohistochemical study of the inflammatory cells was
performed in paraffin-embedded sections using a panel of antibodies
and a standard streptavidin-biotin-peroxidase technique [16]. The
following immunohistochemical markers were employed: T-cell markers
CD45RO, CD3, CD8 (Dako, Glostrut, Denmark), and CD4 (Novocastra,
New Castle, UK); B-cell markers CD20 and CD79a (Dako) and NK cell
markers CD56 and CD57 (Dako). The immunophosphatase technique
(Streptavidin Biotin System) for identification of cytotoxic
granules was performed using the anti-granzyme B, anti-perforin
(Novocastra) and anti-TIA-1 (Immunotech, Marseille, France)
antibodies. The cell count was made using a semi-quantitative
assessment. The percentage of CD4+, CD8+ and lymphocytes with
cytotoxic granules in the inflammatory infiltrate was calculated by
counts on five high magnification fields (640X). The study was
approved by the Research Ethics Committee of Hospital Professor
Edgard Santos and informed consent was obtained from the children’s
mothers or from the persons responsible for the children.
Flow cytometry
Fifty microliters of whole blood was mixed with an equal volume of
1% BSA plus 0.1% sodium azide in PBS and incubated for 30 min on
ice with antibodies against CD4 and CD8 and the corresponding
isotype controls (Coulter-Immunotech). After incubation,
erythrocytes were lysed using a simultaneous fixation and lysis
solution (Becton-Dickinson). A minimum of 10,000 events per sample
were acquired with a FACSort flow cytometer (Becton-Dickinson) and
analyzed using the CellQuest software.
Results
Hyperkeratosis and/or parakeratosis with crusts and acanthosis of
varying degrees were observed in all cases. The acanthosis was
psoriasiform in six biopsies. Mild spongiosis was observed in 10
cases and spongiosis of moderate degree in one. Focal vacuolar
degeneration of the basal layer and pigmented incontinence were
observed in 16 cases. Five cases presented subcorneal pustules and
seven presented collections of degenerated neutrophils within the
stratum corneum (Munro-like abscesses). Lymphocytic epidermotropism
was mild in 11 cases and moderate in four. Small collections of
typical lymphocytes were seen within the epidermis (Pautrier-like
abscesses) (( figure
1 )) in four cases, associated with a minor degree of
spongiosis or without spongiosis. Focal obliteration of the basal
layer was seen in five cases, being of moderate degree in two of
them (( figure 2
)). A linearly arranged layer of single cells in the epidermal
basal layer was seen in two cases. In the dermis, a mild to
moderate infiltration consisting predominantly of lymphocytes was
observed in 18/19 cases and in one it was of marked degree (( figure 2 )). Plasma
cells, neutrophils and rare eosinophils were also observed. The
pattern of dermal infiltration was lichenoid in 10 biopsies, and
superficial perivascular and periadnexial in the others. In two
biopsies a mid-dermis perivascular infiltration was observed
associated with the superficial perivascular pattern. The biopsy of
the papule presented a moderate lymphocytic infiltration associated
with a moderate epidermotropism of lymphocytes, a small
Pautrier-like abscess and linearly arranged single lymphocytes
along the basal layer of the epidermis. The aspects observed were
similar to those detected in the biopsy from the scalp of the same
patient.
The infiltrate consisted predominantely of CD3+ and UCHL-1+ T
cells in all scalp biopsies and in the papular lesion. In six cases
B cells (CD79a+ and CD20+ cells) were also observed. The percentage
of CD8+ and CD4 + cells varied from 53% to 94% and from 4% to 26%
respectively but in one case the percentage of CD4+ cells was
higher (42%) (table 1( Table 1 )). The
epidermal infiltrating lymphocytes were CD8+ (( figure 3 )). Fifteen of 18
cases were positive for TIA-1 and 5/19 for granzyme B. The
frequency of TIA-1 expression ranged from 0.7% to 15% and the
frequency of granzyme B expression ranged from 0.5% to 3%. CD57+
cells were observed in 17/18 cases, corresponding to 0.6% to 17.5%
of the inflammatory cells. In five cases with basal obliteration,
CD57+ cells were seen at the dermo-epidermal junction (( figure 4 )) or within
the epidermis. No CD56+ or perforin+ cells were observed in the 19
patients studied. As shown in table 1, CD4 and CD8 levels in
peripheral blood were highly variable in the seven patients
evaluated (from 25.3 to 48.0, and 19.2 to 30.8, respectively).
However, CD4/CD8 ratios were within the normal range (1.47 ± 0.11,
n = 7).
Table 1 Amount of CD4+ and CD8+ lymphocytes in blood
and skin lesions
|
Blood CD4+ (%)
|
Blood CD8+ (%)
|
Blood CD4/CD8
|
Skin lesions CD4+ (%)
|
Skin lesions CD8+ (%)
|
|
1
|
48.00
|
25.16
|
1.9
|
11.6
|
86
|
|
2
|
25.32
|
19.28
|
1.31
|
11
|
80
|
|
3
|
-
|
-
|
-
|
10
|
73
|
|
4
|
-
|
-
|
-
|
7
|
53
|
|
5
|
38.85
|
22.34
|
1.74
|
11
|
81
|
|
6
|
|
|
|
4.6
|
90
|
|
7
|
40.87
|
27.06
|
1.51
|
10
|
82
|
|
8
|
-
|
-
|
-
|
5
|
86
|
|
9
|
-
|
-
|
-
|
8
|
88
|
|
10
|
40.48
|
30.56
|
1.32
|
8.5
|
80
|
|
11
|
45.89
|
29.53
|
1.56
|
4
|
94
|
|
12
|
-
|
-
|
-
|
8
|
57
|
|
13
|
-
|
-
|
-
|
14
|
83
|
|
14
|
-
|
-
|
-
|
6.5
|
64
|
|
15
|
-
|
-
|
-
|
6
|
81
|
|
16
|
-
|
-
|
-
|
20
|
78
|
|
17
|
-
|
-
|
-
|
26
|
72.5
|
|
18
|
-
|
-
|
-
|
42
|
56
|
|
19
|
46.09
|
30.88
|
1.49
|
10
|
60
|
Comments
The present study was conducted on children born in Salvador,
Bahia, a city in Northeast Brazil with 1.76% of HTLV-I infection in
the general population [17].
The histopathological evaluation of the 19 skin biopsies
obtained showed a chronic dermatitis similar to the pattern
described for atopic dermatitis, seborrheic dermatitis and IDH in
15 cases [5, 18, 19]. However, architectural aspects mimicking the
patch stage of MF were observed in four cases, i.e., collections of
a few lymphocytes within the epidermis (Pautrier-like abscess),
lymphocytic epidermotropism, and/or basal obliteration by
lymphocytes, sometimes with a linear array of lymphocytes along the
basal layer of the epidermis [20-22]. This pattern was associated
with a moderate or marked lymphocytic infiltration in the
superficial dermis, but no atypical lymphocytes or mitoses were
observed. These features are not generally described in atopic or
seborrheic dermatitis [15, 18, 19].
The diagnosis of the patch stage of MF is frequently difficult.
Some investigators state that this diagnosis can be based
exclusively on architectural aspects [20-22], whereas others
believe that only the presence of atypical cells constitutes a
highly reliable feature for the diagnosis of MF because the
architectural aspects in question can be found in biopsy specimens
of benign simulators of MF [23, 24].
The lesions of the present patients disappeared or showed great
improvement during the use of sulfamethoxazole/trimethoprim and,
after drug withdrawal, recurrent lesions were always less severe
and more localized. The patients with architectural aspects
mimicking MF have been followed-up for one or more years (median:
3.75 years) and their lesions have disappeared or improved, showing
that these are benign simulators of this kind of lymphoma. It is
important to make a correct differential diagnosis because ATL may
present, infrequently, the histopathological pattern of MF [25,
26]. Besides, MF may occasionally occur in childhood and puberty
[27, 28].
In atopic and seborrheic dermatitis, T lymphocytes are
predominantly of helper cell phenotype [29-34]. Most of the present
cases (18/19) showed infiltrates predominantly consisting of CD8+
cells, possibly indicating a different pathogenic mechanism. The
predominance of CD8 cells in the skin biopsies could not be
attributed to an inversion of CD4/CD8 ratios in blood, since in
parallel blood samples from seven cases CD4/CD8 ratios were normal
(1.47 +/– 0.11, n = 7), as shown in table 1.
It is known that granule exocytosis mediated by
perforin/granzyme represents the main pathway of CD4 and CD8 T cell
cytotoxicity in humans [35]. The expression of cytotoxic molecules
TIA-1 and granzyme B in a small percentage of cells and the absence
of perforin-positive cells indicate that the majority of CD8+ cells
were not cytotoxic cells but possibly suppressor cells. TIA-1 was
the most frequent cytotoxic molecule observed in the present cases.
TIA-1 is a cytolytic granule-associated protein that may define a
subpopulation of CD8+ T lymphocytes possessing cytolytic potential
but is not related to activation [36] like granzyme and perforin.
In contrast to atopic dermatitis, activated cytotoxic T cells were
rare or absent in the patients studied [35].
CD57+ cells may represent a T cell subset that increases in some
conditions such as acquired immunodeficiency, rheumatoid arthritis,
and after organ transplantation, but that is rare in children [37].
These cells were seen in the majority of the present cases, even
within the epidermis or obliterating the basal layer. It has been
reported that CD57+ T cells produce larger amounts of interferon-γ
than normal T-cells [38]. The distribution of CD57+ cells suggests
that they play a role in the inflammatory process.
No histological or immunological differences were observed
between the scalp and the papular lesions, indicating that the
latter are part of the same process. The predominance of CD8+
cells, the absence of perforin+ cells and the presence of rare
granzyme B+ cells differ considerably from the immunohistochemical
findings of atopic and seborrheic dermatitis [35], possibly
representing additional means of differentiation between IDH and
these dermatites.
Acknowledgments
This research was supported by a grant from Conselho Nacional de
Pesquisa (CNPq) and Fundação de Apoio à Pesquisa na Bahia (FAPESB).
We are indebted to Prof. Neide Ferraz for helpful comments.
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