DISCUSSION
Most of pathophysiology of CSU can be understood from histopathological studies of urticarial lesions.3 Barsilay et al.14 described histopathological changes in urticaria biopsies by demonstrating the presence of numerous cells apart from mast cells, such as eosinophils, lymphocytes, and neutrophils, in the skin.14 In a study conducted by our group in 2016, we demonstrated that urticaria with predominance of eosinophils was related to greater clinical activity of the disease.15 Other authors have described the presence of CD4+ T lymphocytes, variable number of monocytes, basophils, and lymphocytes.2,16 The cytokines involved in the inflammatory skin process in urticaria reveal a mixed cytokine pattern with an increase in IL-4, IL-5, and IFN gamma.16 In 1990, Czarnetzki et al.17 described the presence of macrophages in acute, chronic, and Delayed Pressure Urticaria; and, with the scarce resources available at the time for immunohistochemistry, they observed macrophage cell activation in the lesioned skin.17 Neverthless any previous study has demonstrated the role of macrophage subpopulations (M1/M2 phenotype) in CSU.
In a previous study, we observed a cellular interaction between dendrocytes and mast cells in the microvascular unit of the dermis, using immunoelectronic microscopy.4 The observation allowed us to assume that the macrophage could participate as adjuvant of the immunological response in urticaria; however, their role was not completely established at the time.4 An interaction with macrophages can occur via release of histamine and cytokines such as IL-1 beta, IL-6, and IL-13 in addition to prostaglandin D2 (PGD2) and platelet activating factor (PAF).2 PGD2, PGE2, IL-1 beta, IL-9, and histamine also influence the dendritic cells.18 The activation of macrophages and dendritic cells has been postulated to be involved in the pathophysiology of urticaria.2,18
Macrophages basically perform two functions: (i) killer function, when their function is to eliminate a pathogen or defective cell, which basically depends on M1 populations or (ii) repair, when their functions involve regulation of immune response, angiogenesis, and healing, which depends on M2 macrophages, also termed as “alternatively activated macrophages.6,18,19 In addition to the normal repair function, the M2 macrophage is related to the chronicity of diseases and worse immune responses in the in vitro studies in sepsis, infectious diseases, autoimmune diseases, and vascular diseases.7,9-13,19
The presence of M2 macrophages in tumors is related to a worse prognosis and formation of metastases.19
Few studies have linked the presence of M1 and M2 macrophages in allergic diseases or in diseases in which the participation of mast cells is important, such as urticaria.9,10 In these diseases, M2 macrophages are related to chronicity, worse immune response or worse prognosis.9,10,19,20
The imbalance between TH1/TH2 cytokines has long been considered as a possible mechanism for urticaria. Chen et al.21observed a mixture of TH1/TH2 cytokines in urticaria, in addition to TH17 cytokines.21 In patients with chronic urticaria, there was a predominance of the TH2 pathway, in addition to an increase in GM-CSF.21 The change to M2 macrophages is activated by the presence of IL-10, IL-4, IL-13, GM-CSF, IL-6, corticosteroids, apoptotic debris, and hypoxia, that is, the change is induced by local inflammatory milieu, in order to antagonize and resolve an inflammatory process.5-13,19,22
In the present study, we observed a predominance of the CD163, CD206, and CMAF receptors, compared to pSTAT-1 (M1 macrophage marker), regardless of age, sex, and laboratory tests evaluated, demonstrating that in uncontrolled CSU with maximum doses of second generation anti-H1, in general, the macrophage subtype found is an alternatively activated phenotype (M2).7,8,11,19,21
While studying the immunohistochemical markers in the skin of control individuals, a statistically significant relationship was found with the CMAF marker, which is related to the transcription of cytokines responsible for macrophage and lymphocytic activation.12,22 In studies conducted in mice, CMAF deficiency is directly related to the loss of expression of the F4/80 factor, which regulates the migration of macrophages to peripheral tissues and directly influences IL-4 production.23,24This observation is important, since the expression of CMAF was statistically significant compared to the control individuals, due to the fact that this nuclear transcription factor is constitutionally expressed in a small amount in resident macrophages; however, IL-4 can positively or negatively regulate its production.23
Gabryšová et al.26 demonstrated that CMAF in lymphocytes may be implicated in several normal biological processes and in allergic and autoimmune diseases, because it suppresses the synthesis of IL-2 in CD4+ lymphocytes, activates T helper2 (TH2) cells in allergy, and further activates TH17 lymphocytes, leading to the onset of autoimmune diseases.26 Since our patients had urticaria refractory to antihistamines and high clinical activity of urticaria, a hypothesis could be that a higher expression of CMAF is the result of an exaggerated stimulation mediated by IL-4 and IL-13 cytokines.19,23
CMAF leads to the production of IL-10 and activation of the TH17 pathway, in addition to the perpetuation of TH2-mediated inflammation, which could prolong the inflammatory process and contribute to the maintenance and chronicity of urticaria.20,21,23Another possible explanation would be the continued or intermittent use of systemic corticosteroids by patients with urticaria refractory to antihistamine drugs, which also induces the formation of M2 macrophages with activation of CMAF.1,19,22
The immunohistochemical marker CD206 was negatively associated with the evolution of CSU in our patients. The macrophage mannose receptor (MMR or CD206) is highly expressed in M2 macrophages, strongly induced by IL-4 and IL-13, but not in M1 macrophages.7,19Simultaneously, it is the first receptor to be activated in case of trauma or tissue injury, which leads to the activation of STAT-6, while stimulating the synthesis of TH2 cytokines (IL-4 and IL-13) e IL24.19,20,27 Scmettzer et al28showed that in CSU patients had IgE autoantibodies against IL24 linked with the disease activity. We can infer that in anti-histamines resistant patients we could have a continuous production of IL24 by the M2 macrophage that estimulate the mast cell through IgE anti IL2427,28
We showed a decrease in the concentration of the CD206 in relationship with the course of the disease. One possible explanation could be the fact that more recent studies demonstrate the existence of four subtypes of M2 macrophages.19 We presently know that M2 macrophages can be subdivided into four subpopulations, depending on the stimulus received: (i) M2a, stimulated predominantly by cytokines IL-4 and IL-13 and JAK-1 and JAK-3 receptors, is a high affinity receptor for IgE and increases the concentrations of CD206; (ii) M2b, stimulated by immunocomplexes, increases the secretion of proinflammatory cytokines; (iii) M2c, stimulated by corticosteroid binding to its receptors IL10 and TGF beta; and (iv) M2d, stimulated by high concentrations of IL-10 and tumor factors.9,19,20 CD206 is highly expressed by M2a macrophages but is diminished in the other subtypes and its expression changes according to the influence of the inflammatory milieu; thus, we can assume that an attempt to resolve the inflammatory process is observed in chronic urticaria.
One explanation for M2 macrophages being activated in CSU would be the possibility of elevating IL-25, an IL-17 family cytokine, produced by several cells activated in urticaria, including mast cells, dendrocytes, and eosinophils.29 This could contribute to the presence of activated M2 macrophages in urticaria, indirectly revealed in our study, as it is associated with the increase in CMAF.26,29.
In an immunohistochemical study of eight skin biopsies of urticaria cases, Kay et al.29 observed an increase in the IL-25 expression in mast cells and eosinophils of patients with urticaria. IL-25 belongs to the IL-17 cytokine family and is a potent promoter of TH2 response, and increased expression can be observed in epithelial cells, mast cells, and eosinophils in urticaria lesions.2,29 The observation that mast cells can produce IL-25, followed by an IgE-dependent activation suggests a possible innate immunity pathway that can trigger or amplify TH2 responses.2,29 A second member of the IL-17 family, thymic stromal lymphopoietin (TSLP), is also elevated in mast cells in asthma and mast cell cultures.2 TSLP does not induce mastocytic degranulation; however, it increases the recruitment of mast cells and dendrocytes in the skin.30
Mast cells are negatively regulated via activation of phosphoinositide lipid phosphatases (in general, SHIP 1 and 2) and their deregulation and decreased activity are associated with CSU. The increased activity of SHIP 1 and 2 is related to a deviation in the M1 phenotype of macrophages. Therefore, the deregulation of SHIP could also explain the increase in M2 macrophages.30,31
Recently, the presence of new vessels (neoangiogenesis) has been described in patients with CSU. Kay et al.32 reported that the injured skin of patients with urticaria displays increased dermal vascularization.32 VEGF is a regulatory factor of angiogenesis, inducing endothelial cell proliferation, migration, and secretion of metalloproteinase 1 (MMP1).3,30 Moreover, VEGF promotes the expression of von Willebrand factor, in addition to adhesion molecules such as ICAM-1, VCAM-1, and E-selectin.33 VGEF has been implicated as an induction factor of M2 macrophage polarization.32,33 Macrophage M2 in turn can promote angiogenesis by releasing IL10, TGF-beta, and other cytokines, which results in greater vascularity found in the skin of patients with chronic urticaria.7,33
The present study had limitations, as we were not able to subclassify the type of M2 macrophages, as already done in patients with asthma and contact dermatitis.8,19,20 This subcategory would allow us to observe subtypes of M2 macrophages, which could lead to specific treatments for these patients.
In conclusion, in the CSU patients studied, a predominance of M2 macrophages was observed, with significantly higher CMAF expression, which indicates macrophage activation in patients with CSU. Moreover, we observed a negative correlation between the presence of the CD206 marker and the duration of CSU. Further studies are warranted to focus on therapies targeting M2 macrophage subtypes in patients with urticaria refractory to antihistamines in order to assess their contribution in deciding the best treatment for this disease.
References:
  1. Zuberbier T, Aberer W, Asero R, et al. The EAACI/GA²LEN/EDF/WAO guideline for the definition, classification, diagnosis and management of urticaria.Allergy . 2018;73(7):1393–1414.
  2. Church MK, Kolkhir P, Metz M, Maurer M. The role and relevance of mast cells in urticaria.Immunol Rev . 2018;282(1):232–247. doi:10.1111/imr.12632
  3. Puxeddu I, Pratesi F, Ribatti D, Migliorini P. Mediators of Inflammation and Angiogenesis in Chronic Spontaneous Urticaria: Are They Potential Biomarkers of the Disease?.Mediators Inflamm . 2017;2017:4123694. doi:10.1155/2017/4123694
  4. Criado PR, Jardim Criado RF, Sotto MN, Pagliari C, Takakura CH, Vasconcellos C. Dermal dendrocytes FXIIIA+ phagocytizing extruded mast cell granules in drug-induced acute urticaria.J Eur Acad Dermatol Venereol . 2013;27(1):e105–e112. doi:10.1111/j.1468-3083.2012.04556.x
  5. Yang Z, Grinchuk V, Urban JF Jr, et al. Macrophages as IL-25/IL-33-responsive cells play an important role in the induction of type 2 immunity. PLoS One . 2013;8(3):e59441. doi:10.1371/journal.pone.00594
  6. Wang N, Liang H, Zen K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol . 2014;5:614. doi:10.3389/fimmu.2014.00614.
  7. Shapouri-Moghaddam A, Mohammadian S, Vazini H, et al. Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol . 2018;233(9):6425-6440. doi:10.1002/jcp.26429
  8. Barros MH1, Hauck F, Dreyer JH, Kempkes B, Niedobitek G. Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages. PLoS One. 2013 doi: 10.1371
  9. Saradna A, Do DC, Kumar S, Fu QL, Gao P. Macrophage polarization and allergic asthma. Transl Res . 2018;191:1–14. doi:10.1016/j.trsl.2017.09.002
  10. Takabayashi T, Kato A, Peters AT, Hulse KE, Suh LA, Carter R, Norton J, Grammer LC, Tan BK, Chandra RK, Conley DB, Kern RC, Fujieda S, Schleimer RP. Increased expression of factor XIII-A in patients with chronic rhinosinusitis with nasal polyps.. J Allergy Clin Immunol. 2013;132(3):584-592.e4. doi: 10.1016/j.jaci.2013.02.003.
  11. Palmer MB1, Vichot AA2, Cantley LG2, Moeckel GW1. Quantification and localization of M2 macrophages in human kidneys with acute tubular injury . Int J Nephrol Renovasc Dis. 2014 Nov 7;7:415-9
  12. de Carli ML, Miyazawa M, Nonogaki S, et al. M2 macrophages and inflammatory cells in oral lesions of chronic paracoccidioidomycosis. J Oral Pathol Med . 2016;45(2):141–147. doi:10.1111/jop.12333
  13. Chávez-Galán Leslie, Olleros Maria L., Vesin Dominique, Garcia I. Much More than M1 and M2 Macrophages, There are also CD169+ and TCR+ MacrophagesFront. Immunol., 26 May 2015 | https://doi.org/10.3389/fimmu.2015.00263
  14. Barzilai A, Sagi L, Baum S, et al. The Histopathology of Urticaria Revisited-Clinical Pathological Study. Am J Dermatopathol . 2017;39(10):753-759. doi:10.1097/DAD.0000000000000786
  15. Marques RZ, Criado RF, Machado CD Filho, Tamanini JM, Mello CV, Speyer C. Correlation between the histopathology of chronic urticaria and its clinical picture. An Bras Dermatol . 2016;91(6):760-763. doi:10.1590/abd1806-4841.20165066
  16. M. Caproni, B. Giomi, W. Volpi et al., “Chronic idiopathic urticaria: infiltrating cells and related cytokines in autologous serum-induced wheals,” Clinical Immunology, vol. 114, no. 5 pp. 284–292, 2005
  17. Czarnetzki BM, Zwadlo-Klarwasser GZ, Bröcker EB, Sorg C. Macrophage subsets in different types of urticaria. Arch Dermatol Res . 1990;282(2):93-97. doi:10.1007/BF00493465
  18. Varricchi G, Rossi FW, Galdiero MR, et al. Physiological Roles of Mast Cells: Collegium Internationale Allergologicum Update 2019. Int Arch Allergy Immunol . 2019;179(4):247-261. doi:10.1159/000500088
  19. Shrivastava R, Shukla N. Attributes of alternatively activated (M2) macrophages. Life Sci . 2019;224:222–231. doi:10.1016/j.lfs.2019.03.062.
  20. Suzuki K, Meguro K, Nakagomi D, Nakajima H. Roles of alternatively activated M2 macrophages in allergic contact dermatitis. Allergol Int . 2017;66(3):392–397. doi:10.1016/j.alit.2017.02.015
  21. Chen Q, Zhong H, Chen WC, et al. Different expression patterns of plasma Th1-, Th2-, Th17- and Th22-related cytokines correlate with serum autoreactivity and allergen sensitivity in chronic spontaneous urticaria. J Eur Acad Dermatol Venereol . 2018;32(3):441-448. doi:10.1111/jdv.14541
  22. Tu GW, Shi Y, Zheng YJ, et al. Glucocorticoid attenuates acute lung injury through induction of type 2 macrophage. J Transl Med . 2017;15(1):181. Published 2017 Aug 29. doi:10.1186/s12967-017-1284-7
  23. Cao S, Liu J, Song L, Ma X. The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages. J Immunol . 2005;174(6):3484–3492. doi:10.4049/jimmunol.174.6.3484
  24. Bauquet, A.T., et al., 2009. The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells.Nat. Immunol. 10, 167–175
  25. Nakamura M, Hamada M, Hasegawa K, et al. c-Maf is essential for the F4/80 expression in macrophages in vivo. Gene . 2009;445(1-2):66-72. doi:10.1016/j.gene.2009.06.003
  26. Gabryšová L, Alvarez-Martinez M, Luisier R, et al. c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells [published correction appears in Nat Immunol. 2019 Mar;20(3):374]. Nat Immunol . 2018;19(5):497–507. doi:10.1038/s41590-018-0083-5
  27. Dabitao D, Hedrich CM, Wang F, Vacharathit V, Bream JH. Cell-Specific Requirements for STAT Proteins and Type I IFN Receptor Signaling Discretely Regulate IL-24 and IL-10 Expression in NK Cells and Macrophages. J Immunol . 2018;200(6):2154-2164. doi:10.4049/jimmunol.1701340
  28. Schmetzer O, Lakin E, Topal FA, et al. IL-24 is a common and specific autoantigen of IgE in patients with chronic spontaneous urticaria. J Allergy Clin Immunol . 2018;142(3):876-882
  29. Kay AB, Clark P, Maurer M, Ying S. Elevations in T-helper-2-initiating cytokines (interleukin-33, interleukin-25 and thymic stromal lymphopoietin) in lesional skin from chronic spontaneous (’idiopathic’) urticaria. Br J Dermatol . 2015;172(5):1294–1302. doi:10.1111/bjd.13621
  30. Dobranowski P, Sly LM. SHIP negatively regulates type II immune responses in mast cells and macrophages [published online ahead of print, 2018 Jan 17]. J Leukoc Biol . 2018;10.1002/JLB.3MIR0817-340R. doi:10.1002/JLB.3MIR0817-340R
  31. Bracken SJ, Abraham S and MacLeod AS (2019) Autoimmune Theories of Chronic Spontaneous Urticaria. Front. Immunol. 10:627. doi: 10.3389/fimmu.2019.00627
  32. Kay AB, Ying S, Ardelean E, et al. Elevations in vascular markers and eosinophils in chronic spontaneous urticarial weals with low-level persistence in uninvolved skin. Br J Dermatol . 2014;171(3):505-511. doi:10.1111/bjd.12991
  33. Wheeler KC, Jena MK, Pradhan BS, et al. VEGF may contribute to macrophage recruitment and M2 polarization in the decidua. PLoS One . 2018;13(1):e0191040. Published 2018 Jan 11. doi:10.1371/journal.pone.0191040