Annals ofBurns and Fire Disasters - vol. IX - n. 3 - September 1996

SOME OBSERVATIONS ON MAST CELL NUMBERS IN STEROID-TREATED KELOIDS

Popchristova E, * Mazgalova J.**

Department of Pathology, Tzaritza loanna University Hospital, Sofia, Bulgaria Department of Tissue Conservation, Pirogov Emergency Institute, Sofia

SUMMARY. This study focuses on mast cells in steroid-treated keloids. Ten cases of untreated and steroid-treated keloids were assessed for the number of mast cells. The use of light microscopy morphometric analysis made it possible to establish a decreased number of mast cells in steroid-treated keloids in comparison with that of mast cells in untreated skin lesions. The reduction in the number of mast cells can be considered evidence for the participation of these cells in the stages of keloid healing. The probable ways in which mast cells and other connective tissue cells participate in the processes of steroid keloidal treatment are discussed.

Mast cells, first described over 100 years ago, are well-examined cells of connective tissue. They are located in the connective tissue throughout the body. Cutaneous mast cells are normally present perivascularly in the dermis and subcutaneous tissue, with a distribution ranging from 7000 to 20000 per rmul.' Mast cell granules release vasoactive substances, chemotactic factors, enzymes, proteoglycans and smooth-muscle contracting products .2 Mast cells are of bone-marrow origin' and are in close relationship with other cells, particularly haematopoietic and mesenchymal cells.
Recent studies suggest that mast cells participate in the pathogenesis of atopic and contact dermatitis, fibrosing reactions, bullous pemphigoid, cutaneous disorders, neurofibromatosis, wound healing, polycythemia vera, and psoriasis.' An increase in the number of mast cells was observed in localized sclerodermia,' progressive systemic sclerosis,' and hypertrophic scars and keloids.1,1 Mast cell morphology was noted by Kischer and Bailey' in hypertrophic scars, mature scars and normal skin, and, some years later, mast cell analysis was also performed during the different stages of healing.' Data can be found in the literature regarding the number of mast cells in pressure-therapy treated keloids,' but no data exist referring to the number of mast cells in steroid-treated keloids.
The present study was conducted with a view to determining the number of mast cells in materials from steroidtreated keloids. The aim of the investigation was thus to establish mast cell numbers in skin lesions treated with steroids in comparison with mast cell numbers in untreated keloids.

Materials and methods

A detailed description of the indications, methods of cure, and morphological results of keloid treatment with steroids can be consulted in our paper "Histomorphologic changes in keloids treated with KenacorC.
We used the method of light microscopy, combined with a morphometric analysis. Light microscopic investigation was performed on materials from steroid-treated and untreated keloids placed in 10% neutral paraformaldehyde and embedded in paraffin. Sections were cut and stained with toluidine blue solution 1.5 x 10-'M in MacIlven buffer, and embedded with Entellan for the histological identification of mast cells. The count of mast cells, identified principally by their metachromasia, was performed until two months after staining.
Both morphological and morphometric studies were carried out using a Laboval-type microscope with net eyepiece and stage micrometer (Figs. 1,2) adapted to each other according to the method of Awandilov.
The count of mast cells was performed at a microscopic magnification of 40 x 6.3 x 1.6, where four divisions of the stage micrometer correspond to three divisions of the net eyepiece. As one division of the stage micrometer is equal to 0.01 min, the following measurements are 0.04 divided by 3 = 0.0133 min (one division of the net eyepiece). The area of the smallest square in the eyepiece is thus 0.000177 rimil.
We observed 400 small squares in one field of vision of the microscope at this magnification, i.e. the measuring area in a field of vision was 0.000177 mmI x 400 = 0.0708 min'.
The number of mast cells was established in 50 fields of vision for each slide, i.e. in an area of 0.0708 irmil x 50 = 3.54 min'.
The data for the number of mast cells are presented as mean values for a 1 rum' area of total dermis in untreated and steroid~treated keloids. The relation of the number of mast cells in treated keloids to that in untreated keloids is presented as a percentage proportion (Table 1). The results were subjected to statistical analysis and the significance of P was determined.
The mast cell count was performed in five slides with untreated materials and five slides with steroid-treated keloids.

Fig. 3 - Histomorphological picture of mast cells in untreated keloid. Toluidine blue stain. Original magnification 40 x. Fig. 3 - Histomorphological picture of mast cells in untreated keloid. Toluidine blue stain. Original magnification 40 x.

Results

Data from the mast cell count in total dermis in steroid-treated and untreated keloids are presented in Table I. The mean mast cell density of untreated keloids is significantly greater than that of steroid-treated keloids.
The decreased number of mast cells in treated keloids in comparison with the number in untreated lesions is presented as a percentage relation (mean reduction 29.26%). Analysis of the data showed a significantly decreased mast cell count in the dermis of treated keloids (P >0.001).
The classification of metachromatic stain intensity suggested that mast cells in untreated keloids stained more intensely than those in treated keloids.
Metachromatic staining was very well expressed in some fields of untreated keloid tissue, where the greatest number of mast cells was found. No metachromatic stain was observed in the keloid tissue of steroid-treated keloids, where the metachromatic stain was located only in mast cell granules.
The fields of vision where no mast cells were seen represented 25-30% of the total number of fields of vision for each slide. The highest number of six mast cells for one field of vision was seen in a slide with material from untreated keloids.
In this study, only cells with clearly expressed metachromatic staining of the granules were considered.

Discussion

In our research' on histomorphological changes in steroid-treated keloids we gathered information about mast cells. We regarded our results as evidence for the participation of mast cells in the processes of keloid healing. The data reveal a reduction in the number of mast cells in total dermis in steroid-treated keloids in comparison with untreated skin lesions. Our findings were consistent with the results of Kischer et al.1,1 in their analysis of mast cells in hypertrophic scars. Once again, some years after these detailed investigations, we have devoted our attention to mast cells. By means of their biological mediators, mast cells participate actively in the performance of all connective tissue processes. Data in the literature indicate the role of fibroblasts and increased collagen synthesis in keloids The addition of hydrocortisone or triamcinolone acetate to fibroblast cultures has been shown to retard the growth of fibroblasts." It came to be known that the regulatory control mechanism that controls the rate of collagen synthesis is sensitive to physiological levels of hydrocortisone.

Type of materials N Mast cell density in 1 inni'

p
  1 13.55  
  2 18.13  
Untreated keloids 3 13.04  
  4 10.08  
  5 12.34  
  1 2.26 / 16.67%  
  2 3.02 / 16.65%  
Treated keloids 3 4.20 / 32.20%

0.01 >PA.00 1
  4 4.05 / 40.17%  
  5 5.04 / 40.61%  
Table I - Mast cell density in total dermal zone of treated and untreated keloids

Oikarinen et al.11 suggested in 1978 that ster
oids act by binding on to the receptor sites in human fibroblasts. The addition of the pharmacological agent antihistamine was found to be able to suppress the stimulation of fibroblasts incubated with histamine." Patients with hypertrophic scars and keloids have a significantly in creased tendency to allergy. The histamine content of keloid tissue is higher than that of normal scar tissue" and is related to the increased number of mast cells there. This suggests that the enhanced mast cell activity or count may be a common denominator that predisposes such persons both to allergy and to abnormal wound healing. 17 The large number of biologically active factors released from mast cell granules may stimulate the increased collagen synthesis and fibroblast proliferation."," Fibroblasts themselves have been shown to influence mast cell differentiation and granule synthesis."," Fine regulatory interactions have been shown between the two main types of connective tissue cells, fibroblasts and mast cells. Electron and transmission electron microscopy examinations of fibroblast/mast cell co-cultures suggest that mast cells are capable of extending granule-laden pseudopods to the surface of opposing cells." These mechanisms of transgranulation and degranulation are probably the way in which mast cells expose constituents of their granules to surrounding cells.
Keloid tissue enriched with biologically active products may be accepted as a condition in vivo in which the abnormal histamine content stimulates fibroblast proliferation and increased collagen synthesis. Non-immunological stimuli and/or pharmacological agents might provoke an inhibition of this stimulation and decrease the fibroblast function. There are data in the literature that consider the mechanisms by which a cortisone derivate may depress collagen synthesis. This probably occurred also in our investigation. The way steroids act on mast cells and lead to their decreased number remains unclear to us. We accept that some mast cells might become indistinguishable from surrounding cells as a result of cellular degranulation, during microscopic analysis. Our electron microscopy observations, although devoid of morphological data demonstrate a single mast cell in the dermal zone oi treated keloids.
The main feature of our study is the great reduction in the number of mast cells in steroid-treated keloids. The uniform distribution of mast cells in the total dermis is evidence of our correct clinical treatment. We consider the reduced mast cell count to be an indication of the participation of mast cells not only in the formation of keloids but also in the successive stages of their treatment.

RESUME. Dans cette étude les Auteurs considèrent la question des mastocytes présents dans les chéloïdes traitées avec les stéroïdes. Dix cas de chéloïdes non traitées et traitées avec les stéroïdes ont été évalués pour le numéro de mastocytes. Avec l'emploi de l'analyse morphométrique au microscope optique ils ont observé un numéro réduit de mastocytes dans les chéloïdes traitées avec les stéroïdes par rapport au numéro de mastocytes dans les lésions cutanées non traitées. La réduction dans le numéro des mastocytes est interprétée comme évidence pour la participation de ces cellules dans les phases de la guérison des chéloïdes. Les auteurs discutent enfin le rôle probable des mastocytes et des autres cellules du tissu conjonctif dans les processus du traitement des chéloïdes avec les stéroïdes.

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This paper was received on 8 March 1996.

Address correspondence to: Prof. Janet Mazgalova, Ph.D., Department of Tissue Conservation, Pirogov Emergency Institute, 21 Makedoniya Str., 1606 Sofia, Bulgaria.

 

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