ARTICLE
Inflammatory bowel disease (IBD), is often associated with skin manifestations.
Erythema nodosum (EN) is one of the typical cutaneous lesions commonly
associated with Crohn's disease (CD) and ulcerative colitis (UC). Recent
studies have highlighted the role of bacterial superantigens in IBD [1,
2]. We have recently seen two cases of EN associated with UC and CD. To
study the possible involvement of superantigen and the use of T cell receptor
Vß subsets in the lesional skin associated with IBD, we have analyzed
the T cell receptor Vß receptoire on the infiltrating T lymphocytes
in the lesional skin of EN patients.
Case 1
A 25-year-old woman was admitted complaining of diarrhea which had lasted
over 3 months, fever, arthralgia, and several, painful erythematous lesions
on her lower legs. Colonoscopy showed multiple ulcers and cobblestone
mucosa confirming Crohn's disease. Histologic examination of the erythema
revealed inflammatory cell infiltration into the septum and giant cells
scattered in the subcutaneous tissue. Diet improved both gut symptoms
and cutaneous lesions.
Case 2
A 32-year-old woman was seen after the diagnosis of ulcerative colitis,
which was determined by colonoscopy. She had been treated with prednisolone
and sulphasalazine. As the disease activity went into remission and as
she hoped to fall pregnant, prednisolone was stopped. When she was 22
weeks into pregnancy, a few, painful erythematous lesions developed on
her lower legs. Histologic examination revealed septal panniculitis with
lymphocyte and neutrophil infiltration. Resting induced a remission of
the EN.
Materials and methods
Total RNA was isolated from fifty, 5-µm frozen biopsied tissue
sections of each lesion of EN using RNA zol (Biotex CS 101), which was
then reverse transcribed to cDNA by RAV-2 reverse transcriptase (Takara
Co. Ltd., Kyoto, Japan). PCR analysis of the T cell receptor (TCR) Vß
repertoire was accomplished by using oligonucleotide primers to amplify
specific Vß gene segments paired with a consensus ß-chain
constant region (Cß) primer [3]. As internal controls, a pair of
5' sense C alpha-specific primers and 3' antisense C alpha-specific primers
was also amplified, as described previously by Choi et al. [4].
PCR amplification was started with an initial denaturation step at 94°
C, followed by a 35-cycle profile that consisted of denaturation 94°
C (1 min), annealing 57° C (1 min), and extension at 72° C (1
min and 5 s). The PCR was completed with a final extension step of 7 min
at 72° C. Twenty microliters of PCR products were electrophoresed
in 1.7% agarose gel containing 1% ethidium bromide and visualized under
ultraviolet light.
Results
A PCR-based method was adapted to compare the relative expression of
TCR Vß genes in the lesional skin and peripheral blood. Visualization
with ethidium bromide revealed that expression of Vß 8, 14, 15,
17 and 20 was preferentially detected in EN associated with CD and Vß
14, 15, 16, 17 and 20 in EN associated with UC. Results of densitometric
analysis are shown in Figure 1.
As a control, 3 normal skin samples, which were obtained from the lower
leg at surgery, commonly showed Vß 1, 6 and 14 expression, although
diverse Vß usage was noted (data not shown).
Discussion
Activated T cells are important in the pathogenesis of IBD and a change
in immune activity has been suggested in patients with IBD [5]. Certain
cytokines play a major role in the regulation of IBD [6, 7]. Furthermore,
recent studies using molecular probes and monoclonal antibodies show that
changes in the number of T cells expressing restricted TCR Vß gene
products are due to the involvement of superantigens in the pathogenesis
of such disorders [1, 2, 8, 9]. Superantigens are the protein products
of a number of bacteria and viruses. They stimulate large numbers of T
cells to bind with the TCR in a Vß-specific manner. It is known
that bacterial superantigenic toxins cause acute diarrhoeal illness [10].
Recent studies have demonstrated that intestinal epithelial cells were
capable of presenting superantigens in about 50% of normal individuals
[9]. Vß8+ T cells were elevated in the peripheral blood
and mesenteric lymph nodes of a subset of CD patients as compared with
controls [9], and cultured Vß8+ T cells from these patients
showed decreased cytotoxic activity compared with controls, despite overall
equivalence of CD8+Vß8+ T cell numbers [1].
In this study, our data showed that Vß 14, 15, 17 and 20 were
commonly expressed in EN associated with UC and CD. Vß 8 was predominantly
expressed only in EN associated with CD, but was not detected in UC. Antecedent
throat pain was present in case 1, which may be an indication of the onset
of EN. In case 2, the patient was pregnant, which may have affected immunity
in some way. Our findings suggest that Vß 14, 15, 17 and 20 may
be commonly implicated lymphocyte subsets in EN associated with IBD. The
Vß 8 subset may be involved only in the induction of CD. Further
studies are needed to clarify the possible role of superantigens and Vß
subsets in the induction of EN associated with IBD.
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