Elsevier

Burns

Volume 38, Issue 5, August 2012, Pages 668-676
Burns

Post-burn hypertrophic scars are characterized by high levels of IL-1β mRNA and protein and TNF-α type I receptors

https://doi.org/10.1016/j.burns.2011.12.012Get rights and content

Abstract

Post-burn hypertrophic scars are characterized by increased collagen synthesis and hyperplasia, and may be associated with erythema, pain, dysesthesia, pruritus, and skin border elevation. Although the etiopathogenesis of hypertrophic scarring remains unclear, proinflammatory and profibrogenic cytokines are known to play an important role in general skin dysfunction. This study assessed mRNA expression, proteins, and type I receptors of tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1β) in normal skin, normotrophic and post-burn hypertrophic scars. Skin biopsies were obtained from 10 hypertrophic and 9 normotrophic scars, and 4 normal skin sites. Only post-burn scars covering more than 10% of the body were included. Ex vivo histopathological analysis evaluated scar maturity, in situ hybridization assessed mRNA expression, and cytokine protein and cytokine/cell colocalization were performed using single- and double-label immunohistochemistry, respectively. IL-1β is overexpressed in hypertrophic scars at the post-transcriptional level, associated primarily with keratinocytes and CD1a+ cells. Type I receptors for TNF-α are overexpressed in blood vessels of hypertrophic scars. The coordinated overexpression of IL-1β and TNF-α type I receptor may maintain the fibrogenic phenotypes of hypertrophic scars, even those in “remission”.

Introduction

Burns constitute a high-interest research area due to the physiological importance of the skin. The functional, aesthetic, and psychological consequences of burn scars may dramatically influence patients’ quality of life [1]. Among other important characteristics that determine a scar's state of activation, the number of T lymphocytes and macrophages in the inflammatory infiltrate is a likely determinant in scar development [2], [3], [4], [5], [6]. A correlation has also been proposed between the quantity, function, and activity of immune cells in the inflammatory infiltrate, and fibroblast to myofibroblast differentiation via the production and expression of various cytokines [7], [8], [9]. These cytokines influence the development of pathological scars, resulting in irreversible changes to tissue architecture [8], [10].

Post-burn hypertrophic scars (HSc) are characterized by chronic inflammation, increased collagen synthesis, higher cellular growth (hyperplasia), and increased cell turnover (dead cells are replaced by younger cells) [1], [11]. These characteristics are manifested clinically by erythema, pain, dysesthesia, pruritus, and skin border elevation. Despite the large volume of research on HSc, its etiopathogenesis has yet to be clearly elucidated.

Transforming growth factor-beta (TGF-β) directly triggers the proliferation of fibroblasts and/or stimulates the production of connective tissue [12], [13], [14]. Other cytokines, such as platelet-derived growth factor, the fibroblast growth factor family, interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-α), are produced primarily by activated macrophages. Some of these cytokines are also produced by many other cell types, such as lymphocytes, endothelial cells, and fibroblasts [15], [16], [17]. TNF-α regulates many cellular responses during wound repair, such as proliferation, differentiation, inflammation, scarring, and apoptosis. TNF-α is present in two molecular forms from a unique gene: as a 26-kDa membrane protein (mTNF) and as a 17-kDa soluble protein (sTNF) derived from the membrane form by proteolytic release via the TNF-α converting enzyme [18]. To perform its biological function, TNF-α binds to type I (TNFR1, 55 kDa) and type II (TNFR2, 75 kDa) receptors that can activate different signaling pathways [19]. The effects generated through the mitogen-activated protein (MAP) kinase pathway are crucial during scarring processes [20].

Interleukin-1β (IL-1β) mediates immune regulation and inflammatory response. It is produced primarily by activated macrophages, but is also expressed by B and T lymphocytes, as well as epithelial, endothelial, and mesenchymal cells [21]. IL-1β is generated as a precursor (pro-IL-1β) and converted to its mature form by the IL-1β converting enzyme (ICE or caspase-1); it is thought to be active only as a mature protein. IL-1β interacts with the IL-1 receptor complex formed by the receptor (IL1R1) and an accessory protein. IL-1R heterodimerizes and interacts with myeloid differentiation primary response gene 88 (MyD88); they join with toll interacting protein and IL-1R–associated kinases 1 and 4. The proximity of these molecules to the receptor facilitates interaction through association of their death domains. In signaling, the receptor joins TNF receptor-associated factor 6, activating MAP kinases (TGF-β-activated kinase 1, MAP kinase 6) and subsequently triggering the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), Jun N-terminal kinase (JNK), and MAP kinase p38, among others [22]. Despite structural differences, IL-1β cooperates with TNF-α [23] to execute certain processes, such as control of collagen synthesis [21].

In this study, we used in situ hybridization (ISH) and immunohistochemistry (IHC) to examine the expression and tissue localization of TNF-α and IL-1β mRNA, proteins, and type I receptors in HSc, normotrophic scars (NSc), and normal skin (S) with the aim of understanding cytokine regulation and the participation of these factors in abnormal scarring.

Section snippets

Tissue collection

All biopsies were obtained during reconstructive or cosmetic surgical procedures, with the patients’ informed consent. We collected 4 S samples, 9 NSc samples, and 10 HSc samples. All scar tissue samples were obtained from burned patients; S specimens were obtained from unburned areas. Only NSc and HSc tissues collected from lesions at least 1 year after trauma that had damaged 10–20% of the patients’ total body surface were included. None of the patients showed evidence of infection or any

Histomorphological differences related to hypertrophic scarring

Macroscopically, only HSc exhibited a raised morphology, which was frequently indurated, erythematous and hyperpigmented. Histopathological analysis showed no difference in the number of lymphocytes between scar groups; these results are consistent with those of a previous study [27]. NSc and HSc were both characterized by slight infiltrates of dermal lymphocytes that formed small groups of perivascular cells in the papillary dermis, and occasionally in the reticular dermis. A few other

Discussion

TNF-α and IL-1β are two of the most pleiotropic and widely distributed cytokines. They are considered essentially proinflammatory, but have also been described as profibrogenic [13]. Because both play relevant roles in the growth and maintenance of pathological scars and other cutaneous fibrogenic diseases (i.e., scleroderma), their expression (particularly that of TNF-α) has been reported controversially to increase in HSc [10], [15], [17], [30], [31]. Although IL-1 has not been fully

Conflict of interest

All authors must disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work.

Acknowledgments

The authors wish to thank Edward Tredget, MD, MSc. for his critical review of the manuscript, María Teresa Gorráez de la Mora, MD from the Department of Pathology, Centro Médico Nacional “20 de Noviembre”, ISSSTE, for her technical assistance; and Marco A. Marín, MD and colleagues for collecting tissue samples from patients of the rural surgery program of the Mexican Burn Association (Asociación Mexicana de Quemaduras A.C.). We also acknowledge Jennifer Piehl, PhD from Write Science Right for

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  • Cited by (0)

    1

    Both authors contributed equally to this work.

    2

    Previous address: Laboratory of Connective Tissue, División de Investigación Biomédica, CMN “20 de Noviembre”, ISSSTE, Mexico City, Mexico.

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