ARTICLE
Dendritic cells (DC) are the most potent antigen-presenting cells (APC)
and are believed to be crucial for the initiation of T cell response to
foreign antigens [1]. DC in the skin capture and process antigens, undergo
activation, migrate to lymphoid organs and secrete cytokines to initiate
the immune response. Fully mature and activated DC are characterized by
the expression of CD83, a member of the immunoglobulin superfamily [2].
Langerhans cells (LC) are thought to represent a subset of DC distinguished
by their expression of the CD1a antigen and their exact relationship to
CD83+ DC is not fully understood. CD83+ DC may lack
or express less CD68 (lysosomal antigen), which are associated with monocytes
and macrophages [1].
It has been reported recently that CD83+ DC are in intimate
contact with T lymphocytes in normal skin, suggesting that communication
between these two cell types in situ may be involved in the immune
response to cutaneous antigens [3]. A recent study described a small number
of CD83+ DC in human leprosy lesions [4]. The subset of CD83+
DC is likely to play a major role in initiating an antimicrobial immune
response in the skin.
Sporotrichosis is a granulomatous mycotic infection caused by Sporothrix
schenckii. Granuloma formation, a critical event in the immune response
against S. schenckii, is thought to be a result of the Th1 response
in the host [5, 6]. Although changes in the number or localization of
mature and activated CD83+ DC might be expected in the granulomas
of sporotrichosis, no data are yet available. To investigate the presence
of CD83+ DC in sporotrichosis, we applied an immunohistochemical
technique to formalin-fixed, paraffin-embedded biopsy sections after antigen
retrieval.
Material and methods
Specimens and reagents
The subjects of this study were five patients with sporotrichosis who
had been referred to Kyushu University Hospital. Samples were obtained
by skin biopsy with the informed consent of the patients. Formalin-fixed,
paraffin-embedded sections were examined in this study.
Mouse monoclonal antibodies against CD83 (clone: HB15A, IgG2b, Immunotech,
Marseille, France), CD1a (clone: O10, IgG1, Immunotech) and CD68 (clone:
KP-1, IgG1, DAKO, Glostrup, Denmark) were used as primary antibodies.
Control mouse IgG1 and IgG2b were purchased from Pharmingen, San Diego,
CA, USA.
Immunohistochemistry
The sections were stained using a Histofine SAB-AP kit (Nichirei, Tokyo,
Japan) according to the manufacturer's recommendation. A series of preliminary
experiments was done to optimize the antigen retrieval system. Based on
the results, the skin sections were treated with 0.1% trypsin for 60 min
at 37° C prior to immunostaining for CD83. Heat pretreatment was
performed by incubating the sections in 10 mM citrate buffer (pH 6.0)
in a pressure cooker at 110° C for 5 min prior to immunostaining
for CD68 and CD1a. The sections were incubated with primary antibody overnight
at 37° C. We used new fuchsin for the Histofine SAB-AP kit as a substrate,
and the sections were counterstained with hematoxylin. The control isotype-matched
mouse IgG1 and IgG2b antibodies always showed negative staining.
Results
CD83+ cells in granulomatous lesions
Immunohistochemical staining using anti-CD83 monoclonal antibody showed
that CD83+ DC existed within and around the granulomas, comprising
approximately 0.2 to 0.8% of total inflammatory cells. The most common
distribution pattern of CD83+ DC appeared to be as clusters
with mononuclear lymphoid cells within the granulomas (Fig.
1). In addition, CD83+ DC were occasionally scattered
around the granulomas (Fig. 2).
High-intensity staining of CD83 antigens was detected not only on the
surface, but also in the cytoplasm of DC. Most CD83+ cells
were found to be large, irregularly shaped cells with large nuclei and
dendrite formation (Fig. 3).
No CD83+ DC were observed in the epidermis overlying the sporotrichosis
granulomas.
CD1a+ cells in granulomatous lesions
The number and distribution of CD1a+ cells in the epidermis
of sporotrichosis samples were similar to those of epidermal Langerhans
cells (LC) in normal skin. A considerable number of CD1a+ cells
were detected in the papillary dermis and around the granulomas, though
CD1a+ cells were rarely found within the granulomas.
CD68+ cells in granulomatous lesions
There were large numbers of CD68+ monocytes and macrophages
in the granulomas intermingling with lymphoid cells. All the multinucleated
giant cells were CD68+.
Discussion
DC are efficient and effective APC that play a major role in initiating
the primary immune response [7]. DC have been found in almost every tissue,
and account for less than 1% of all mononuclear cells in peripheral blood
[1]. Peripheral blood and tissue DC take up antigens, undergo activation
and subsequently migrate to the draining lymph nodes where they initiate
the immune response. High levels of expression of CD83, detected with
the antibody HB15, have been found on activated and mature DC [2]. Thus,
high-level expression of CD83 is currently considered to be the most specific
marker of activated and mature DC. In vivo, high expression of
CD83 is a valuable marker for activated and mature DC in kidney [8] and
prostate [9] cancers. A recent study described a small number of CD83+
DC in human leprosy lesions [4]. CD83+ DC is therefore expected
to be of great importance in the antimicrobial immune response in the
skin.
Although the immunological mechanisms involved in the prevention and
control of sporotrichosis are not fully understood, Th1 response to S.
schenckii may be one of the host defense mechanisms. Recent studies
have investigated the immune response to sporotrichosis in terms of the
production pattern of cytokines. We previously reported that peripheral
blood mononuclear cells from patients with sporotrichosis produce high
levels of IFN-gamma in response to stimulation with sporotrichin [5].
Using reverse transcription-polymerase chain reaction, the presence of
IFN-gamma mRNA was detected in the skin lesions of sporotrichosis [6].
It is of great importance to identify the in situ status of CD83+
DC in granulomas of sporotrichosis. In this study, we have demonstrated
the existence in vivo of a population of CD83+ DC within
and around the cutaneous granulomas of human sporotrichosis. This novel
finding indicates that activated and mature DC may play a role in the
immune response to sporotrichosis.
Immunohistochemical staining for CD83 was done by using formalin-fixed,
paraffin-embedded skin biopsy sections according to a reported method
[10], with some modifications. A series of preliminary experiments was
done to optimize the antigen retrieval system for CD83 staining, and pretreatment
with trypsin was found to be effective.
DC have recently been shown to be a source of
IL12, which leads to generation of Th1 responses [11]. Thus, DC could
serve as both the APC and the IL12-producing initiator of the Th1 response
[12]. The presence of activated and mature CD83+ DC within
and around the infected granulomatous tissue of patients with sporotrichosis
is likely to stimulate Th1 responses that promote effective control of
the infection.
The molecular signals controlling the development of activated and mature
CD83+ DC in sporotrichosis are not yet known. That direct S.
schenckii infection of DC may be required for inducing CD83+
DC in vivo is an intriguing possibility. This possibility is supported
by a report describing activation of human peripheral blood-derived DC
by infection with live Mycobacterium tuberculosis [13].
In contrast, a considerable number of CD1a+ cells were detected
around the granulomas, though they were rarely found within the granulomas.
We did not conduct double label immunostaining in this study, but it seems
unlikely that CD83+ DC in sporotrichosis lesions coexpress
CD1a. It is unclear whether CD83+ DC in sporotrichosis are
derived from blood DC, dermal DC, or epidermal LC. As there were large
numbers of CD68+ monocytes and macrophages in the granulomas,
the CD83+ DC may be derived from CD68+ cells.
There is little information available regarding CD83+ DC
in other skin diseases, and we cannot say whether the observed frequency
of CD83+ DC in patients with sporotrichosis represents a high
or a low level. Further study is needed to clarify the functional implications
of these cells in the pathogenesis of sporotrichosis.
CONCLUSION
Acknowledgements
This work was supported by grants from the Ministry of Education, Science,
Sports and Culture of Japan, and also from the Ministry of Health and
Welfare of Japan. Dr. H. Duan is kindly supported by a postdoctoral fellowship
program for foreign researchers of Japan Society for the Promotion of
Science. We thank Mr. T. Kanemaru at Morphology Core, Faculty of Medicine,
Kyushu University for his help.
Article accepted on 15/5/01
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