Skin fibrosis associated with keloid, scleroderma and Jorge Lobo's disease (lacaziosis): An immuno‐histochemical study

Abstract

Fibrosis is a common pathophysiological response of many tissues and organs subjected to chronic injury. Despite the diverse aetiology of keloid, lacaziosis and localized scleroderma, the process of fibrosis is present in the pathogenesis of all of these three entities beyond other individual clinical and histological distinct characteristics. Fibrosis was studied in 20 samples each of these three chronic cutaneous inflammatory diseases. An immunohistochemical study was carried out to explore the presence of α‐smooth muscle actin (α‐SMA) and vimentin cytoskeleton antigens, CD31, CD34, Ki67, p16; CD105, CD163, CD206 and FOXP3 antigens; and the central fibrotic cytokine TGF‐β. Higher expression of vimentin in comparison to α‐SMA in all three lesion types was found. CD31‐ and CD34‐positive blood vessel endothelial cells were observed throughout the reticular dermis. Ki67 expression was low and almost absent in scleroderma. p16‐positive levels were higher than ki67 and observed in reticular dermis of keloidal collagen in keloids, in collagen bundles in scleroderma and in the external layers of the granulomas in lacaziosis. The presence of α‐actin positive cells and rarely CD34 positive cells, observed primarily in keloids, may be related to higher p16 antigen expression, a measure of cell senescence. Low FOXP3 expression was observed in all lesion types. CD105‐positive cells were mainly found in perivascular tissue in close contact with the adventitia in keloids and scleroderma, while, in lacaziosis, these cells were chiefly observed in conjunction with collagen deposition in the external granuloma layer. We did not find high involvement of CD163 or CD206‐positive cells in the fibrotic process. TGF‐β was notable only in keloid and lacaziosis lesions. In conclusion, we have suggested vimentin to be the main myofibroblast general marker of the fibrotic process in all three studied diseases, while endothelial‐to‐mesenchymal transition (EndoMT) and mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role.

1. INTRODUCTION

Fibrosis is the excess deposit of extracellular matrix (ECM) components, especially collagen, in localized or diffuse form, in organs and tissues, which can cause an increase in volume, hardening and healing. It is a common physiological response of many tissues and organs subjected to chronic injury. A wide variety of stimuli, including persistent infections; autoimmune reactions; allergic responses; and chemical, physical and mechanical insults can trigger a fibrogenic response. ¹ , ² , ³

Keloid, lacaziosis (Jorge Lobo's disease or lobomycosis) ⁴ and scleroderma (localized or morphea) are cutaneous diseases with fibroses in their pathogenesis beyond their distinct clinical and histological characterizations. Keloids are caused by injury to the cutaneous reticular dermis including trauma, insect bite, burn, surgery, vaccination, skin piercing, acne, folliculitis, chicken pox and herpes zoster infection. ⁵ Lacaziosis is a chronic granulomatous fungal infection of the skin and subcutaneous tissue characterized by keloid‐like lesions. The etiologic agent is Lacazia loboi, an uncultivated fungus of the order Onygenales and the family Ajellomycetaceae. ⁶ The pathogenesis of scleroderma is obscure. It has been suggested to be triggered by a viral or bacterial infection, possibly Borrelia burgdorferi, ⁷ although some authors refute this. ⁸ , ⁹ , ¹⁰ According to Careta and Romiti, ¹¹ inflammatory processes with a probable autoimmune basis and an embryonic origin, such as genetic mosaicism, seem to be more clearly associated with the aetiopathogenesis of scleroderma. Several studies have considered activation of T CD4+ lymphocytes and Tregs lymphocytes CD4 + CD25high + Foxp3+ as key stimuli promoting the vascular abnormalities and fibrosis observed in scleroderma. ¹² , ¹³ , ¹⁴

General pathology concepts could imply that development of keloid, lacaziosis and scleroderma involves chronic inflammatory reaction that provokes a common host tissue repair. Repair is often used for surface epithelium (healing) and parenchymal and connective tissue (fibrosis). ECM plays a central role in this repair process, instructing cells or reinforcing the synthesis of their primordial mesenchymal molecules. In fact, ECM mechanically stabilizes tissue damage by mobilizing cytokines, chemokines and important repair triggers transforming growth factor beta (TGF‐β), platelet‐derived growth factor, vascular endothelial growth factor and fibroblast growth factor. ¹⁵ , ¹⁶ Furthermore inflammatory macrophages (activated residents or those from inflammatory monocytes) could be the origin of the collagen deposits via assisting in the chemotaxis of other inflammatory cells participating in the synthesis of ECM molecules, and in particular collagen. ¹⁷ Among the cells that are attracted and characterize the proliferation phase of the repair process are resident fibroblasts and monocytes of bone marrow origin that differentiate into fibroblasts, pericytes and endothelial and smooth muscle cells. ¹⁸ The new ECM must contain collagen fibres that maintain the mechanical strength of the tissue. ¹⁹

The aim of this study was to investigate some fibrotic and related marker expression using   immunohistochemistry, and thus identify the cell and tissue markers of myofibroblasts, mesenchymal cells, macrophages and the cytokine TGF‐β expressed in three distinct cutaneous diseases: keloids, lacaziosis and scleroderma.

2. MATERIALS AND METHODS

2.1. Samples

Twenty skin paraffinized skin biopsies samples each of keloids, lacaziosis and scleroderma were retrieved from the archive of the Dermatopathology Laboratory of the Dermatological Clinic Division of Hospital das Clínicas FMUSP. These had been obtained for diagnostic purposes or were the products of surgical excision for treatment (keloids). Keloid samples were obtained from 10 males and 10 females of mean age 28.7 and 26.6 years, respectively. Scleroderma samples (morphea) were obtained from 11 males and nine females mean age 40.4 and 51 years, respectively. All lacaziosis samples were taken from males, but the age data (any clinical data) were not included in the records. It is important to note that we have distinguished keloids from hypertrophic scars. According to our histological analysis, keloids exhibit fibrogenesis with the presence of young collagen fibres and older collagen fibres, including those with hyaline alteration, arranged in a coiled arrangementis (‘tumoriform characteristics’). It is distinguishable from the hypertrophic scar where there is aberrant deposition of dense collagen fibres in the same phase of the maturation process (Limandjaja et al). ²⁰

2.2. Histology, immunohistochemistry and morphometrical analysis

Paraffinized skin samples were cut into 3–4 μm sections for conventional histology (haematoxylin and eosin – H&E) and imunohistochemical assays. An immuno‐streptavidin–peroxidase method staining was carried out following the protocol described: histological slides were dewaxed and hydrated and incubated in peroxide 3% solution for 30 min at room temperature (except TNF‐alfa protocol assays, we used methanol peroxidase solution). Then, slides were incubated for 20‐min in a water bath (98°C) in citrate buffer solution, pH 6.0. The slides were covered with blocking solution (skimmed milk powder diluted in PBS – 12 g of milk in 200 ml of PBS) and incubated in a humid chamber for 20 min at room temperature. The specific primary antibody (described in Table 1) was added in sufficient quantity to cover the fragments, and the slides were incubated for 18–24 h in a humid chamber, at 4°C. For the next step, ultra vision large volume detection system anti‐polyvalent, HRP—ready to use—Lab Vision was applied. Reactions were visualized by applying the DAB solution for 5 min, with Harris' Haematoxylin counterstaining for 1 min. ²¹ For quantitative analysis of immunostained cells, images were obtained using an optical microscope Olympus BX40 coupled to a microcomputer, and quantification of immunostained cells was performed using AxioVision 4.8.2 software (Zeiss). Ten microscopic (×40 objective) fields of each histological section for each target antigen were examined. The immunostained cells (stained in brown) were counted. The number of cells per mm² was determined by the ratio of the immunolabelled cells to the area of each image.

TABLE 1.

Monoclonal antibodies for immnunohistochemistry

Antigen	Supplier	Clone	Isotype	Specifity (human antigens)	Titration
α‐Actin	Dako	1A4	mIgG2a, kappa	Smooth muscle, myofibroblasts, myoepithelial cells	1.100
Vimentin	Dako	V9	mIgG1 kappa	Intermediate filament (IF) of the cytoskeleton of vertebrate cells	1.100
Desmin	Dako	D33	mIgG1 kappa	Smooth muscle cells (skeletal and cardiac)	1:50
CD31	Dako	JC/70A	IgG1	Endothelium vascular, haematopoietic progenitor	1.100
CD34	Dako	QBEnd/10	mIgG1 kappa	Endothelial cell membrane	1.100
CD105	Serotec	SN6h	IgG1	Monocytes, cell surface MJ7/18 antigen Endoglin	1:20
FoxP3	eBioscience	FJK‐16S	IgG2a, kappa	Forkhead box protein 3 – high level in CD25 + CD4 positive regulatory T cells	1:50
TGF‐b1(v)	Santa Cruz	sc‐146	Rabbit polyclonal IgG	Epitope mapping at the C‐terminus of TGFβ1	1.100
CD68	Dako	PG‐M1	mIgG1 kappa	Epitope on the macrophage‐restricted form of the CD68 antigen	1.100
CD 163	Dako	10D6	IgG3 kappa	Non determined epitope of CD163 protein scavenger receptors of macrophages	1.100
CD 206	Spring	5C11	IgG1	Mannose receptors – Macrophages	1:100

2.3. Statistical analysis

For the quantitative analyses, the t‐test was conducted on the Gaussian distribution data, and the Mann–Whitney test on the non‐Gaussian distribution data. All analyses were made using GraphPad Prism 5.0 software.

2.4. Ethics approval

The Institutional Review Board of the Universidade de São Paulo Medical School Hospital approved this study (Protocol #3.534.258). All procedures were conducted in accordance with the Helsinki declaration of 1964 and its later amendments or with comparable ethics standards. The study is based on samples originally obtained for diagnostic purposes and retrieved from the files of the dermatopathology laboratory of the institution. Therefore, the Institutional Review Board deemed informed consent from each patient to be unnecessary.

3. RESULTS

3.1. Histopathology

Keloid and lacaziosis samples displayed thickened epidermis. In scleroderma, the epidermis showed more histological variation, from atrophic or nearly normal, to slightly thicker than usual. A related rectification of the epidermal layer (flattening of the epidermis and replacement of the papillary and reticular dermis by a scar tissue) was generally found in all skin samples (Figure 1A). The main lesions were observed in the reticular dermis, especially aspects of collagen deposition. Keloids were recognizable at low magnification as localized connective tissue proliferation in the reticular dermis, well circumscribed, always separated from the epidermis by the papillary dermis. At higher magnification, keloid tissue showed large bundles of thick, homogeneous, hyaline collagen fibres, generally referred to as keloid collagen (keloid nodule) (Figure 1B,C). The bundles were not organized parallel to the epidermis, but in an accumulation of fibroblasts and cells such as mononuclear inflammatory cells, including macrophages, lymphocytes and plasma cells. Skin appendages were only observed at the scar periphery, with none inside the scar. Important to say that hypertrophic scars are distinguished from keloids because they are confined to the limit of the area of injury that caused them, which is not the case with keloids.

FIGURE 1.

(A–H) Skin sections of patients with keloid, lacaziosis and scleroderma, (A) keloid showing a related rectification of the epidermal crests (asterisks). Note eosinophilic thickness collagen fibres (arrowheads) and a perivascular inflammatory cells exudate (arrows) (bar = 32 μm); (B) higher magnification of (A) showing large bundles of thick, hyaline and homogeneous collagen fibres called in the literature ‘keloid collagen’ (KC) (bar = 16 μm). (C) KC higher magnification of (A) showing spindle shaped cells (fibroblasts) (arrows) and eosinophilic thickness collagen fibres (arrowheads) (64 μm); (D) lacaziosis showing an inflammatory infiltrate comprising mononuclear cells and multinucleate giant cells (GC) (Langhans giant cell). Numerous yeast‐like cells of Lacazia loboi are present (arrows) (bar = 64 μm); (E) Large bundles of thick, homogeneous and hyaline collagen fibres (arrowheads) and spindle shape cells (fibroblasts) mixed to inflammatory cells as well (bar = 64 μm); (F) scleroderma showing thickening and homogenization of collagen bundles in the dermis. A focal chronic inflammatory reaction could be seen around skin appendages and blood vessels (arrowheads). (G) higher magnification showing this cellular exudate (arrowhead) and atrophical adnexal structures as eccrine glands (asterisks) (64 μm); (H) a typical straight edge of the dermal–subcutaneous interface was observed (dashed lines) (64 μm). Haematoxylin–eosin (HE) staining. Ep (Epithelium); Papillar Dermis (PD), Reticular Dermis (RD).

In lacaziosis, the dermis showed an extensive chronic inflammatory reaction comprising multiple granuloma formations with an inflammatory infiltrate consisting mainly of macrophages, epithelioid cells (activated macrophages with pink granular cytoplasm and indistinct boundaries)–and multinucleate giant cells, including both foreign body and Langhans giant cells containing numerous yeast‐like Lacazia loboi cells (Figure 1D). These aggregates of macrophages, epithelioid cells and giant cells were surrounded by a collar of lymphocytes and plasma cells. Older granulomas with a rim of fibroblasts were also present in combination with cellular exudative granulomas. Thus, moderate to intense collagen deposition was detected around granulomas (Figure 1E).

In scleroderma, the reticular dermis showed thickening and homogenization of collagen bundles that expanded to subcutis associated with vascular alterations and inflammatory cell infiltrate. A mild to moderate chronic inflammatory reaction could be seen around eccrine sweat glands, hair follicles and blood vessels (Figure 1F,G). Atrophy of adnexal structures as pilosebaceous units and eccrine glands was frequently observed. A common straight edge of the dermal–subcutaneous interface throughout the chronic inflammatory process was observed (Figure 1H).

3.2. Immunohistochemistry

3.2.1. Alfa‐Actin (α‐SMA), vimentin, desmin and TGF‐β

Alpha‐actin‐positive cells were mainly observed in smooth muscle cells of blood vessels walls of the papillary and reticular dermis in all three lesions studied. In some cases, α‐SMA‐positive cells consisting of spindle‐shaped stromal cells (myofibroblasts) were observed in the reticular dermis (Figure 2A). Vimentin expression was more marked than α‐SMA cell labelling. Well‐defined vimentin‐positive fibroblasts were observed in both papillary and reticular dermis in keloid (Figure 2B). The same morphological aspect of vimentin‐positive fibroblasts was also observed in scleroderma and lacaziosis as well. Moreover, in lesions of lacaziosis, a granulomatous inflammatory disease, we observed displayed intense vimentin‐positivity in granulomas cells too, especially giant cells and epithelioid macrophages.

FIGURE 2.

A–F: (A, B) a case of keloid to represent alpha‐Actin and vimentin immunohistochemical stanning, respectively; (A) higher magnification of a keloid nodule area showing endothelia vessel wall positive cells (arrows), but we can also observe some α‐SMA positive cells consisting of spindle‐shaped stromal cells (myofibroblasts) (arrowheads) in keloid collagen (KC) (reticular dermis) (16 μm); (B) compare vimentin expression to alpha‐Actin expression. Spindle‐shaped stromal cells (myofibroblasts) much more markedly visible than α‐Actin cell labelling throughout reticular dermis (16 μm). Immuno‐streptavidin–peroxidase method staining, Haematoxylin‐Harris counterstain. Figure C alfa‐Actin (α‐SMA) * a,b keloid versus lacaziosis (p = .0039); *a,c keloid versus scleroderma (p < .0001) and * b, lacaziosis versus scleroderma (P = .0664) Figure D vimentin * a,b keloid versus scleroderma (p < .001); a,b lacaziosis versus scleroderma (p < .001); b, keloid versus lacaziosis (p = .9905); * Figure E a case of keloid to represent TGF‐beta positive cells consisting of spindle‐shaped stromal cells (fibroblasts) (arrows) throughout the reticular dermis mixed to thick, homogenous and hyaline collagen fibers (asterisks) (16 μm); Figure F, keloid versus lacaziosis (p > .999); a,b keloid versus scleroderma (p < .0001); a,b lacaziosis versus scleroderma (p = .0007).

The α‐SMA expression was significantly higher in keloids than in lacaziosis (p = .0039) and scleroderma (p < .0001). Lacaziosis and scleroderma α‐SMA expression did not significantly differ (p = .0664) (Figure 2C). Vimentin expression was higher in keloid and lacaziosis in comparison to scleroderma (p < .0001). However, it was equal between keloid and lacaziosis (unpaired t test, p = .9905) (Figure 2D). Desmin and cytokeratin expressions were not detected in either papillary or reticular dermis cells, with the exception of smooth muscle erector pili of hair follicles and epithelial cells (epidermis, hair follicles and eccrine sweat glands).

In all three cutaneous diseases, diffuse staining was observed mostly in TGF‐β‐positive cells consisting of spindle‐shaped cells (fibroblasts) throughout the reticular dermis (Figure 2E). The expression of TGF‐β in keloid and lacaziosis lesions was higher than in scleroderma (p < .0001 and p = .0007, respectively), but it is similar between keloid and lacaziosis (p > .999) (Figure 2F).

3.2.2. CD31, CD34 and CD105

The expression of the endothelial/mesenchymal markers CD31, CD34 and CD105 was observed mainly along the blood vessel wall of the papillary or reticular dermis. With rare exceptions, we observed mesenchymal stromal cell populations throughout the dermal compartment, as well as in adnexal appendages adventitia (Figure 3A–C). We did not find a significant difference of CD31 expression among the three lesions studied (Figure 3D). However, CD34 expression was significantly lower in keloids than in lacaziosis and scleroderma samples (p < .0001), with similar expression in lacaziosis and scleroderma (p > .9999) (Figure 3E). CD105 expression was significantly lower in keloid compared with lacaziosis (p = .0013), but similar to scleroderma (p = .9853). It was higher in lacaziosis in comparison to scleroderma (p = .0402) (Figure 3F).

FIGURE 3.

A–F: (A–C) a case of keloid to represent CD31, CD34, CD105, CD105 positive cells, respectively, localized along the blood vessels wall in the dermis (arrows). Except in a few cases as seen in (C), we observed some mesenchymal stromal cell population throughout the dermal compartment (arrowheads) (16μm). Immuno‐streptavidin–peroxidase method staining, Haematoxylin‐Harris counterstain. Figure D, CD31 Expression: * keloid versus lacaziosis (p > .9999); * keloid versus scleroderma (p > .9999); * lacaziosis versus x scleroderma (p = .08697). Figure E, CD34 Expression * a,b keloid versus lacaziosis (p > .9999); Figure F, CD105 Expression * a,b keloid versus lacaziosis (p = .0013); * a, keloid versus scleroderma (p = .9953); * b,c lacaziosis versus scleroderma (p = .0402).

3.2.3. CD68, CD163 and CD206

Expression of the macrophage markers CD68, CD163 and CD206 was found throughout the dermis. Immunostained cells exhibited macrophage morphology and were observed within inflammatory cell exudate (Figure 4A,C). These cells showed moderate to intense expression in mononuclear exudate in some keloid and scleroderma lesion samples. CD68‐positive cells were readily observed in lacaziosis, but, in keloid and scleroderma, were only occasionally involved in the inflammatory response. Although CD163‐ and CD206‐positive cells were observed in higher absolute numbers than CD68‐positive ones, the latter cells were more easily observed in keloid and scleroderma. CD 206 stained cells were observed mainly around blood vessels, not in close proximity to the endothelium layer (Figure 4C). Under morphometrical analysis, CD68 expression was higher in lacaziosis than in keloid and scleroderma (p < .0001), but no difference was observed between keloid and scleroderma lesions (p > .9999) (Figure 4D). CD163 and CD206 levels did not differ among the studied cutaneous diseases (Figure 4E,F).

FIGURE 4.

A–F: (A–C) a case of keloid to represent CD68, CD63, CD206, respectively. (A,B) CD68 and CD163 positive cells, respectively keep a macrophage morphology and they were easily found occasionally distributed mixed composing the inflammatory cellular exudate (arrows). In (C), note CD206 positive cells was mainly distributed around blood vessels, but not strictly close to endothelium layer maintaining regularly distributed along external layers as well (arrowheads) (32 μm). Immuno‐streptavidin–peroxidase method staining, Haematoxylin‐Harris counterstain. Figure D. CD68 expression: * a,b lacaziosis versus keloid (p < .0001); a,b lacaziosis versus scleroderma (p < .0001); a, keloid and scleroderma cases (p = .9999).Figure E: CD163 expression: * keloid versus lacaziosis (p < .2452); * keloid versus (P < .7628); * lacaziosis versus (p < .6190). Figure F: CD206 expression: * keloid versus lacaziosis (p > .9999); *keloid versus scleroderma (p = .1275); * lacaziosis versus scleroderma (p = .0623).

3.2.4. Ki67, p16 and FOXP3

We investigated whether the major collagen producing cells were actively proliferating (Ki67) or senescent (p16). p16 is known as a tumour suppressor protein that acts by inhibiting the cyclin‐dependent kinases CDK4 and CDK6 from phosphorylating the retinoblastoma protein, resulting in G1 cell‐cycle arrest. As such, p16 has been implicated in cellular senescence. ²⁰ Ki67‐positive cells consisting of spindle‐shaped stromal cells (myofibroblasts) and, in some cases, leukocytes, were observed distributed sparsely throughout the reticular dermis. The detection of positive cells increased in the presence of a mononuclear exudate. The expression of p16 was greater than Ki67 in all samples (Figure 5A,B). The expression of FOXP3 was primarily observed in mononuclear cells composed of inflammatory exudate (lymphocytes) located along the papillary dermis or around blood vessels or adnexal appendages of reticular dermis. FOXP3‐positive spindle‐shaped stromal cells (fibroblasts) were observed throughout the reticular dermis (Figure 5C).

FIGURE 5.

A–F: (A,B) a case of scleroderma to represent KI67 and p16 immunohistochemical cellular characterization. (A) Any ki67 positive cells could be found in the dermis associated to fibroblasts (arrows) or to thick and homogenous collagen fibres (asterisks); (B) On the other side, many p16 positive cells throughout the fibrosis process; and homogenous collagen fibres (asterisks) as well (16 μm). (C) FOXP3 positive nuclei of cells consisting of spindle‐shaped stromal cells (fibroblasts) throughout the reticular dermis with homogenous collagen fibres (asterisks) could be seen (arrows) (16μm). Immuno‐streptavidin– peroxidase method staining, Haematoxylin‐Harris counterstain. Figure D Ki67 expression: a,b *keloid versus lacaziosis p < .9999. Figure E p16 expression: 17 a, *keloid versus lacaziosis p = .3112; a,b keloid versus scleroderma p = .0002; b, *lacaziosis versus scleroderma; p > .9999; Figure F Foxp3 expression * a,b keloid versus × lacaziosis p = .001; * a,b scleroderma versus lacaziosis p = .0071; * a, keloid versus scleroderma p > .9999.

Quantitative analyses revealed higher p16‐positive cell density in tissue compared with Ki67 cell density/field (Figure 5D,E). Quantitative analyses revealed higher p16 expression in keloid than in lacaziosis (p < .0001) and scleroderma (p = .0002) (Figure 5D). Scleroderma showed almost no Ki67‐positive cells, significantly lower than in keloid (p = .0016) and lacaziosis (p < .0001) (Figure 5E). In general, there were few positive cells in all cases, with keloid and scleroderma lesions exhibiting higher FOXP3 expression than lacaziosis (p = .0011 and p = .0071, respectively). Levels in keloid and scleroderma lesions did not differ (p > .9999) (Figure 5F).

4. DISCUSSION

In fibrosis, tissue repair is rapid, as it mainly uses ECM to repair damage, as opposed to true tissue or organ regeneration that restores the appropriate cell and ECM component proteins, such as collagen. ²² In examining collagen deposition in three skin fibrotic diseases, we found greater deposition of thickened collagen fibres mainly in keloid and scleroderma, as expected. The major producers of collagen fibres are myofibroblasts, which can‐cause an imbalance in ECM synthesis that manifest as fibrosis. ²³ Fibroblast differentiation into myofibroblasts may include induction of expression of α‐SMA and high levels of the intermediate filament protein vimentin. Herein, we observed higher expression of vimentin than α‐SMA in the three fibrotic cutaneous diseases studied. α‐SMA cell positivity was primarily observed in smooth muscle cells of blood vessels walls of the papillary or reticular dermis, not in keloid nodules. It was also restricted to eccrine glands myoepithelial cells and vessel walls. However, higher α‐SMA expression was seen in keloids which could indicate less reactive myoepithelial cell proliferation than in scleroderma. ²⁴ In addition, higher vimentin expression than α‐SMA antigen could be indicate vimentin as an important cell myofibroblasts marker. Based on previous literature, studies of skin wound repair, vimentin has been shown to function as a signalling integrator in tissue regeneration and healing. ²⁵ , ²⁶ In fact, Walker et al., ²⁵ in an elegant study, showed cytoskeletal vimentin being released into the extracellular space during injury and suggested that the extracellular vimentin pool links specifically to the mesenchymal leader cells and thus plays an essential role in signalling their fate change to myofibroblasts.

Vascular alterations have been reported in keloid, lacaziosis and scleroderma with loss of blood vessels causing tissue hypoxia that leads to fibrosis and potentially to endothelial lesions. ²¹ , ²⁷ Since myofibroblasts could originate from the endothelial‐to‐mesenchymal transition (EndoMT) and mesenchymal stem cells (MSC), we characterized selected EndoMT markers, including CD31, CD34 and CD105 transition to MSCs. ²⁸ Endothelial CD31‐ and CD34‐positive cells were readily observed, mainly in blood vessels throughout the reticular dermis, but absent in stromal cells of connective tissue. The majority of tissue samples showed low presence of either blood vessels or angiogenesis in association with areas of fibrosis. In fact, we did not observe CD31 expression differences among the three cutaneous diseases samples, but CD34 expression was lower in scleroderma. In scleroderma, endothelial cell damage has been reported as a result of viral agents, cytotoxic T cells, antibody‐dependent cellular toxicity, anti‐endothelial cell antibodies and ischemia‐reperfusion injury. ²⁹ Lee et al., ³⁰ described CD34⁺ stromal cells in mid and deep dermis, and they observed an inverse correlation between α‐SMA and CD34 stromal stains in both layers. Herein, we saw similar results ‐ as evidenced by the lack of stromal cell CD34 positivity.

In contrast to CD34, in keloid and scleroderma samples, CD105‐positive cells with spindle‐shaped morphology were mainly observed in perivascular tissue. According to Majesky, ³¹ these cells may represent stromal cells exhibiting MSC properties in close contact with the vessels' adventitia. In lacaziosis, a few spindle‐shaped CD105‐positive cells were observed, chiefly within the external granuloma layer. Although the presence of CD105‐positive cells was low in all lesion types, quantitative analysis revealed lower expression in lacaziosis than in keloid and scleroderma. Rinkevich et al. ³² reported that local fibroblasts, not circulating cells, are responsible for the connective tissue seen in fibrosis. Our observation of mild CD105 cellular expression may confirm this, since mesenchymal cells (precursors collagen producer cells) could be also derived from bone marrows. Satoh et al., ¹⁸ for example, described a potential atypical monocytes (Ceacam1 + Msr1 + Ly6C − F4/80 − Mac1+ monocytes) as potential mesechymal cell origin.

Macrophages appear to play origin‐specific roles in repair. ¹⁷ In the skin, the early inflammatory phase of injury response depends on circulating monocyte‐derived M1 macrophages that express CD68. In later phases, these macrophages are replaced by a specific reparative population of M2 macrophages that express CD163 and CD206 and other cellular markers. ³³ We found significant CD68 expression in lacaziosis only within the granulomatous exudate. CD68 expression was low in most keloid and scleroderma samples as a consequence of scarcity of inflammatory cells. There were also few positive cells labelled CD163 and CD 206 showed a paucity of positive cell labelling as well.

In all skin samples studied there were few FOXP3 positive cells. Scleroderma samples demonstrated less FOXP3 cells than keloid and lacaziosis ones. It was reported that Treg lymphocytes reduced collagen synthesis by keloid fibroblasts in experimental conditions Murao et al., ³⁴ As mentioned, TGF‐β‐dependent signalling regulates scarring, inducing fibroblast proliferation and myofibroblast differentiation and increasing collagen synthesis, but TGF‐β inhibits FOXP3 expression. ³⁵ , ³⁶ Our immunohistochemistry results did not show high expression of FOXP3 in any cutaneous disease studied. This could be associated with high TGF‐β expression in keloids and lacaziosis but this observation was not confirmed for scleroderma, which showed no TGF‐β expression. This result of scleroderma samples is concordant with Antiga et al. ¹³ These authors reported few TGF‐β‐positive cells in both systemic sclerosis and morphea skin lesions. However these results are subject to, potential misinterpretation because fibrotic lesions are often dynamic undergoing a period of inflammation and subsequent resolution with fibrosis and finally remodelling. The origin of skin samples from a single point in the evolution of the fibrosis process where expression of some of the markers may have been higher eg TGF‐b and then reduced as the disease and response to it evolve. In this regard, however, the three pathological processes studied were all from comparable siutations ‐ well‐established lesions, at the stage of diagnostic clinicopathological characteristics.

Finally, we could also speculate an association of FOXP3 with expression of p16, a senescence marker. ²⁰ , ³⁵ FOXP3 is a transcriptional repressor of the oncogenes HER/ErbB2 and Skp2 and has recently been reported to upregulate the expression of the tumour suppressor p21. ³⁶ The expression of FOXP3 in senescence is regulated by p53 upon DNA damage and subsequently regulated by p21, producing reactive oxygen species that promote cell senescence. ³⁷ We can report that FOXP3 expression appears to be related to p16 expression in keloids, since keloid tissue showed higher expression of p16 than did lacaziosis and scleroderma.

In conclusion, we could suggested vimentin to be the main myofibroblast general marker in the fibrotic process in all three studied diseases. So far, we have concluded that endothelial‐to‐mesenchymal transition (EndoMT), mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role. However, all studied samples represent one point in time in the evolution of lesions where they open a discussion about concepts such as no macrophage involvement may not be accurate if the early evolving lesion expresses markers, which change as the lesion matures.

CONFLICT OF INTEREST

The authors declare no conflict of interest in this work.

Tafuri WL, Tomokane TY, Silva AMG, et al. Skin fibrosis associated with keloid, scleroderma and Jorge Lobo's disease (lacaziosis): An immuno‐histochemical study. Int J Exp Path. 2022;103:234‐244. doi: 10.1111/iep.12456