Annals of Burns and Fire Disasters - vol. IX - n. 4 - December 1996

EXPRESSION OF FUNCTIONAL INTERLEUKIN 2 RECEPTOR IN POST-BURN HYPERTROPHIC SCARS

Castagnoli C,(1) Trombotto C.,(2) Stella M.,(3) Magliacani G.,(3) Teich Alasia S.(1)

(1)Piedmont Foundation for Studies and Research in Burns, Turin, Italy
(2)Centre for Immunogenetics and Experimental Oncology, National Research Council, Italy
(3)Department of Plastic Surgery and Burn Unit, Trauma Centre, Turin

SUMMARY. Immunoperoxidase staining of skin sections obtained from 14 hypertrophic scars, 8 normotrophic scars and 6 samples of normal skin was performed using monoclonal antibodies (MoAbs) against p55 and p75 chains of the interleukin 2 receptor (IL-2R), An anomalous expression of keratinocytes of IL-2 high affinity receptors was clearly detected in all tested hypertrophic scar specimens. Human leukocyte antigen class 11 (HLA-DR) and intercellular adhesion molecule type I (ICAM-1) were ectopically expressed on the same cells. In contrast, CD36 antigen was detected only in the stratum granulosum of epithelium and was absent in the basal layers of the epidermis. A correlation was found between IL-2R, ICAM-1 and HLA-DR molecule expression and the presence of abundant infiltrates of activated T lymphocytes and macrophages, suggesting that this anomalous distribution was induced by cytokines produced by infiltrating cells. In the dermis of hypertrophic scar samples a positive staining with anti-IL-2R p55 (a chain) and p75 (P chain), ICAM-1, and HLADR MoAbs was detected on fibroblasts and endothelia. The production of IL-2R has not been previously reported for keratinocytes and fibroblasts. These results emphasize the role played by immune mechanisms in hypertrophic scarring and suggest the involvement of cellmediated immune phenomena.

Hypertrophic scars are the result of alterations that occur in normal wound healing. They are characterized by the continued production of collagen over a long period of time, hyperplasia, increased cell turnover, and abundant leukocyte infiltrates in the dermis and epidermis.
It has recently been suggested that immunological factors play a pivotal role in the pathogenesis of these abnormalities: this hypothesis is supported by the ectopic expression of such scars and by a local alteration in the biosynthesis of some cytokines.
Many inflammatory dermatoses (allergic contact dermatitis, lichen planus, psoriasis) and the graft versus host disease are characterized by an ectopic expression of HLA class 11 and ICAM-1 molecules on epidermal keratinocytes associated with the presence of abundant activated T lymphocytes infiltrating the skin.
IL-2R is expressed on lymphocytes, natural killer cells, monocytes, and some non-haemopoietic cells. Its functions include the promotion of proliferation and certain forms of cellular maturation.' It has also been established that the IL2/IL-2 receptor system plays a pathogenic role in several human diseases and animal disease models.
The present study attempts to identify pathogenic factors involved in hypertrophic scarring. Leukocytes infiltrating the scars were analysed for the presence of IL-2R and its correlation with other activation markers such as HLADR, ICAM- I and CD36 molecules.

Biopsies were taken after informed consent from 14 patients, aged 8-60 yr, who underwent plastic surgery under general anaesthesia for the correction of extensive hypertrophic scars consequent to thermal injury. None of the patients was treated with immunomodulating agents before surgery.
Selected patients had developed pathological scars that despite continuous compressive therapy were still present at least 2 yr after trauma (long-term hypertrophic scars). Control specimens of normal skin were obtained from these patients during corrective plastic surgery . Normotrophic scars were obtained from informed consent patients undergoing surgery for aesthetic reasons.

Five-millimetre punch biopsies of hypertrophic (N=14) and normotrophic (N=8) scars and of normal skin (N=6) were obtained from different anatomical sites. Tissue samples were snap-frozen in cold isopentane (-70 'C) and mounted in Oct 4583 embedding compound. Five-micron-thick cryostat seelions were cut in serial sections and transferred to microscope slides. The slides were air-dried and stored at -30 'C.

A number of monoclonal antibodies were used in this work. HLA-DR was detected with HOT 214 12 (a gift from Dr S. Ferrone, New York Medical College, N.Y., USA), which recognizes a DR-localized monomorphic determinant also shared by DP products; and AA3.84,11 which recognizes an epitope common to DR, DQ and DP molecules. These two MoAbs are referred to as anti-DR MoAbs, although anti-DR cannot be distinguished from anti-DP reactivity.

1. The IL-2R p55 chain was detected with the antiCD25 MoAb (IgGl; Becton-Dickinson) and the anti-Tac MoAb (IgG1 Ortho Diagnostic System). The two MoAbs showed the same reactivity pattern, and were used indifferently.
2. The IL2R p75 chain was detected with the anti-IL2R beta p75 MoAb (IgGl, Endogen USA).
3. The anti-CD36 MoAb NL07 (IgM) was the kind gift of Dr Massimo Alessio, San Raffaele Scientific Institute, DIBIT, Milan.
4. T cells were detected with MoAb Leu-4+5b (lgG2a, Becton-Dickinson) which recognizes CD3+CD2 molecules.
5. Macrophages were identified by positive reactivity with MoAb Leu-M5 (IgG2b; Becton-Dickinson) directed against CD 11 c specificity.

The indirect immunoperoxidase technique was performed on cryostat fissue sections fixed in absolute acetone for 10 min. Serial sections were overlaid with 20 pI of different MoAbs at the appropriate dilution for 45 min. After three washings with cold phosphate-buffered saline for 5 min., 20 pI of rabbit anti-mouse IgG (Dako, Copenhagen, Denmark) diluted 1:50 were added for 45 min; after washing, 20 pl of peroxidase-anti-peroxidase complex (Dako, Copenhagen, Denmark) were added for 60 min. Tissue cross-reaction of the second antibody was inhibited by pre-treating tissue sections with normal rabbit serum. After further washings, 100 VI of AEC (Sigma) substrate were added for 20 min. Endogenous peroxidase activity was inhibited by the addition of methyl alcohol and 0.03% hydrogen peroxide. The specificity of the immunostaining was evaluated by replacing the primary antibody with non-immune mouse ascites, resulting in an absence of any immunoreaction. Slides were examined under a microscope in double-blind manner.

Stained cells were counted in two or three sequential sections of each specimen, considering separately three different compartments: epidermis, subpapillary dermis, and reticular dermis. Positively labelled cells were counted in high-magnification fields (x 280) with at least 100 cells counted in the different fields of the first section; corresponding fields were analysed in sequential sections. Activated T cells were estimated in sequential sections stained respectively with anti-CD3+CD2, anti-CD25, and anti-HLA class 11 MoAbs. The percentage of activated T cells was calculated as the ratio between positively stained cells with anti-IL-2R and HLA class 11 MoAbs and the total number of infiltrating T cells in the corresponding fields of the first section.

The presence of activated infiltrating cell subpopulations was evaluated first. A much higher number of infiltrating T cells and macrophages was seen in all specimens of hypertrophic scars than in normotrophic scars of normal skin. Activated T cells (IL-2R+, HLA-DR+) were 70% (71 ± 14) of total T cells in hypertrophic scars as against about 40% (44 ± 23) in normotrophic scars and 10% (14 ± 12) in normal skin samples (Table 1). Activated T cells were present as a cluster of a few cells in the epidermis and as dense agglomerates in the subpapillary dermis.
The results were similar in all specimens from hypertrophic scars and controls, independently of the anatomical localization and the extent of the lesion.

A positive reaction with MoAbs anti-fL-2R p55 and p75 was detected in sequential sections on keratinocytes in all hypertrophic samples; positive keratinocytes were distributed in more or less abundant foci, mainly close to the basal membrane (Figs. lab and Fig. 2a) but also on suprabasal layers. HLA-DR and ICAM-1 molecules are ectopically expressed in the same areas on keratinocyte cell membrane. Near these foci of positive keratinocytes it is possible to detect the presence of infiltrating macrophages and lymphocyte cell membrane. In contrast, CD36 molecules were detected only in the stratum granulosum of the epithelium and were completely absent in the basal layers of the epidermis.
The control-tested specimens of normotrophic scars and normal skin were always negative on keratinocytes (Fig. I c).

In 10 out of the 14 hypertrophic samples a positive staining with anti-IL-2R p55 and p75 MoAbs was detected also on 10% of the total number of fibroplasts seen in hypertrophic tissues. (Fig. 2b).
The ectopic expression of DR and ICAM- I molecules on 30% of dermal fibroblasts was confirmed in all hypertrophic samples (Table 11). HLA DR, ICAM- I and CD36 molecules were highly expressed in the vascular endothelium.
IL-2R+ (p55 and p75), DR+ICAM-1 or CD36 fibroblasts were never seen in normal skin and normotrophic scar sections (Fig. Ic). These results are summarized in Table IL

Fig. la - Immunoperoxidase staining with MoAb anti-CD25 (IL-2R p55) of sections from hypertrophic scar: foci of strongly positive keratinocytes can be seen. Fig. 1b - Immunoperoxidase staining with MoAb antiAL-2R beta chain (p75) of sequential section: similar staining pattern can be seen (magnification x 280). Fig. lc - Immunoperoxidase staining with normal sldn: keratinocytes are negative and no positive infiltrating cells can be seen (magnification x 140).

Fig. 2a - Immunoperoxidase staining with MoAb-anti CD25 (IL-2R) of sections from hypertrophic scar: epidermis with positive keratinocytes. Fig. 2b - Immunoperoxidase staining with MoAb-anti CD25 (IL-2R) of sections from hypertrophic scar: dermis with positively stained fibroblasts (magnification x 280).

The multichain IL-2R system is pivotal in the regulation and function of multiple cells in the immune system and it plays a major role in the activation, differentiation and proliferation of multiple lineages of haematopoietic cells.
IL-2R is composed of at least two subunits, the p55 (u. chain) and p75 (P chain) glycoprotein. The p55 subunit binds IL-2 with low affinity (Kd 10-1) and the p75 binds with intermediate affinity (KdIO-'), whereas a dimeric receptor composed of both p55 and p75 binds IL-2 with high affinity (Kd 10-12).
IL-2R 8 is responsible for signal transduction, whereas IL-21Z (x has the primary function of creating a high affinity receptor by association with the P chain IL-2R, a growth factor for lymphocytes, is present on all classes of lymphocytes, monocytes and granulocytes. Not only is IL-2R expressed more widely than originally supposed on different types of haernotopoietic cells but its presence has also been observed on a wide variety of non-hematopoietic normal or malignant cells of epithelial or mesenchymal origin. Cells may also express p55 alone, p75 alone, or both subunits. The p55 IL-2R seems to be expressed on thymic stromal cells and on oligodendrocyte progenitor cells. IL-2R+ (p55) cells are found also at the local site of autoimmune reactions,' for example in the thyroid of obese strain chickens, in the salivary gland of patients with Sj6gren's syndrome, in chronic active plaques in brains of patients with multiple sclerosis, in the synovial fluid of RA patients, and in human lung carcinoma." A recent work has demonstrated that on intestinal epithelial cell lines IL-2R B and 7 chains appeared to be functional.
The direct effects of IL-2R and its ligand IL-2 on nonlymphoid cells, which have been observed both in vivo and in vitro, suggested two possibilities, firstly that IL-2 not

Note: First section stained with anti-T cells MoAb; sequential section with anti-IL-2R and anti-HLA DR MoAbs. only is a growth factor for lymphocytes but also has much wider and more diverse effects on non-lymphoid tissue cells, including changes in these cells' sensitivity to other cytokines, and secondly that it regulates the expression of other surface molecules, such as HLA-DR and ICAM-1 molecules, involved in cell-to-cell interactions.
The results reported here indicate that high affinity IL2R can be expressed on keratinocytes and fibroblasts in hypertrophic scars, which display many features of a typical inflammatory immune reaction site, such as the presence of abundant activated dermal and epidermal infiltrating T lymphocytes and the ectopic expression of DR and ICAM-1 molecules on keratinocytes and fibroblasts .2, 1 The expression of IL-2R on epithelial cells and fibroblasts may permit a highly plastic pathway of non-lymphoid celllymphocyte interaction, which may prove critical for the function of the skin immune system.
The functional consequences in vivo of MHC class 11, ICAM-1 and IL-2R expression by keratinocytes have not been clarified. Gaspari and Katz" suggest on the basis of experimental data that HLA class 11 positive keratinocytes can participate in some immune reactions. The co-expression on keratinocytes of IL-2R, ICAM-1 and HLA-DR strongly suggests a role of these cells in antigen presentation. It is also known that interferon-gamma and other cytokines released by activated T cells and macrophages can induce HLA-DR and IL-2R.1 The correlation between the expression of HLA-DR, ICAM-1 and IL-2R on keratinocytes and the presence of infiltrating activated T cells in the epidermis suggests that factors released by activated lymphocytes are responsible for the results described here.

The past decade has seen an exponential increase in the number of publications reporting the presence of number of positively reacting specimens various immunoregulatory molecules in the epidermis. It has recently been suggested that epidermal keratinocytes, by virtue of their capacity for producing cytokines, are an integral component of the immune system.` Keratinocytes in fact produce several inflammatory cytokines: IL-1 cc and P, IL-3, IL-6, IL-8, G-CSI, M-CSF, and GM-CSE The production of IL-2 and the expression of IL-2R have not been previously reported for keratinocytes.
Immune mechanisms, particularly cell-mediated immune phenomena, thus seem to be primarily involved in hypertrophic scarring.
The study of the immunological factors involved in hypertrophic scaring will contribute to a better understanding of normal wound healing and facilitate development of new strategies for the management of pathological scars and other fibrotic conditions.

RESUME. Les Auteurs ont effectué la coloration avec l'immunoperoxydase de sections cutanées obtenues de 14 cicatrices hypertrophiques, 8 cicatrices normotrophiques et 6 échantillons de peau normale, avec l'emploi des anticorps monoclonaux (MoAbs) contre les chaînes p55 et p75 du récepteur de l'interleukine 2 (IL-2R). Ils ont observé une évidente expression anormale des kératinocytes des récepteurs d'IL-2 à haute affinité dans tous les prélèvements de cicatrice hypertrophique analysés. Le groupe Il des antigènes des leucocytes humains (HLA-DR) et le type 1 de la molécule d'adhésion intercellulaire (ICAM-1) étaient exprimés ectopiquement sur les mêmes cellules. Au contraire, l'antigène CD36 a été observé seulement dans la couche granuleuse de l'épithélium tandis qu'il était absent dans les couches basales de l'épiderme. Les Auteurs ont trouvé une corrélation entre l'expression des molécules d'IL-2R, ICAM-1 et HLA-DR et la présence de nombreux infiltrats de lymphocytes T activés et de macrophages, ce qui semble indiquer que cette distribution anormale est provoquée par les cytokines produites par les cellules infiltrantes. Dans le derme des échantillons de cicatrice hypertrophique une coloration positive avec anti-IL-2R p55 (chaîne a) et p75 (chaîne P), ICAM-1 et HLA-DR MoAbs a été observée sur les fibroblastes et les endothéliums. La production d'IL-2R n'a pas été précédemment décrite dans la littérature pour les kératinocytes et les fibroblastes. Ces résultats soulignent le rôle joué par les mécanismes immuns dans la cicatrisation hypertrophique et semblent indiquer la participation de phénomènes immuns à médiation cellulaire.

Acknowledgments. This work was supported by the Fondazione Piemontese per gli Studi e le Ricerche sulle Ustioni (Piedmont Foundation for Studies and Research in Bums). Dr Claudia Trombotto was supported by a fellowship from the same Foundation. The helpful suggestions of Prof. Patricia Momigliano Richiardi during the preparation of the manuscript are gratefully acknowledged.

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