ARTICLE
Discoid lupus erythematosus (DLE) and Jessner's lymphocytic infiltration
of the skin (LIS) are chronic inflammatory diseases which are both characterized
by patchy dermal infiltrates of activated T cells. The infiltrates consist
mainly of CD4+ T helper cells and contain only a few CD8+
cytotoxic T cells [1]. However, epidermal involvement with focal hydropic
degeneration of the basal cell layer, atrophy or hypertrophy of the prickle
cell layer and hyperkeratosis is only found in DLE. These alterations
of the keratinocytes as well as the epidermal infiltration by some CD4+
and, to a lesser extent, CD8+ lymphocytes, are as yet poorly
understood and cannot be related to the deposits of immunoglobulins and
complement at the dermal-epidermal junction in lesional skin of DLE [2].
The findings suggest that the keratinocytes of DLE and LIS show different
cellular and inflammatory properties in these otherwise quite similar
inflammatory processes of the skin.
In the present study, we have therefore investigated the phenotype of
the epidermal keratinocytes in skin biopsies from typical DLE and LIS
by immunohistochemistry using monoclonal antibodies against major histocompatibility
class II molecules (HLA-DR) and the intercellular adhesion molecule-1
(ICAM-1). Both surface molecules were recently shown to be involved in
the generation and maintenance of intraepidermal lymphocytic infiltration
in various types of T cell-mediated skin inflammation [3-5]. Moreover,
all biopsies were stained with the monoclonal antibody (MoAb) 27E10 against
the heterodimer of the calcium-binding proteins MRP-8 and MRP-14. This
antigen represents an activation and differentiation marker for tissue
macrophages in inflammation [6] and seems also to be a valuable tool for
evaluating the cellular properties of inflammatory keratinocytes [7, 8].
Material and methods
Biopsy material
The study was performed on diagnostic skin biopsies from patients with
clinically typical, previously not treated, DLE (n = 11) and LIS (n =
10). Control biopsy material from normal skin (n = 5) was obtained during
unassociated routine operations. All specimens were divided and fixed
in formaldehyde for routine histopathology and in liquid nitrogen for
immunofluorescence testing and immunohistochemical staining. Histological
examinations revealed typical alterations for DLE and LIS. In DLE, diagnosis
was confirmed by direct immunofluorescence showing granular deposits of
IgM and IgG, respectively, at the dermal-epidermal junction [2]. All biopsies
from LIS were negative on direct immunofluorescence. None of the patients
had antinuclear antibodies.
lmmunohistochemistry
Cryostat sections of 4 µm thickness were fixed in acetone at 4°
C. The sections were incubated with monoclonal antibodies (MoAbs) against
HLA-DR, ICAM-1, and the 27E10 epitope. For the detection of HLA-DR, two
different MoAbs were used. The reaction patterns and sources of the applied
antibodies are listed in Table
I. Positive reactions were visualized by a two step immunoperoxidase
technique using goat-anti-mouse IgG (Dianova, Hamburg, Germany) as described
in detail elsewhere [9]. The specificity of labelling was confirmed by
replacing the monoclonal antibodies with equal amounts of non-specific
mouse IgG of the same idiotype.
Evaluation of immunostaining
The pattern and intensity of keratinocyte staining were evaluated by
a semiquantitative histological score. The evaluations were performed
by two independent investigators with very similar assessments of the
staining patterns on comparison of the obtained results.
Results
The specimens of normal skin showed no positive staining of the epidermis
with the MoAbs against 27E10, HLA-DR, and ICAM-1, as outlined in Table
II.
Discoid lupus erythematosus
The epidermis displayed intense band-like staining for the 27E10 antigen
(Fig. 1a). This pattern
was specifically confined to lesional skin, with the surrounding uninvolved
epidermis failing to stain with the MoAb 27E10. The basal keratinocytes
were generally less strongly labelled than the suprabasal and upper epidermal
cells. The latter cells were characterized by a positive reaction of the
cell membrane and outer cytoplasm. Only 1 out of the 11 DLE biopsies showed
no epidermal labelling. Remarkably, this biopsy did not demonstrate the
keratinocyte alterations typical for DLE, and was histologically characterized
by a pronounced atrophy of all epidermal layers.
The stainings for ICAM-1 yielded a positive reaction of the outer membranes
of the basal keratinocytes. There was usually a focal epidermal expression
which was most pronounced in areas with intraepidermal lymphocytes (Fig.
1b). Moreover, this staining pattern was only found in 6 out of
the 11 specimens. Two biopsies displayed a very weak, if any, focal staining.
Three out of the 11 biopsies showed no ICAM-1 expression of the epidermal
and follicular keratinocytes, although their histology was quite typical
for DLE.
The two different MoAbs against HLA-DR did not reveal a positive reaction
of the epidermal keratinocytes in any of the biopsies. Even those areas
of the epidermis which were infiltrated by lymphocytes did not stain for
HLA-DR.
Lymphocytic infiltration
There was no epidermal staining for the 27E10 epitope in 8 out of the
10 biopsies (Fig. 2a).
Two biopsies revealed an intense labelling of the keratinocytes which
was comparable to the findings in DLE. These biopsies showed, however,
pathological changes of the keratinocytes in as much as the epidermis
was acanthotic and hyperkeratotic in the one case, and atrophic in the
other case.
The stainings for ICAM-1 were similar to 27E10. Eight specimens revealed
no positive reaction of the keratinocytes (Fig.
2b). The two biopsies, characterized by acanthotic and atrophic
epidermis respectively and by 27E10 expression, also demonstrated ICAM-1
labelling of the cell membrane, especially in the basal keratinocytes.
No HLA-DR expression of the epidermis was found in the LIS biopsies.
Discussion
The present study shows pronounced differences in the inflammatory and
cellular properties of keratinocytes in DLE and LIS which may help to
understand the pathophysiological mechanisms of these T cell-mediated
inflammatory skin diseases.
The adhesion molecule ICAM-1 was found to be expressed by the epidermis
in 8 out of 11 biopsies of DLE. There was an often intense but focal staining
of the cell membrane of the basal and suprabasal keratinocytes, and this
staining was closely related to the presence of intraepidermal lymphocytes.
In contrast, the biopsies from LIS did not show epidermal ICAM-1 expression,
except for 2 cases which, however, were histologically characterized by
pathological alterations of the epidermis not usually seen in this disease.
It was demonstrated that the expression of ICAM-1 on keratinocytes plays
a pivotal role in various inflammatory skin diseases by mediating epidermal
T lymphocyte trafficking [3-5, 10, 11]. The staining results of this and
a very recent study [12] on DLE confirm these findings, in as much as
intraepidermal lymphocytes with lymphocyte function associated antigen-1
expression were predominantly found in areas with keratinocyte ICAM-1
expression. The only focal staining of lesional epidermis can be explained
by transient expression.
In contrast to other studies on DLE [1, 12-14], we surprisingly observed
neither in DLE nor in LIS positive HLA-DR staining of the lesional epidermis.
The negative reaction in all 11 cases of active DLE is probably due to
the well-known transient epidermal expression of HLA-DR in inflammation
and the timepoint for obtaining the biopsies [15]. This indicates that
the keratinocyte HLA-DR expression does not play a significant role in
mediating the intraepidermal infiltration and maintenance of lymphocytes
in DLE.
The 27E10 antigen was recently shown to be expressed by keratinocytes
in various inflammatory processes of the epidermis [7]. This surface antigen,
first described in inflammatory macrophages [6], was identified as the
biologically active heterodimer of the calcium-binding proteins MRP-8
and MRP-14 [17, 18]. The complex formation of these proteins is a calcium-dependent
process which is known to interfere with calcium homeostasis, biosynthetic
functions and structural organization of cells [8, 19]. All present data
thus indicate that the 27E10 antigen is a marker for a distinct type of
cellular activation and also seems to be associated with a changed terminal
differentiation. In monocytes, inflammatory activation and adherence to
extracellular matrix components, e.g. fibonectin and collagen lead
to 27E10 expression [20]. Recently, evidence was provided that calcium-induced
differentiation of keratinocytes is associated with complex formation
of MRP-8 and MRP-14 (27E10 antigen) [8]. Whether epidermal 27E10 expression
plays a major pathogenic role in inflammatory and neoplastic dermatoses
such as lichen planus, cutaneous graft versus host disease and
mycosis fungoides [16] or is secondary to the inflammatory infiltration
of the epidermis remains to be determined.
The present study demonstrates that immunostaining
with the moAb against the 27E10 antigen revealed a strong positive reaction
of the epidermis in biopsies from DLE, whereas biopsies from typical LIS
were negative. The staining in DLE was most pronounced in the suprabasal
and upper epidermal layers and was also seen in lesional areas showing
dermal lymphocytic infiltrates but lacking histological alterations of
keratinocytes and inflammatory infiltrates of the epidermis. In contrast,
no epidermal expression of 27E10 was observed in the skin surrounding
the lesions. Similar staining results of the epidermis in DLE were previously
obtained with a polyclonal antiserum against a MRP-8 and MRP-14-containing
complex [21] and the moAb MAC387 which recognizes MRP-14 [22]. A predominant
suprabasal staining of 27E10 has been reported by Schlegel-Gomez in inflammatory
periodontal disease [23]. Because the gingival epithelial cells showed
constant 27E10 expression independent of the stage of inflammation, they
concluded that epithelial staining at least in this disease might be a
primary event. In accordance with these findings, studies of Crohns' disease
revealed that epithelial cells adjacent to ulcerative lesions in the terminal
ileum displayed a strong staining for 27E10 [24]. However, 27E10 positivity
could not clearly be separated from inflammatory infiltration of the tissue.
Although not proven, the immunohistochemical investigations on DLE suggest
that keratinocyte expression of these calcium-binding proteins is independent
of the inflammatory infiltration of the epidermis and might represent
a primary phenomenon of the disease. The attractive hypothesis of abnormal
keratinocytic activation and premature terminal differentiation in DLE
is supported by a recent study showing epidermal hyperproliferation with
irregular expression of the differentiation marker involucrin and filaggrin
in the lower epidermis of the disease [25]. One might speculate that the
changed terminal differentiation is related to the well-known increased
sensitivity of the keratinocytes to sunlight in DLE [26] since ultraviolet
(UV) radiation is able to affect the differentiation status of even normal
keratinocytes. In particular, UVB light was very recently shown to upregulate
the keratin pattern typical of terminal differentiation of the cells [27].
Moreover, we could demonstrate that UVB radiation induces programmed cell
death by apoptosis in keratinocytes [28], a process that is thought to
be involved in the differentiation process of the epidermis [29] and to
play a role in initiating the inflammatory autoimmune response in systemic
lupus erythematosus [30-32].
CONCLUSION
Lymphocytic infiltration of the epidermis in DLE is most likely mediated
by keratinocyte expression of ICAM-1, but not HLA-DR. This lymphocyte-homing
capacity of the keratinocytes may be related to, or even induced by, the
state of activation and irregular terminal differentiation of the cells,
which in turn may represent the central pathological event of this as
yet poorly understood process of cutaneous inflammation. In addition,
the 27E10 antibody provides a new reliable immunohistological marker for
the often difficult differential diagnosis between LIS and DLE.
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