Mast Cells in Non-neoplastic Skin Lesions

8 Indian Medical Gazette JANUARY 2013 Original Article Mast Cells in Non-neoplastic Skin Lesions Meenakshyee M. Joshi, Lecturer, Archana C. Buch, Professor, N. K. Panicker, Professor, S. S. Chandenwale, Professor Department of Pathology, Padmashree Dr. D. Y. Patil Medical College, Pimpri, Pune. Abstract Background: Mast cells are found in tissues throughout body, particularly in proximity to surfaces that interface with the external environment. They are found at all levels of dermis, where they are grouped around blood vessels, nerves and appendages. Increase in mast cell number have been reported in various cutaneous diseases. Aims: We aim to identify and analyse the morphological aspects of distribution of mast cells and their value in non-neoplastic skin lesions. Material and Methods: Ninety seven biopsies of various skin lesions were studied for mast cells by Toluidine Blue at ph 3.9. Result: All the skin lesions showed significant increase in mast cell number. Psoriasis showed maximum number of mast cells (127/mm 2 ) followed by Lichen planus (108/mm 2 ). Among the cases of cutaneous bacterial infections, mast cells were maximum in leprosy cases (99/mm 2 ) and lowest in cutaneous tuberculosis. A comparative decrease in number of mast cells was seen from lepromatous leprosy to tuberculoid leprosy. A wide range of morphological alterations of mast cells were observed in these skin lesions. Psoriasis and bullous pemphigoid cases showed maximum spindle cell morphology. Conclusion: Tissue mast cell number, distribution and morphology vary in different skin lesions depending on the degree of mast cell activation. This may have a bearing on diagnosis and management. Keywords mast cells, non-neoplastic skin lesions Introduction Mast cells are dynamic cells playing central role in allergic inflammation, protective immune response and other inflammatory responses. They are found in all levels of skin including dermis, around blood vessels, nerves, appendages, at dermoepidermal junction (DEJ) and also in subcutaneous tissue 1,2,3. Variations in morphologic, biochemical and/or functional characteristics of mast cells from different anatomical sites have been reported raising the possibility that mast cells of different phenotypes may have different functions in health and disease. In the present study, we attempt to evaluate mast cell infiltration with respect to morphology, distribution and quantity in non neoplastic skin lesions using simple histologic techniques. Material and Methods The study included total 103 skin biopsies, 97 from various non neoplastic skin lesions and 6 from normal appearing skin as control. The tissues were fixed in 10% formalin, processed by routine paraffin embedding technique. Two sections were obtained from each block, one section was stained by hematoxylin and eosin and was evaluated histologically with clinical correlation. The other section was stained with freshly prepared Toluidine blue stain. The Toluidine blue stained sections were evaluated for qualitative and quantitative aspects of mast cells (Leica Q Win V 3 software analysing system). Mast cells were counted per field at 400x magnification, 1pixel, zoom 0.27x Address for correspondence: Dr Archana Buch, B 603, Gold Coast, Ivory Estates, Someshwarwadi, Pune 411 008. E-mail : drarchanabuch@yahoo.co.in

Indian Medical Gazette JANUARY 2013 9 using image analyser. The area selected included DEJ, perivascular, periadenexal, and areas of cellular infiltrate. Mast cells were categorised as granulated and degranulated. The granulated mast cells were further categorized morphologically as round, spindle or oval. The result of the study are expressed as the mean mast cell count per mm 2. These were analysed statistically by using unpaired t test(t value), annova (F value) and Z test (z value) and p-value (probability) was calculated. Observation Ninety seven non-neoplastic skin lesions were grouped into six different categories and mean mast cell counts in these are presented in Table 1. In normal skin, mast cells were seen predominantly in perivascular and periadenexal location. They were sparse around DEJ and in dermis. They were round to oval in shape and intact in nature. The mean mast cell count was 33.33/mm 2 and a S.D of 8.16. The mast cell morphology and distribution in various non neoplastic skin lesions are presented in Table 2. Since only few cases of vasculitis, atrophoderma, keloid, hypertrophic scar and dermatitis herpetiformis were included in the study, they have been grouped as others for statistical analysis. Skin lesions were maximum in 2nd and 3rd decade of life with M:F ratio 1.94:1. There was slight increase in mast cell number as age increased. The maximum number of mast cell count was seen in lesions over trunk as compared to other sites. Among the spectrum of leprosy cases, mast cell count

10 Indian Medical Gazette JANUARY 2013 was maximum in lepromatous leprosy (LL) and minimum in borderline tuberculoid (BT). Reactional leprosy cases of erythema nodosum leprosum (ENL) revealed mast cell count similar to borderline lepromatous leprosy (BL). Older lesions of Psoriasis had less mast cells than earlier lesions. In Lichen planus, mast cell- nerve contact was not observed. Round cell morphology of mast cells (Fig. 1a) was seen predominant in leprosy, lichen planus, lichen simplex chronicus. Spindle cells (Fig. 1b) were seen maximum number in cases of psoriasis followed by bullous pemphigoid. Keloid had all variety of mast cells with more of spindle cell morphology. Oval cells (Fig. 1c) were seen more in leprosy, psoriasis and atopic dermatitis. Most of the skin lesions showed both degranulated and granulated mast cells (Fig. 1d). Lichen Planus and leprosy had more degranulated mast cells (Fig. 1d). Favoured location of mast cell distribution in leprosy cases were periappendageal (Fig. 2a) and perivascular (Fig. 2b). Cutaneous tuberculosis had a predilection for Fig. 1 1a: Round mast cell in dermis, 1b: Spindle mast cell in dermis, 1c: Oval mast cell at dermoepidermal junction, 1d: Degranulated mast cell at dermoepidermal junction. (Toluidine blue, 400x) periappendageal areas while lichen planus and erythema nodosum leprosum had more perivascular and dermal collections (Fig. 2c) respectively. A few cases of leprosy showed mast cells around granulomas (Fig. 2d).

Indian Medical Gazette JANUARY 2013 11 highlights the role of mast cells in causing the histopathological changes of ENL. The reactional leprosy group (ENL) had more mast cell count in dermis as compared to non-reactional leprosy. This finding may indicate the important role of mast cells in dynamic changes in the cell mediated immune response in leprosy and leprosy reactions 5,6,7. Fig. 2 2a: Round, oval, spindle (arrow) and degranulated (arrowhead) mast cells in periappendageal area, 2b: Round mast cell (arrow) and degranulated mast cell (arrowhead) in perivascular area, 2c: Oval mast cell in dermoepidermal junction, 2d: Spindle mast cell around granuloma in leprosy. (Toluidine blue, 400x) Discussion In the present study, the mast cell density showed progressive increase over immunological spectrum of tuberculoid leprosy to lepromatous leprosy except borderline cases. The interesting difference in mast cell count between polar and the borderline cases, was that the lepromatous area of the borderline cases showed lower counts compared to that of the polar lepromatous cases. On the contrary, in the tuberculoid area, the borderline cases showed higher counts compared to polar tuberculoid leprosy (TT). Aroni et al had similar findings 4. This suggests that, periodic follow up of indeterminate and borderline lesions for mast cell count might help in predicting stability of the lesions. In LL, there is predominance of T-helper cell type 2 (TH2) response of anti inflammmatory cytokines as against T- helper cell type 1(TH 1) response of proinflammatory cytokine liberation seen in TT leprosy. Mast cells are believed to be associated with a variety of cytokines such as TNF-α, IL-1, IL-4, IL-6 and GM-CSF, most of which belong to TH 2 cytokine response. Predominance of TH 2 response in LL thus, explains the presence of raised mast cell counts in it 5. Erythema nodosum leprosum cases revealed mast cell density similar to those seen in BL leprosy cases. Maximum number of mast cells were observed in dermis, which Mast cells in tuberculosis inflammation have received little attention. We found an increase in mast cell count, but to a lesser extent as compared to other non neoplastic skin lesions. In any case, appearance of caseation heralds a decline in mast cell count. Mast cell products such as histamine, heparin and TNF-α are mitogenic for fibroblasts and endothelial cells. Hence, the increase in vascularity in lepromatous leprosy and scarring in cutaneous tuberculosis may be attributed to these mediators from increased mast cells in these lesions 5. Lower mast cell count in tubercular granulomas as compared to tuberculoid leprosy may help in distinguishing these two entities which can mimic each other. We found significant increase in number of spindle, oval and round shapes and degranulation in mast cells in cases of psoriasis. This is relevant in the pathogenesis of psoriasis. Earliest manifestation of psoriasis is swelling and widening of intercellular junction of endothelial cells. This is followed by the appearance of mast cells around post-capillary venules showing degranulation 8. Mast cells secrete TNF-α which induces expression of ICAM-1 which is considered to be an important initiator of leukocytekeratinocyte interaction in skin inflammation 9. Jiang WY et al have noted that increase levels of IL-8 in keratinocytes of psoriatic plaques play a contributing role in migration of mast cells to lesion sites 10. Degranulated and spindle shaped active mast cells releases powerful serine proteinases, tryptase and chymase which promotes inflammation. It was also noted that in older psoriatic lesions, the number of mast cells were significantly lower than in newly formed psoriatic lesions. It decreases progressively also with effective treatment 11. Hence, mast cell density may reflect the age of lesion, relapse and response to treatment. In lichen planus, though there was significant increase in mast cell infiltration, no increased mast cell-nerve contact was noted. This finding is similar to that of

12 Indian Medical Gazette JANUARY 2013 Naukkarinen A et al who suggested that mast cell number were increased in both lesional and symptom free skin in lichen planus but there was no increase in mast cell-nerve contacts. They suggested that the massive inflammatory reaction in lichen planus is not neurogenic 11. We also did not notice increase in mast cell-nerve contact and thus, support the above findings. The initiation of atopic dermatitis is driven by activation of TH-2 cells, leading to increase levels of IL-4, IL-10, IL- 13 and IgE. IgE contributes to the inflammatory cell infiltrate by several mechanisms. Acute skin inflammation is associated with a predominance of IL-3 and IL-4 expression 12. Thus, mast cells play an important role in atopic dermatitis. Lichen Simplex Chronicus is a prototype of atopic dermatitis. We observed increase in mast cells in these lesions. Mast cells secrete fibrogenic cytokines which explains increase the number of fibroblast activity and vertically oriented collagen bundles in these lesions. The increased number and morphological alterations (round and spindle shapes) in bullous pemphigoid observed by us is similar to that of Dvorak et al 13. They also observed a sequence of histopathologic events which began with mast cell alteration and proceeded to infiltration by various inflammatory cells. Renata et al also observed increased degranulation of mast cells in developing lesions of bullous pemphigoid together with progressive eosinophillic infiltration 14. Our findings were similar. Mast cell derived IL-5 could be a potent mediator in attracting and stimulating eosinophils at the blister site. Lee et al observed an increase in number of mast cells in patients with active pruritic keloid. Mast cells were closely associated with myofibroblasts with frequent direct cell contacts. The filopodia of mast cells were seen intimately applied along the cell membrane of myofibroblast. Degranulated mast cells were also seen and mast cell granule were seen lying free in interstitium. The surrounding interstitial matrix showed edema. It is apparent that the interaction between mast cells, their products and myofibroblasts, has an important role in pathogenesis of keloid 15. Conclusion The role of mast cells in health and disease is significant. Distinct alterations of mast cells are also noted in the various non neoplastic skin lesions. Mast cell density, distribution and morphology may be useful in identifying various skin lesions, understanding their evolution and modify treatment protocols. References 1. Metcalfe D.D., Baram D., Mekori Y.A. Mast cells. Physiol Rev. 77(4):1033-1079, 1997. 2. Beutler E., Litchman M.A., Barry S.C., Kipps T.J. Production, biochemistry, and functions of basophils and mast cells In: editors. Williams hematology, 5th ed. New York: McGraw-Hill; p 805-865, 1995. 3. Ramdas Naik, Pai M.R., Bautwal P.B., Shankar Narayana, Nayak K.S., Gandhi A. Study of mast cells in non neoplastic skin lesions. Indian J Pathol Microbiol. 46: 173-175, 2003. 4. Kyriaki Aroni, George K., Liossi Anna, Dimitris Panteleous. An investigation of mast cells in two basic leprosy groups. Int J Leprosy. 61:634-635, 1993. 5. Bagwan I.N., Khandekar M.M., Khadana P., Jadhav M.V., Deshmukh S.D. A study of mast cells in granulomatous lesions of skin with special emphasis on leprosy. Indian J Leprosy. 76(1): 31-37, 2004. 6. Cree I.A., Coghill G., Beck J.S. Mast cells in leprosy skin lesions. J Clin Pathol. 43:196-200, 1990. 7. Mahasavariya P., Jiamton S., Manonukul J., Khemngern S. Mast cells in leprosy and leprosy reactions. International J of Dermatol. 39(4):274-277, 2000. 8. Mobini N., Toussaint S., Kamino H. Noninfectous Erythematous, Papular, and Squamous Diseases. In: Elder E. David, editor. Histopathology of the skin. 10thed. Philadelphia: Lippincott Williams & Wilkins; p 175-176, 2010. 9. Brody I. Mast cell degranulation in the evolution of acute eruptive guttate psoriasis vulgaris. J Invest Dermatol. 82(5):460-464, 1984. 10. Toruniowa B., Jablonska S. Mast cells in initial stages of psoriasis. Arch Dermatol Res. 280(4):189-193, 1998.

Indian Medical Gazette JANUARY 2013 13 11. Naukkarinen A., Harvima I.T., Aalto M.L., Harvima R.J., Horsmanheimo M. Quantitative analysis of contact sites between mast cells and sensory nerves in cutaneous psoriasis and lichen planus based on a histochemical double staining technique. Arch Dermatol Res. 283:433-437, 1991. 12. Wu H., Brandling-Benett A.H., Harrist J.T. Noninfectious Vesicobullous and Vesicopustular Diseases. In: Elder E. David, editor. Histopathology of the skin. 10thed. Philadelphia: Lippincott Williams & Wilkins; p 242, 2010. 13. Dovark A.M., Mihm M.C. Jr, Osage J.E., Kwan T.H., Austen K.F., Wintroub B.U. Bullous pemphigoid, an ultrastructural study of inflammatory response: eosinophil, basophil and mast cell granule changes in multiple biopsies from one patient. J Invest Dermatol. 78(2):91-101, 1982. 14. Renata Kaminska, Naukkarinen A., Glinski W., Horsmanheimo M., Harvima I.T. Mast cells in developing subepidermal bullous diseases: Emphasis on tryptase, chymase and protease inhibitors. Acta Derm Venerol. 79:351-355, 1999. 15. Lee Y.S., Vijayasingam S. Mast cells and myofibtoblasts in keloid: a light microscopic, immunohistochemical and ultrastructural study. Ann Acad Med Singapore. 24(6):902-905, 1995.