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Anais Brasileiros de Dermatologia

On-line version ISSN 1806-4841

An. Bras. Dermatol. vol.79 no.6 Rio de Janeiro Nov./Dec. 2004

http://dx.doi.org/10.1590/S0365-05962004000600004 

CLINICAL, EPIDEMIOLOGICAL, LABORATORY AND THERAPEUTIC INVESTIGATION

 

Expression of the cytokeratins in infectious and parasitic skin diseases associated with epidermal hyperplasia*

 

 

Maria Christina Marques Nogueira-CastañonI; Tullia Cuzzi MayaII; René Garrido NevesIII

IAdjunct Professor – Department of Morphology, UFJF, MG)
IIAdjunct Professor, Department of Pathology, UFRJ, RJ
IIITitular Professor, UFRJ, RJ

Correspondence

 

 


ABSTRACT

BACKGROUND: Cytokeratins (K) are the major structural proteins of epithelial cells and they display the greatest heterogeneity of all intermediate filament proteins. The study of many isolated cytokeratins by immunomarcation enables the structural verification of the cytoskeleton in many neoplastic and inflammatory diseases.
OBJECTIVE: To verify the immunohistochemical pattern of cytokeratin expression in infectious and parasitic diseases associated with squamous hyperplasia.
METHODS: Histological sections obtained from formalin-fixed and paraffin-embedded tissue from cromomycosis, paracoccidioidomycosis, leishmaniasis and condylomata acuminata lesions were marked with the DEK10, LL025, LL002 and AE1 antibodies by the immunoperoxidase technique (avidina-botina).
RESULTS: Different degrees of epidermal hyperplasia were observed predominantly or exclusively in the following four diseases: absence of immunoreactivity to DE-K10 in areas of intense epidermal hyperplasia and delayed K10 immunohistochemical staining in areas of moderate to discreet/absent hyperplasia; superbasal expression pattern for K16, regardless of the degree of hyperplasia as well as superbasal epitops discharging for LL002 (C14) and AE1 (C10, 14, 16, 19).
CONCLUSIONS: The modifications indicate that regardless of the nature of the etiologic agent and degree of hyperplasia, changes in keratinocyte differentiation and proliferation may occur.

Key-words: epidermis; hyperplasia; keratin.


 

 

INTRODUCTION

Cytokeratins (K) are proteins present in all epithelial cells. Their association leads to the formation of an arc of intermediary filaments making up the main component of the epithelial cytoskeleton.1 Classified as either type I (acid) or type II (neutro-basic), they are often co-expressed in pairs, in which the keratin filaments are heteropolymers with equimolar quantities of an acid (type I) and base (type II) protein.2,3

The pertinent production of monoclonal anticytokeratin antibodies has grown. Their biochemical characterization and the study of immunohistochemical revelation patterns in normal human tissues suggest they represent a significant contribution in their application to diagnostic pathology and the overall investigation field.4-7

In the normal human epidermis, keratinocytes follow a maturation pattern during their migration from the basal to the corneal layer. This is accompanied with changes to the synthesis of cytokeratins. Accordingly, the authors of the present study found cytokeratins 5 and 14 in the basal compartment and cytokeratins 1 and 10 in the superbasal compartment. This distribution constitutes a characteristic pattern of keratinizing squamous epithelium.8,9,10

In several cutaneous diseases, the cytokeratin expression pattern in the epidermis is altered. In those characterized by epidermal hyperproliferation, as for example psoriasis, a few epidermal tumors as well as cutaneous ulcers in the healing phase, the keratinocyte differentiation pattern is interrupted and/or substituted by an alternate modified pattern.11 Alterations include the premature appearance of certain terminal differentiation markers, delays in the synthesis of others and the expression of cytokeratins that are not found in the healthy epidermis, like for example K6 and K16.12-15

In the present study, the expression of individual cytokeratins was investigated in the epidermis of infectious and parasitic dermatoses that are often associated with hyperplastic epithelial morphological alterations (like American tegumentary leishmaniasis, paracoccidioidomycosis, cromomycosis, and condylomata acuminata). The objective is to seek subsidies in order to better understand epidermal differentiation and maturation in these diseases.

 

MATERIAL AND METHODS

The present paper is a retrospective study of formalin-fixed (10%) and paraffin-embedded cutaneous lesion fragments of leishmaniasis, paracoccidioidomycosis, cromomycosis and condylomata acuminata (10 cases of each disease). Four-micra thick histological sections were placed in laminated slides with 3-aminopropyltriethoxysilane (according to the manufacturer's, Sigma A-3648's, guidelines). They were deparaffinized in xilol, re-hydrated in alcohol solutions and in water. The inhibition of endogenous peroxydase was made in successive baths of hydrogen peroxide solutions. At first, the material was incubated with normal horse serum for monoclonal antibodies AE1, LL002 and DEK-10, and bovine albumin for monoclonal antibody LL025. The aim was to reduce the non-specific links by blockage, thereby avoiding the presence of background coloring during photographic development. The primary antibodies used, which were all monoclonal, their specificities, dilutions, incubation time and temperature, whether requiring pre-treatment or not, their commercial sources and bibliographic references, are all presented in chart 1.

 

 

For antibodies AE1, LL002 e LL025, the authors proceeded to an antigenic recuperation by means of incubation in a citrate buffer solution (Merk 244 and 6586), heated in a microwave oven (Shap RH-4A33 operating at a frequency of 2.45 GHZ, at the maximum energy level of roughly 750 W) for two five-minute cycles for markers AE1 and LL002, and six minutes for marker LL025.

The material was then incubated with biotinylated antibody (Vectastain ABC kit-mouse IGg-1), a drop of solution/stock in 10 ml of Dako TBS for 30 minutes at 37ºC in a humid chamber, and with an avidina-botina complex conjugated with peroxydase (Vectastain ABC kit) for 30 minutes, in a humid chamber. The product of the reaction was revealed with DAB (diamine-benzene), and the sections counterstained with Harris hematoxylin, then dehydrated and mounted in a synthetic medium. The dilution of primary and secondary antibodies was done in a Tris-HCl 0.05M pH 7.6 in BSA buffer (bovina albumin serum, Sigma Immunochemical A2153) at 5% with sodic acid 0.015M. As positive controls for antibodies DEK10, AE1 and LL002, histological sections of normal skin from the axillary region were utilized. For the monoclonal antibody LL025, histological sections of basal cell carcinoma were used, as recommended by the commercial manufacturer, and other cutaneous diseases were introduced. They were known to be related to cellular proliferation, such as psoriasis, lichen planus and keratoacanthoma. The latter showed a strong expression of K16, in accordance with the referenced consultations.16,17 An omission of the primary antibody was utilized as a negative control for the reaction.

 

RESULTS

The 40 analyzed cases showed variations in epidermal thickness throughout the examined fragment. The following variations were recorded: intense (pseudoepitheliomatous) hyperplasia; moderate (regular) hyperplasia; discreet or absent hyperplasia (epidermis with usual thickness); intense hyperplasia associated with moderate hyperplasia; discreet or absent hyperplasia in association with moderate hyperplasia; discreet or absent hyperplasia associated with moderate and intense hyperplasia.

With the use of negative and positive controls, a brown stain could be observed in the cytoplasm of the epithelial cells. The marking pattern in this sample (when positive) was also cytoplasmatic with all of the antibodies utilized. The results obtained are indicated in chart 2.

 

 

In the cromomycosis, paracoccidioidomycosis and leishmaniasis samples, K10 expression was negative in areas of intense hyperplasia. However, at the sites of moderate hyperplasia, there was a predominance of superbasal positivity associated with the delayed expression (Figure 1) and a normal epidermis pattern in the areas of discreet or absent hyperplasia. K16 was overwhelmingly positive for the epidermis in the whole sample, regardless of the degree of hyperplasia (Figure 2). Similarly, K14 showed intense and uniform expression over the whole epidermal thickness, regardless of the degree of hyperplasia (Figures 3, 4, 5 and 6). Monoclonal antibody AE1, which immunoreacts selectively with the epitops of the basal cells in the normal epidermis, showed a superbasal pattern in all cases, regardless of the degree of hyperplasia (Figure 7). However, variations in positivity and intensity of the reaction in the basal compartment did occur. The latter depended on the degree of hyperplasia present.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In the condylomata acuminata, K10 was predominantly positive in the areas of intense and moderate hyperplasia. However, there was a delayed expression and variations in reaction intensity. In areas of discreet or absent hyperplasia, the pattern was akin to a normal epidermis. K16 showed a predominance of positivity in the areas of intense and moderate hyperplasia. Yet the latter saw a weaker reaction intensity. Indeed, in the areas of discreet or absent hyperplasia, it was negative. K14 showed intense positivity over the entire epidermal thickness in areas of intense hyperplasia (Figure 8). This also occurred in areas of moderate hyperplasia, though with weaker intensity. The AE1 antibody showed immunoreactivity to be predominantly positive and intense all over the epidermal thickness in the areas of intense hyperplasia. Likewise, the epidermis pattern in areas of moderate, discreet or absent hyperplasia was normal.

 

 

DISCUSSION

The results demonstrated a clear sign of changes in cytokeratin expression with the monoclonal antibodies used in all examined diseases. There was also a relation between the expression of cytokeratins and the various degrees of epidermal hyperplasia present.

The occurrence of epidermal modifications in the expression of K10, which is considered to be one of the molecular markers for terminal differentiation of keratinocytes,12 indicates and reinforces previously held evidence regarding which such modifications represent the presence of alterations in the keratinization, even in the initial lesion phases when an exuberant epithelial hyperplasia does not yet exist.19,20

Monoclonal antibody LL025, monospecific for K16, is intimately related to the hyperproliferation state of the keratinocyte,12 which is an antigen absent in the normal interfollicular epidermis. It showed overwhelming positivity in the whole sample, regardless of the degree of hyperplasia observed, and even in the areas of the epidermis that had normal thickness.

In hyperproliferative diseases and after experimental traumatisms, increased K16 expression has been described as anticipating epidermal proliferation, followed by a drop in K10 expression.21,22 This was also demonstrated in the present study in the areas of the epidermis that are practically normal or at least within the limits of normality, as well as in discreet hyperplasia areas. Nonetheless, K16 expression backs up and reinforces the point of view that the keratinocyte responds to a supposed stimulus with an alternate pattern of differentiation. However, a disease-specific alteration does not appear because identical changes are induced in the normal keratinocytes when migration and mitosis prove necessary.23

The results of this study show explicitly that, even prior to epithelial morphological evidence, alterations may already be observed in the intermediary filaments of cytokeratins. These findings fully agree with the literature. That is, the K16 expression is not a consequence of hyperproliferation, but indicates that the latter had been triggered or had already appeared.22,23

There was no case in which monoclonal LL002 antibody, which immunoreacts with K14, revealed the pattern of restricted marking (positive basal and eventually positive parabasal cells) observed with the normal epidermis.24 All of the cases showed intense and uniform marking over the whole thickness of the epidermis regardless of the degree of hyperplasia. This has also been described in states of hyperproliferation during epidermal regeneration, that is, pan-epidermal positivity.24

This fact, in association with the positivity of K16 and the absence or delay of K10, reinforces the proposal that K14 and K16 appear to be other cytokeratin polypeptides that substitute the K10 in the superbasal layer of the regenerating epidermis. These changes are assumed to be a reflection of the newly formed epidermis's immaturity insofar as K13 and K18 (normal in superbasal cells of the fetal epidermis) also present as positive.21

Monoclonal antibody AE1 also exhibited a different expression pattern to the one observed in the material control, with a positive cytoplasmatic and intense reaction in the superbasal compartment of all cases, regardless of the degree of hyperplasia. However, variations in reaction positivity and intensity were observed in the basal layer, depending on the degree of hyperplasia present.

These results demonstrated that not only do the basal keratinocytes in the affected epidermis express a differentiation pattern altered by monoclonal antibody AE1, but so do the superbasal ones.

Earlier studies focusing on the immunoreactivity of the molecular AE1 marker in various diseases, by means of immunofluorescence and peroxidase-antiperoxidase techniques in frozen histological sections of normal and abnormal human skin, demonstrated that on normal skin and on vulgar ichtyosis, i.e. a non proliferative epidermal disease, this antibody reacted with the epidermal basal cells. Very different immunoreactivity patterns were observed in various other epidermal diseases, like psoriasis, verruca vulgaris, seborrheac keratosis, actinic keratosis, as well as in certain epidermal neoplasias, like basocellular and epidermoid carcinoma, in addition to Bowen's disease, which showed superbasal positivity for this antibody with some variations in reaction intensity.25,26

While the biochemical bases for understanding these reactivity patterns are still in need of elucidation, the results presented herein show evidence that the expression of cytokeratins may perhaps be affected by various evolving states of the disease.

 

CONCLUSIONS

The altered expression of epidermal cytokeratins in inflammatory diseases of an infectious and parasitic nature, such as cromomycosis, paracoccidioidomycosis, leishmaniasis and condylomata acuminata, reflect the occurrence of modifications in the differentiation and proliferation of keratinocytes, regardless of the nature of the stimulus (fungus, protozoa or virus).

The positivity found for K16, a marker of hyperproliferating keratinocytes, associated with the delayed expression of K10, a terminal differentiation marker of the keratinocyte, is an indicator that points to a change in the expressive profile of the epidermal cytokeratins. This was substituted by a pattern of cellular hyperproliferation in areas morphologically defined as hyperplasia as well as those with no morphological evidence of increased mitotic activity. However, there is reciprocity between the terminal keratinization markers and hyperproliferation markers. When K16 is positive, there is usually a delay in K10 expression, that is, there is a change in the expression of superbasal keratinocytes. The latter failed to exhibit the immunocytological pattern of cytokeratins in association with terminal differentiation (K1, K10) and a substitution by cytokeratins related to cellular hyperproliferation (K6, K16).

Even in the absence of hyperplasia, that is, an epidermis with normal thickness whether it is a perilesional area or an epidermis superjacent to the inflammatory process, there were alterations in the expression pattern of cytokeratins, mainly K16 and K10. This predicts a more precocious hyperproliferation pattern in future morphologic alterations, meaning that these alterations would be more of a "cause" than a "consequence" of the hyperproliferative state.

Changes in the expression pattern of cytokeratins function as an immunocytological indicator suggesting that some inductive factor, probably present in the dermal conjunctive tissue and/or in the inflammatory infiltrate triggered by the parasite, acts primarily at the level of the cytoskeleton. Only later does it go on to manifest itself with the aspect of cellular hyperproliferation (hyperplasia).

 

 

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Correspondence to
Maria Christina Marques Nogueira-Castañon
Rua Florival Cherem Cruzeiro 300 - Serro Azul
36036-390 Juiz de Fora MG
Telefone: (32) 3235-6650
E-mail: castañon@terra.com.br

Received on July 07, 2004
Approved by the Consultive Council and accepted for publication on August 27, 2004

 

 

* Study carried out at the Post-Graduate Department in Dermatology, HUCFF/Federal University of Rio de Janerio, RJ