Aziz Ghahary

HYPERTROPHIC SCARS, KELOIDS, AND CONTRACTURES: THE CELLULAR AND MOLECULAR BASIS FOR THERAPY

Keloids, hypertrophic scars, and contractures are a result of aberrations of the normal wound healing process. An understanding of the cellular and molecular events that are implicated in the development of these fibroproliferative disorders will allow for optimization of wound healing. In turn, treatment choices can be based on the most current scientific information available.

Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells.

Peripheral blood fibrocytes are a newly identified leukocyte subpopulation that displays fibroblast-like properties. These blood-borne cells can rapidly enter the site of injury at the same time as circulating inflammatory cells. We hypothesize that circulating fibrocytes represent an important source of fibroblasts for healing of extensive burn wounds where it may be difficult for fibroblasts to migrate from the edges of uninjured tissue. In this study we identified and quantified fibrocytes among the adherent cells cultured from human peripheral blood mononuclear cells (PBMC) obtained from 18 burn patients and 12 normal individuals, based on their ability to express type I collagen. Our results showed that adherent cells cultured from PBMC of burn patients differentiated to fibrocytes more efficiently than did those from normal individuals. The percentage of type I collagen-positive fibrocytes was significantly higher for patients than for controls (89.7 ± 7.9% versus 69.9 ± 14.7%, p < 0.001). This percentage was consistently higher for patients with a ≥30% total body surface area burn until 1 year, with the highest percentage appearing within 3 weeks of injury. A positive correlation was found between the levels of serum transforming growth factor-β1 (TGF-β1) and the percentage of fibrocytes developing in the cultures of PBMC derived from these patients. We also demonstrated that fibrocytes were derived from CD14+ cells but not CD14− cells. Conditioned medium from CD14− cells was, however, required for fibrocyte differentiation, whereas direct contact between CD14− and CD14+ cells was not necessary. Treatment of the cell cultures with TGF-β1 enhanced the development of collagen-positive cells, whereas the inclusion of neutralizing anti-TGF-β1 antibodies in the CD14− conditioned medium suppressed fibrocyte differentiation. These data suggest that the development of fibrocytes is up-regulated systemically in burn patients. Increased TGF-β in serum stimulates the differentiation of the CD14+ cell population in PBMC into collagen-producing cells that may be important in wound healing and scarring.

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-γ showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-γ-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-γ-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-γ alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-γ. Coculture of eosinophils with an IFN-γ-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

Identification of fibrocytes in postburn hypertrophic scar.

Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast‐like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two‐dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte‐specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte‐specific protein‐1 and to the N‐terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual‐labeled spindle‐shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen‐presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

transforming Growth Factor-β in Thermally Injured Patients with Hypertrophic Scars: Effects of Interferon α-2b

Hypertrophic scarring is a common dermal fibroproliferative disorder that leads to poor quality wound healing, prolongs rehabilitation, and increases morbidity following major thermal and other injuries to the deep dermis. Local and systemic transforming growth factor (TGF)-beta has been implicated as a fibrogenic cytokine in the pathogenesis of many fibrotic disorders, whereas interferon (IFN) alpha-2b may improve the pathologic features of dermal fibrosis directly or by antagonizing the effects of TGF-beta and histamine. Nine patients with severe hypertrophic scarring were evaluated for 8 weeks before treatment with subcutaneous recombinant IFN alpha-2b, 2 x 10(6) IU three times per week for 24 weeks. Clinical assessment was performed using standardized photography, a burn scar assessment tool, and serial scar volume measurements. Monthly measurements of serum TGF-beta and plasma Ntau-methylhistamine were made prior to, during, and after IFN alpha-2b therapy and compared with 27 age-matched controls. Serial biopsies of the hypertrophic scars and normal skin were performed for evaluation of mast cell numbers. Significant improvement in scar assessment occurred in 7 of 9 patients, and 3 of 9 demonstrated significant reductions in scar volume with interferon therapy beyond that occurring during the 8-week control period. For the entire group, mean rates of improvement were significantly better during interferon therapy with no recurrence following treatment. Before interferon therapy, serum TGF-beta was significantly higher in the burn patients with hypertrophic scarring than in a control population (123.04 +/- 36.48 vs. 56.85 +/- 8.38 ng/ml, p < 0.05). Within 3 months of IFN alpha-2b therapy, serum TGF-beta levels fell significantly and remained within the normal range during therapy and after interferon therapy was stopped. Plasma Ntau-methylhistamine levels were also significantly elevated in the hypertrophic scar patients as compared with age and sex-matched controls (153.6 +/- 92.07 vs. 48.3 +/- 28.9 pg/ml, p < 0.05), and significant reductions were achieved with interferon therapy and maintained after interferon was discontinued. Paired biopsies of hypertrophic scarring and normal tissue demonstrated increased numbers of mast cells in hypertrophic scars compared with normal uninjured skin from the same patients (2.65 +/- 1.63 vs. 1.04 +/- 0.62 cells/high power field, p < 0.001); however, no significant change in mast cell content of the hypertrophic scars accompanied interferon therapy. Patients with severe hypertrophic scarring demonstrate increased levels of serum TGF-beta and plasma Ntau-methylhistamine following thermal injury. A significant clinical improvement in scar quality and volume occurred during IFN alpha-2b therapy, which was associated with normalization of serum TGF-beta and plasma Ntau-methylhistamine levels. A double-blind, placebo-controlled trial will be required to further assess the usefulness of subcutaneous treatment with IFN alpha-2b for the treatment of hypertrophic scarring.

Hypertrophic scar tissues and fibroblasts produce more transforming growth factor-β1 mRNA and protein than normal skin and cells

Transforming growth factor‐β1 is a well‐known fibrogenic cytokine produced by many types of cells including dermal fibroblasts. To investigate whether this fibrogenic cytokine is involved in development of hypertrophic scar, transforming growth factor‐β1 gene expression was evaluated in small skin samples. Because a sufficient quantity of normal skin from patients with hypertrophic scar is not readily available, a reverse transcription‐polymerase chain reaction technique was used. Quantitation of gene expression by reverse transcription‐polymerase chain reaction is difficult partly due to the lack of suitable complementary RNA standards. We have established a convenient, reliable procedure to construct an internal standard for transforming growth factor‐β1 starting with a gene specific polymerase chain reaction product. After digestion of the polymerase chain reaction product with endonuclease, a small piece of cDNA from human procollagen α1(I) cDNA with compatible ends was inserted into the polymerase chain reaction‐DNA fragment. The recombinant cDNA was re‐amplified by polymerase chain reaction and subcloned into a plasmid containing bacteriophage T7 and T3 promoters. Complementary RNA was prepared from the recombinant plasmid and amplified by reverse transcription‐polymerase chain reaction together with the tissue or cellular RNA. After amplification, the products were electrophoresed in an agarose gel containing ethidium bromide. The bands for internal standard and transforming growth factor‐β1 mRNA were scanned, digitized, and plotted against the amount of internal standard complementary RNA added in the reverse transcription‐polymerase chain reaction. The number of mRNA molecules/cell was calculated. We examined the transforming growth factor‐β1 mRNA in hypertrophic scar tissue and in normal skin and found that hypertrophic scar tissues expressed five‐fold more transforming growth factor‐β1 mRNA than normal skin per unit of wet weight. We used this procedure to quantitate transforming growth factor‐β1 mRNA expression in 5 pairs of fibroblast cultures derived from hypertrophic scar and normal skin. The results showed that hypertrophic scar fibroblast cultures contain significantly more molecules of mRNA for transforming growth factor‐β1 than normal cells (116 ± 6 vs. 97 ± 7, p = 0.017, n = 5). These results were supported by Northern analysis for transforming growth factor‐β1 mRNA in the cells and enzyme‐linked immunosorbent assay for TGF‐β1 protein in fibroblast‐conditioned medium. In conclusion, hypertrophic scar tissue and fibroblasts produce more mRNA and protein for transforming growth factor‐β1, which may be important in hypertrophic scar formation. The construction of the gene specific internal standard for reverse transcription‐polymerase chain reaction is a simple and reliable procedure useful to quantitate gene expression in a small amount of tissue or number of cells.

Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-β in human post-burn hypertrophic and mature scars

The distributions of the small proteoglycans, decorin and biglycan and the large proteoglycan, versican, in normal skin and post‐burn hypertrophic and mature scars, were compared using monoclonal and polyclonal antibodies to the core proteins. Biglycan and verscan were virtually undetectable in normal dermis but readily seen in hypertrophic scars. Staining for decorin was strong throughout the dermis in normal skin but restricted to the deep dermis and a narrow zone under the epidermis in hypertrophic scar—areas which did not stain for versican. Decorin was absent or reduced in the nodules in these specimens. In mature post‐burn scars, staining for all three proteoglycans demonstrated an intensity that was intermediate between that in normal dermis and that in the nodules of the hypertrophic scars. Transforming growth factor‐β was present in the nodules of hypertrophic scars but the deep dermis of these specimens stained most intensely for this cytokine which was also found in the dermis of mature scars but was not detectable in normal dermis. The apparent co‐distribution of decorin and transforming growth factor‐β suggests that this proteoglycan may play an active role in the resolution of the scars. Changes in proteoglycan type and distribution could possibly account, at least in part, for the derangement of collagen and the altered physical properties of hypertrophic scar tissue.

Transforming Growth Factor-beta mRNA and Protein in Hypertrophic Scar Tissues and Fibroblasts: Antagonism by IFN-alpha and IFN-gamma In Vitro and In Vivo

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.

Healing of Burn Wounds in Transgenic Mice Overexpressing Transforming Growth Factor-β1 in the Epidermis

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Role of keratinocyte–fibroblast cross‐talk in development of hypertrophic scar

The ability to generate or repair injured tissue is essential to the continuity of human life. As in all other organs, wound healing in the skin is a dynamic process involving tissue response to different types of insults. This process involves a continuous sequence of signals and responses in which platelets, fibroblasts, epithelial, endothelial, and immune cells come together outside their usual domains to orchestrate a very complex event that results in tissue repair. These signals, which are mainly growth factors and cytokines, orchestrate the initiation, continuation, and termination of wound healing. An imbalance in the synthesis and release of these cytokines and growth factors at the wound site, therefore, may result in either retarded wound healing, as is seen in diabetic patients and the elderly population, or overhealing wounds such as fibroproliferative disorders frequently seen following surgical incision, traumatic wounds, and severe electrical and thermal injury. In general, regardless of the site of injury, in any phase of the dynamic healing process, a fine balance between synthesis of extracellular matrix and degradation by a large family of enzymes, known as matrix metalloproteinases, is required for maintaining the structural integrity of healing tissue. The availability of new models such as organotypic co‐culture systems have allowed us to gain new insight into the cell–cell interactions at both cellular and molecular levels. Recent evidence indicates that mesenchymal–epithelial interactions play a critical role in regulation of skin homeostasis and this cross‐talk is mediated by soluble factors acting as autocrine/paracrine regulators of fibroblast and keratinocyte growth, function, and differentiation. In this review we address the question of how keratinocyte–fibroblast interaction plays a role in controlling the expression of key extracellular matrix molecules such as matrix metalloproteinases, which are critical in the healing process following any types of insults to the skin.