Dermatology: Practical and Conceptual Research | Dermatol Pract Concept 2020;10(4):e2020106 1 Dermatology Practical & Conceptual Diminished Expression of Galectin-3 Around Blisters in Bullous Pemphigoid: An Immunohistochemistry Study Maryam Aghighi1, Bruce R. Smoller2 1 Department of Pathology, Robert Wood Johnson Barnabas Health, Livingston, NJ, USA 2 Department of Pathology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA Key words: bullous pemphigoid, galectin-3, blister formation Citation: Aghighi M, Smoller BR. Diminished expression of galectin-3 around blisters in bullous pemphigoid: an immunohistochemistry study. Dermatol Pract Concept. 2020;10(4):e2020106. DOI: https://doi.org/10.5826/dpc.1004a106 Accepted: June 12, 2020; Published: October 26, 2020 Copyright: ©2020 Aghighi and Smoller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-4.0), which permits unrestricted noncommercial, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: None. Competing interests: The authors have no conflicts of interest to disclose. Authorship: M.A. and B.S. contributed significantly to the manuscript. B.S. designed the study. M.A. and B.S. analyzed and interpreted the data. M.A. wrote the initial draft of the manuscript. M.A. and B.S. edited and finalized the manuscript. Corresponding author: Maryam Aghighi, MD, Department of Pathology, Robert Wood Johnson Barnabas Health, 94 Old Short Hills Road, Livingston, NJ 07039, USA. Email: maryam.aghighi@rwjbh.org Background: Bullous pemphigoid (BP) is a subepidermal blistering disorder caused by autoantibodies directed against hemidesmosomal proteins. Many patients with BP demonstrate circulating IgE auto- antibodies. Although the role of IgE in the pathogenesis of BP is unknown, a correlation between IgE antibodies and eosinophilia has been observed. Soluble CD23 and galectin-3 are the main elements of the IgE group. The roles for CD23 in BP as a potential biomarker and IgE production regulator have been characterized, but no studies have evaluated any roles for galectin-3 in this disease. Objective: In this study, we evaluated galectin-3 expression in BP as a first step in assessing its role in the pathogenesis of this autoimmune blistering process. Patients and Methods: Sixty specimens diagnosed as BP were stained with antibodies to galectin-3. The percentages of nuclear and cytoplasmic galectin-3 expression and staining intensity were evaluated. Results: There was a significant difference in galectin-3 cytoplasmic and nuclear expression within ke- ratinocytes immediately surrounding and above the blisters: (1) cytoplasmic (mean = 17.2% ± 2.4%) vs adjacent unaffected skin (mean = 66.7% ± 2.0%, P < 0.0001) and (2) nuclear (mean = 1.9% ± 0.4%) vs adjacent unaffected skin (mean = 13.2% ± 1.2%, P < 0.0001). Conclusions: Lower expression of galectin-3 around blisters in BP may suggest a role as an adhesion molecule. Loss of galectin-3 may add to the extension of blister formation by initiating cell-extracellular matrix disassembly and may be involved with the associated dermal inflammation and the eosinophil chemotaxis. Further studies will be necessary to elucidate the result of this observed loss on disease pathogenesis. AbstrACt 2 Research | Dermatol Pract Concept 2020;10(4):e2020106 showed the highest intensity with very dark brown-colored stain. Interobserver concordance was high, as staining differ- ences were prominent. All statistical calculations were per- formed using Microsoft Excel software. An alpha level of P < 0.05 was used to indicate significant differences. results All included specimens had been previously diagnosed as BP based upon the patients’ clinical findings, histologic findings of a subepidermal blister that contained eosinophils in the blister cavity, and the demonstration of linear staining with IgG and C3 along the dermal-epidermal junction on contem- poraneously received biopsies analyzed with direct immun- ofluorescence. Galectin-3 showed lower expression above and lateral to the blisters compared to adjacent unaffected skin. We ob- served these changes in galectin-3 expression in both cyto- plasmic and nuclear patterns around the blisters and adja- cent unaffected skin. The galectin-3 expression was diffuse in adjacent unaffected skin with high intensity in both the cy- toplasm and nucleus of keratinocytes. However, expression was diminished and irregular, with lower intensity above and lateral to the blisters. We observed a strong expression of galectin-3 in the inflammatory infiltrate and the surround- ing vessels underlying the blisters, as well as the sebaceous glands near the blisters. Out of 60 biopsies analyzed, this difference was noted in galectin-3 cytoplasmic expression of 56 biopsies (93.3%) and failed to demonstrate the difference in only 4 biopsies (6.7%). Similarly, galectin-3 nuclear expression of 59 biop- sies (98.3%) demonstrated this difference, while only 1 bi- opsy (1.7%) did not show the difference. Figure 1, A-D illustrates an example in which 10% galectin-3 positivity is noted in a cytoplasmic pattern with the intensity of 1+ and 1% nuclear expression with an inten- sity of 1+ above (Figure 1 B) and lateral (Figure 1 D) to the blister. These were significantly lower than adjacent unaf- fected skin (Figure 1C), demonstrating 70% galectin-3 pos- itivity in a cytoplasmic pattern with an intensity of 2+ and 10% nuclear expression with the intensity of 2+. Figure 2, A-D demonstrates that galectin-3 was ex- pressed in 5% and 1% in cytoplasmic and nuclear patterns respectively with the intensity of 1+ above (Figure 2B) and lateral (Figure 2D) to the blister, which were significantly lower than adjacent unaffected skin (Figure 2C) with 70% and 20% galectin-3 positivity in cytoplasmic and nuclear patterns respectively with the intensity of 3. Similarly, Figure 3, A-D shows 10% cytoplasmic and 1% nuclear galectin-3 positivity with the intensity of 1+ above (Figure 3B) and lateral (Figure 3D) to the blister in cyto- Introduction Bullous pemphigoid (BP) is a subepidermal disorder de- scribed by autoantibodies against hemidesmosomes in the dermal and epidermal junction, usually in elderly patients. BP usually starts with pruritis and urticaria and subsequently progresses to the development of blisters on the skin caused by subepidermal separation through the basement mem- brane at the level of the lamina lucida [1]. In addition to the IgG deposition seen along the dermal-epidermal junction (variably with depositions of IgM, IgA and C3, as well), most patients with BP present an autoimmune response by generating IgE autoantibody. Although the role of IgE in the pathogenesis of BP is unknown, a correlation between IgE antibodies and eosinophilia has been observed [2]. Sol- uble CD23 and galectin-3 are 2 main elements of the IgE group. While the role of soluble CD23 in BP as a potential biomarker, IgE production regulator and disease severity predictor has been characterized [3], no studies about pu- tative roles for galectin-3 in BP have been performed [4]. Galectin-3 is a beta galactoside binding protein that is essen- tial in the cell-to-cell or matrix adhesion. It broadly exists in the nucleus and cytoplasm of various cell types or may be found extracellularly on the cell surface [5]. As no stud- ies have been reported on the role of galectin-3 immunohis- tochemical profile in pemphigoid disease, we attempted to evaluate the galectin-3 expression in patients with BP in the current study. Materials and Methods This retrospective study was approved by the institutional review board of our institution. We included 60 specimens from 55 patients diagnosed with BP between 2017 and 2020. Five patients had 2 such biopsies. The biopsy specimens were processed in formalin, dehy- drated through graded alcohol washes, embedded in paraf- fin and sliced on a microtome. Tissue slices on glass slides were stained with Cell Marque mouse monoclonal galectin-3 (MilliporeSigma, Inc.) using an Autostainer Link 48 (Agilent Technologies, Inc) with Low pH antigen retrieval and EnVi- sion FLEX detection kit. The antibody was used in a dilution of 1:25. The immunohistochemical analysis was performed under a light microscope (Olympus BX40F). The percentage of galectin-3 nuclear and cytoplasmic expression around blister and adjacent unaffected skin were evaluated both qualitatively and semi-quantitatively by the authors and compared using a t-test. The intensity of galectin-3 around the blister and adjacent unaffected skin was evaluated semi-quantitatively using the range of 1+ to 4+ and compared using a t-test. The value of 1+ showed the lowest intensity with very light brown-colored stain, and 4+ Research | Dermatol Pract Concept 2020;10(4):e2020106 3 to differentiation and maturation of the keratinocytes [6,7]. Galectin-3 has an important function in cancer development and in inflammatory and immune responses. It has intracel- lular and extracellular functions, such as playing a crucial role in cell-to-cell interactions. Galectin-3 is expressed in keratinocytes, which is believed to have an impact on the functionality of immune cells in response to inflammatory skin conditions. The role of galectin-3 in wound healing and various in- flammatory skin diseases, such as atopic dermatitis and pso- riasis, has been characterized previously. It has been reported that intracellular galectin-3 has an impact on the expression of cell surface and extracellular matrix receptors, which sub- sequently affects cell-matrix interactions and cell migration. This process has a significant contribution to the healing of wounds [8]. Galectin-3 is a pro-inflammatory mediator in atopic der- matitis, needed for the inflammatory response to epicutane- ous antigens via its impact on T cells [9]. Galectin-3 expres- sion in dermal capillaries has a role in the reorganization of the capillary system and the involvement of inflammatory cells in psoriasis [10]. Additionally, it has been shown that plasmic and nuclear patterns, which were significantly lower than adjacent unaffected skin (Figure 3C) with 60% and 10% galectin-3 positivity with the intensity of 3+ in cyto- plasmic and nuclear patterns. Statistically, there was a significant difference in galec- tin-3 cytoplasmic expression within keratinocytes immedi- ately surrounding and above the blisters (mean = 17.2% ± 2.4%) compared to those in the adjacent unaffected skin (mean = 66.7% ± 2.0%, P < 0.0001, Figure 4A). Similarly, galectin-3 keratinocyte nuclear expression around blisters (mean = 1.9% ± 0.4%) was significantly lower than in ad- jacent unaffected skin (mean = 13.2% ± 1.2%, P < 0.0001; Figure 4B). In addition, the cytoplasmic intensity of galec- tin-3 around blisters (mean = 1.42 ± 0.08) was significantly lower than adjacent unaffected skin (mean = 2.6 ± 0.06, P < 0.0001; Figure 4C). Discussion Galectin-3 is a β-galactoside binding lectin that is found in epithelial cells, including keratinocytes. It is expressed in the nucleus and cytoplasm of normal keratinocytes and related Figure 1. Lower expression of galectin-3 around the blister compared to adjacent unaffected skin. (A) Galectin-3 stain of a subepidermal blister of bullous pemphigoid (×20), (B) lower expression of galectin-3 above the blister (×200), (C) adjacent unaffected skin (×200), (D) lower expression of galectin-3 lateral to the blister (×200). A B C D 4 Research | Dermatol Pract Concept 2020;10(4):e2020106 α6β4, wherein integrin α6β4 functions as a laminin-332 re- ceptor. Galectin-3 stimulates the relationship of laminin-332 and integrin α6β4 [14]. Galectin-3 also promotes epidermal growth factor receptor causing genesis of cell surface pro- teins. This process is essential to control the cell signal path- way and migration [15]. Blister formation in BP happens as a result of the loss of epithelial cell-extracellular matrix adhesion at the hemi- desmosomal junction. Since galectin-3 in keratinocytes regu- lates cell-extracellular matrix adhesion, its lower expression around blisters in BP may disrupt the hemidesmosomal ad- hesion complex, resulting in blister formation. While autoantibodies directed against BP1 and 2 an- tigens trigger the blister formation, loss of galectin-3 may play a role in the extension of blister formation by initiating cell-extracellular matrix disassembly at the level of the lam- ina lucida of the basement membrane zone and may also be involved with the inflammatory response and eosinophil chemotaxis underlying the blisters, further accentuating the extent of blister formation. galectin-3 has a role in melanocytic and non-melanocytic skin cancers [11]. Galectin-3 promotes cell adhesion, pro- gression and metastasis in melanoma, and its expression is higher in abnormal melanocytes, in contrast to benign, banal melanocytes [12]. Higher expression of galectin-3 in basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) has been observed [11]. It may interact with regulatory genes and influence the development and progression of BCC and SCC. The cytoplasmic expression of galectin-3 is correlated with tumor size in SCC [6]. There has been no report regarding the role of galectin-3 in blister-forming skin disorders such as BP. Urticarial le- sions may be one of the first presentations in BP and progress to recurrent chronic blisters. Eosinophils are involved in the pathogenesis of BP and essential for anti-BP2 IgE-mediated blistering. Since galectin-3 is the main element of the IgE group, galectin-3 may have a role in the formation of blisters in this disorder. Hemidesmosomes are essential structures in keratino- cytes for epithelial cell-extracellular matrix connections [13]. Hemidesmosomes are composed of plectin 1a and integrin Figure 2. Lower expression of galectin-3 around the blister compared to adjacent unaffected skin. (A) Galectin-3 stain of a subepidermal blister of bullous pemphigoid (×20), (B) lower expression of galectin-3 above the blister (×200), (C) adjacent unaffected skin (×200), (D) lower expression of galectin-3 lateral to the blister (×200). A B C D Research | Dermatol Pract Concept 2020;10(4):e2020106 5 Figure 3. Lower expression of galectin-3 around the blister compared to adjacent unaffected skin. (A) Galectin-3 stain of a subepidermal blister of bullous pemphigoid (×20), (B) lower expression of galectin-3 lateral to the blister (×200), (C) adjacent unaffected skin (×200), (D) lower expression of galectin-3 above the blister (×200). Figure 4. Comparison of galectin-3 expression in blister and adjacent unaffected skin. (A) cytoplasmic expression (P < 0.0001), (B) nuclear expression (P < 0.0001), (C) intensity (P < 0.0001). Gal-3 = galectin-3. A C D B A B C 6 Research | Dermatol Pract Concept 2020;10(4):e2020106 skin. J Dermatol Sci. 2011;64(2):85-91. DOI: 10.1016/j.jderm- sci.2011.07.008. PMID: 21889881. 7. Chen H-Y, Lo C-H, Li C-S, Hsu DK, Liu F-T. Galectins and cutaneous immunity. Dermatol Sin. 2012;30(4):121-127. DOI: 10.1016/j.dsi.2012.10.002. 8. Cao Z, Said N, Amin S, et al. Galectins-3 and-7, but not galec- tin-1, play a role in re-epithelialization of wounds. J Biol Chem. 2002;277(44):42299-42305. DOI: 10.1074/jbc.M200981200. PMID: 12194966 9. Saegusa J, Hsu DK, Chen H-Y, Yu L, Fermin A, Fung MA, Liu F-T. Galectin-3 is critical for the development of the allergic inflammatory response in a mouse model of atopic dermati- tis. Am J Clin Pathol. 2009;174(3):922-931. DOI: 10.2353/ ajpath.2009.080500. PMID: 19179612. 10. Lacina L, Plzakova Z, Smetana K, Stork J, Kaltner H, Andre S. Glycophenotype of psoriatic skin. Folia Biol (Praha). 2006;52(1-2):10-15. PMID: 17007105. 11. Kapucuoglu N, Basak PY, Bircan S, Sert S, Akkaya VB. Immunohistochemical galectin-3 expression in non-melanoma skin cancers. Pathol Res Pract. 2009;205(2):97-103. DOI: 10.1016/j.prp.2008.09.001. PMID: 18951731. 12. Prieto VG, Mourad-Zeidan AA, Melnikova V, et al. Galectin-3 expression is associated with tumor progression and pattern of sun exposure in melanoma. Clin Cancer Res. 2006;12(22):6709- 6715. DOI: 10.1158/1078-0432.CCR-06-0758. PMID: 17121890. 13. Koster J, Geerts D, Favre B, Borradori L, Sonnenberg A. Analysis of the interactions between BP180, BP230, plectin and the integrin α6β4 important for hemidesmosome assembly. J Cell Sci. 2003;116(2):387-399. DOI: 10.1242/jcs.00241. PMID: 12482924. 14. Walko G, Castañón MJ, Wiche G. Molecular architec- ture and function of the hemidesmosome. Cell Tissue Res. 2015;360(2):363-378. DOI: 10.1007/s00441-014-2061-z. PMID: 25487405. 15. Kariya Y, Kawamura C, Tabei T, Gu J. Bisecting GlcNAc residues on laminin-332 down-regulate galectin-3-dependent keratinocyte motility. J Biol Chem. 2010;285(5):3330-3340. DOI: 10.1074/jbc.M109.038836. PMID: 19940114. Conclusions We demonstrated lower expression of galectin-3 around the blisters in BP specimens. The pathogenesis of the blis- ter formation may be associated, at least in part, with al- tered expression of galectin-3. Further studies are required to elucidate the result of this loss on the pathogenesis of the observed histologic findings and determine the exact patho- genesis of blister formation. Acknowledgments We would like to express our special thanks of gratitude to Ms. Sierra Kovar who helped us in collecting the cases and preparing and staining the slides. references 1. Schmidt E, Della Torre R, Borradori L. Clinical features and practical diagnosis of bullous pemphigoid. Immunol Allergy Clin. 2012;32(2):217-232. DOI: 10.1016/j.iac.2012.04.002. PMID: 22560135. 2. 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