EUROPEAN TISSUE REPAIR SOCIETY

SCAR CONTROL II

INTERLEUKIN-4 IN THE CONTROL OF SCARRING

WOUND repair is the result of a spatially- and temporally-controlled sequence of events, resulting in a limited activation of cells. Uncontrolled activation of connective tissue cells in wounds may result in hypertrophic scars or keloids.¹

Many growth factors and cytokines are implicated in the fibroblast activation necessary for new connective tissue formation.² Epidermal Growth Factor (EGF), Platelet- Derived Growth Factor (PDGF), Insulin-like Growth Factor-I (IGF-I)and Fibroblast Growth Factors (FGF) 1 and 2 are the main factors able to stimulate fibroblast migration and/or proliferation. Transforming Growth Factor-â (TGF-β), Connective Tissue Growth Factor, Insulin-like Growth Factor 1 and, as it was shown in our laboratory and others, Interleukin-4 (IL-4), are potent activators of extracellular matrix synthesis. An imbalance in the production of some of these cytokines and growth factors may be involved in hypertrophic scars and keloids.

Human IL-4 is a 15 kDa glycoprotein, composed of 129 amino acids.³ It was initially characterized as a B-cell growth and differentiation factor. Its gene is located on chromosome 5 (q23–31). It is secreted by T-lymphocytes of the Th-2 subgroup, mast cells, basophils, and eosinophils infiltrated into connective tissues. High-affinity receptors are found on B and T-cells, polymorphonuclears, monocytes/ macrophages and also on connective tissue cells such as fibroblasts and endothelial cells.

Interleukin-4 is a potent activator of dermal fibroblasts and is over-expressed in lesions where extracellular matrix synthesis is increased

Previous studies from our laboratory and others have shown that IL-4 is a potent activator of collagen synthesis in fibroblast cultures.^4–6 Significant stimulation of extracellular matrix synthesis was found for concentrations of IL-4 as low as 1 ng/ml (10 U/ml). It occurred at a pretranslational level and was found for type I collagen, type III collagen, fibronectin (Figure 1) and also for some specific proteoglycans, such as decorin7 which stabilizes collagen fibrils in the dermis.

Interestingly, we observed that IL-4 is strongly expressed in scleroderma skin.⁸ Intense staining with anti- IL-4 antibodies was observed in the deeper layer of the dermis in scleroderma lesions. The staining was particularly intense near dermal capillaries. The localization of the labelling suggested that fibroblasts were the cells responsible for the increased production of IL-4 in scleroderma skin. Immuno-histochemistry and northern blot analysis of scleroderma fibroblast cultures confirmed that these cells were able to strongly express IL-4 (Figure 2), suggesting an autocrine auto-activation of fibroblasts in scleroderma lesions. The implication of IL-4 in the development of fibrosis was also supported by experiments of Ong et al ⁹ who demonstrated that administration of neutralizing anti-IL-4 antibodies prevented dermal collagen deposition in a mouse model of scleroderma. Moreover, the IL-4 level has been reported to be increased in serum from patients with scleroderma.¹⁰

The role of IL-4 in normal wound healing

In order to demonstrate the role of IL-4 in wound healing, we performed several in vivo experimental studies in mice.¹¹ On day 0, full thickness wounds (8 mm diameter) were made on the back of series of Swiss mice, using punchbiopsies. In a first series of mice, the expression of IL-4 in the wound area was studied by immunohistochemistry,

Figure 1: IL-4 stimulates type I collagen, type III collagen and fibronectin gene expression in fibroblast cultures. Confluent cells were incubated for 24h in Dulbecco’s modified Eagle’s medium supplemented with 1% fetal bovine serum and 0 (controls), 10 or 100 units IL- 4 per ml (0, 1, and 10 ng/ml). RNAs were extracted and specific mRNAs for a1(I), a1(III) collagens, and fibronectin were analyzed by northern blots.

Figure 2: Expression of IL-4 is increased in scleroderma skin and scleroderma fibroblast cultures.

Expression of IL-4 was analyzed in scleroderma skin by immunohistochemistry (left) and in scleroderma fibroblast cultures by immunohistochemistry (middle) and northern blot (right). Intense staining was observed in scleroderma skin and scleroderma fibroblast cultures (original magnification, x 250) using immuno-peroxidase staining with a monoclonal anti-human IL-4 antibody (Genzyme). Specific IL-4 mRNA was strongly increased in scleroderma skin fibroblasts (SCD), compared to normal skin fibroblasts (N). The housekeeping gene Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mRNA was used as reference.

using a rat-anti-mouse IL-4 monoclonal antibody (Genzyme), after 1, 2, 4, 8, 12, and 21 days post-wounding. A second series of mice received a topical application on the wound of either normal saline (controls) or IL-4 (250 ng per day for 4 days). Finally, in a third series of experiments, mice received the application of an IL-4 antisense oligonucleotide (50 µg in 50 µl saline mixed in 0.04g low viscosity carboxymethylcellulose). This oligonucleotide (5'- GGGGTTGTGTCGCAT-3') was able to completely suppress the translation of the murine IL-4 mRNA¹² Control mice received the application of an IL-4 sense oligonucleotide (5'-ATGCGTCTCAACCCC-3') at the same dose. Wound area was then measured and biopsies were collected for histological examination, morphological and image analysis of the wounds at days 1, 2, 4, or 7 postwounding.

Immunohistochemical study of the distribution of IL- 4 expression during wound healing showed an intense staining of the deeper layers of the skin under the wound. It appeared as soon as day 1, was maximal at days 2–4 (Figure 3) and disappeared completely by 3 weeks.

IL-4 application on the wound significantly accelerated wound closure. Mean wound areas ± SEM at day 4, expressed as percentages of initial wound areas at day 0, were 83.0 % ± 7.9 in the control vs 54.9% ± 8.6 in the IL- 4 treated mice. The number of inflammatory cells, measured by image analysis after staining by haemateinerythrosin safran, was increased in IL-4-treated wounds in comparison with controls (108 ± 16 cells per 10000 µm² vs 78 ± 12, p < 0.01).

On the other hand, topical IL-4-antisense oligonucleotide application induced a strong decrease of IL-4 expression under the wound and a significant delay in wound closure (wound area at day 7: 41.3 ± 8.4 mm² in the controls vs 73.0 ± 14.2 mm² in the IL-4 antisense treated mice, p < 0.02). The antisense oligonucleotide application significantly decreased cell density in the wounds (23.5 ± 2.9 cells per 10000 µm² vs 51.4 ± 12.4 in the controls). It also induced a severe disorganisation of the collagen bundles and decreased by 38% (p < 0.001) the area fraction occupied by collagen fibers as determined by image analysis after sirius red staining.

Figure 3: IL-4 is transiently expressed during normal wound healing in mice.

IL-4 expression was studied by immunohistochemistry in mice wounds. Immunoperoxidase staining was performed with a monoclonal rat anti-mouse-IL-4 antibody (Genzyme) on normal unwounded skin (control) and on wounded skin at day 4 after injury. Staining for IL-4 is indicated by arrows (original magnification, x 100). Intense staining appeared under the wound at day 1, was maximal at days 2-4, and disappeared completely by 3 weeks.

Figure 4: Expression of IL-4 is increased in human keloids.

Immunohistochemistry of normal human skin (left) and keloid (right). Immunoperoxydase staining with a monoclonal anti-human IL-4 antibody (Genzyme) demonstrated intense staining of fibroblastic cells, mainly in the lower dermis, around dermal capillaries (original magnification, x 250).

Topical application of IL-4 (250 ng) completely reversed the negative effect of the IL-4 antisense treatment on wound closure.

Expression of IL-4 is increased in human keloids

In order to check whether IL-4 might play a role in the formation of keloids, we performed immunohistochemical analysis of six normal skin and six keloids scars. No significant labelling with the anti-IL-4 antibody was found in normal skin. On the other hand, intense labelling of fibroblastic cells was found in the lower dermis of all the keloid scars, localized mainly around dermal capillaries (Figure 4). Such a strong and sustained expression suggests an uncontrolled autocrine activation of fibroblasts in these lesions.

Discussion

How the wound healing process is controlled is still incompletely understood. Regulation of the deposition of extracellular matrix components in vivo is complex: it involves interactions among many different cell types and between cells and extracellular matrix macromolecules, and necessitates the coordinated expression of many different soluble mediators that either stimulate or inhibit the process. Keloids may be caused by a dysregulation of this sequence of events, particularly by an uncontrolled expression of cytokines and growth-factors. Roles for TGFb1,¹³ basic FGF,¹⁴ and IL-15 (Castagnoli et al., 1999) in the development of hypertrophic scars and keloids have been previously suggested .

The capacity of IL-4 to enhance the production of collagen and other extracellular matrix macromolecules synthesis^4,5,6,15 (Serpier et al., 1997) makes it a good candidate for the activation of connective tissue cells in hypertrophic scars and keloids. It may also be the case in scleroderma lesions, where a strong expression of IL-4 by fibroblastic cells has been reported.8 We have shown that IL-4 is a potent activator of the wound repair process¹¹ and that it is overexpressed in pathological scars.¹⁶ Taken together, our data suggest that uncontrolled autocrine activation of fibroblasts by IL-4 might be one of the factors involved in the formation of keloids.

Acknowledgements

The authors’ research was supported by grants from CNRS and the University of Reims Champagne-Ardenne. The invaluable contribution of many co-workers, especially Dr Catherine Fertin, Dr Véronique Salmon-Ehr, Dr Hervé Serpier, Pr Tony Godeau and Pr Philippe Birembaut is gratefully acknowledged.

References

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Authors:
Laurent Ramont and François-Xavier Maquart
Laboratory of Biochemistry,
CNRS FRE 2534, Faculté de Médecine,
51 rue Cognacq-Jay,
51095 Reims cedex, France.
E-mail: fmaquart@chu-reims.fr