Heather A. Shankowsky

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.

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.

Deep dermal fibroblasts contribute to hypertrophic scarring

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-β1 and TGF-β1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more α-smooth muscle actin (α-SMA), had higher α-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS.

Fibrocytes from burn patients regulate the activities of fibroblasts

Wound healing requires an elaborate interplay between numerous cell types that orchestrate a series of regulated and overlapping events. Fibrocytes are a unique leukocyte subpopulation implicated in this process. One role proposed for these cells in wound healing is to synthesize extracellular matrix. Interestingly, using mass spectrometry to quantify hydroxyproline, we discovered that the capacity of fibrocytes from normal subjects or from burn patients to produce collagen is much less than that of dermal fibroblasts. Therefore, we investigated whether fibrocytes could play an indirect, regulatory, role in the healing of burn wounds by affecting the functions of dermal fibroblasts. Dermal fibroblasts treated with medium conditioned by burn patient fibrocytes, but not by those derived from normal subjects, showed an increase in cell proliferation and migration. Using confocal microscopy, flow cytometry, and immunoblotting, we found the level of α‐smooth muscle actin (α‐SMA) expression to be increased in these treated dermal fibroblasts, which also showed an enhanced ability to contract collagen lattices. To determine whether these effects could be attributed to transforming growth factor β (TGF‐β1) or to connective tissue growth factor (CTGF), we measured total TGF‐β1 levels in the conditioned medium by an enzyme‐linked immunosorbtion assay and assessed levels of CTGF mRNA and protein in fibroblasts and fibrocytes by reverse transcription‐polymerase chain reaction and Western blotting. The results showed significantly higher levels of TGF‐β1 and CTGF produced by burn patient fibrocytes. In addition, the application of a TGF‐β1 neutralizing antibody significantly reduced the effect of burn patient fibrocyte medium on dermal fibroblast proliferation, migration, and collagen lattice contraction. Our results suggest that in healing burn wounds, fibrocytes could regulate the activities of local fibroblasts.

Quality-of-life and outcome predictors following massive burn injury

Background: Quality of life is a major criterion when decisions regarding resuscitation, reconstruction, and rehabilitation of patients with massive burn injuries are being considered. There has been little research focusing on quality of life following burn injuries involving more than 50 percent total body surface area in the adult population. The authors’ goals were to describe quality of life and identify specific clinical and functional indices that predict good quality of life following massive burn injuries. Methods: Using a prospective study design, 47 patients who survived a massive burn between 1980 and 2001 were recruited from a single burn unit. Clinical data were collected from hospital records, function was assessed using the Abbreviated Burn-Specific Health Scale, and quality of life was assessed using the Short Form-36 survey. The Short Form-36 scores were compared with population norms. Univariate and multivariate regression analyses were used to identify factors predicting Short Form-36 scores. Results: Overall, the mean age was 28 ± 1.8 years, 96 percent (45 of 47) were men, and the mean burn size was 64 ± 2.1 percent total body surface area. Compared with Canadian population norms, burn patients had significantly lower Short Form-36 scores in the domains of role phys-ical (69.1 versus 82.1, p = 0.0067) and general health perception (67.2 versus 77.0, p = 0.00014). At the time of injury, the amount of total full-thickness burn predicted follow-up Short Form-36 physical summary scores (R2 = 15 percent, p < 0.001). At the time of follow-up, addition of the patient’s hand function significantly contributed to the prediction of Short Form-36 physical summary scores (R2 = 44 percent, p < 0.001). At the time of injury, the age of the patient predicted follow-up Short Form-36 mental summary scores (R2 = 25 percent, p < 0.001). At the time of follow-up, addition of the patient’s perceived level of social support significantly contributed to the prediction of Short Form-36 mental summary scores (R2 = 44 percent, p < 0.001). Conclusions: Survivors of massive burn injury reported a good quality of life in most Short Form-36 domains. The authors identified the size of the total full-thickness injury and the age of the patient as factors available at the time of injury that predict quality of life. The addition of hand function and the patient’s perceived level of social support at the time of follow-up improved prediction of quality of life. Accordingly, this information on quality of life after massive burn injury could aid in decision making at the time of resuscitation, reconstruction, and rehabilitation.

Profile of the paediatric burn patient in a Canadian burn centre

Five hundred and eighty-three children (0-18 years old), consisting of 33.4 per cent of all burn inpatients, were admitted to the University of Alberta Hospitals over an 11-year period (January 1978 to December 1988). Demographic and outcome variables, in addition to aetiological factors, were examined. 48.4 per cent of burns occurred in children less than 4 years of age, with males predominating in every age group (P less than 0.001). Children had smaller burns, a higher incidence of scalds, less inhalation injuries and a lower mortality compared to adult burn patients admitted over the same time period (P less than 0.05). There was a low incidence of confirmed child abuse by burns (1.4 per cent). High-risk environments identified were the home (74.6 per cent of burns) and recreational settings (12.4 per cent of burns), mainly occurring around campfires. Native children were overrepresented in the burn population compared to the general population by a factor of approximately 10:1. Scald prevention, high-risk environments (home and recreational), high-risk populations (male and natives) and unsafe practices with flammable liquids (petrol in particular) should be emphasized in paediatric burn prevention programmes.

Accelerated wound healing in leukocyte-specific, protein 1-deficient mouse is associated with increased infiltration of leukocytes and fibrocytes.

Wound healing is a complex process involving the integrated actions of numerous cell types, soluble mediators, and ECM. Recently, a newly identified cell type, the fibrocyte, has been reported to contribute to wound healing and fibrotic conditions such as hypertrophic scarring. We previously established leukocyte‐specific protein 1 (LSP1) as a marker for fibrocytes. LSP1 is an F‐actin binding protein and substrate of p38 mitogen‐activated protein kinase and protein kinase C, and has been reported to be important in leukocyte chemotaxis. We examine the biological roles of LSP1 in skin wound healing using Lsp1−/− null mice. These animals showed accelerated healing of full‐thickness skin wounds, with increased re‐epithelialization rates, collagen synthesis, and angiogenesis. Healing wounds in Lsp1−/− mice had higher densities of neutrophiles, macrophages, and fibrocytes. Along with increased leukocyte infiltration, levels of macrophage‐derived chemokine expression, TGF‐β1, and VEGF were all up‐regulated. These results demonstrate that the absence of LSP1 promotes healing of skin wounds. The primary mechanism seems to be an increase in leukocyte infiltration, leading to locally elevated synthesis and release of chemokines and growth factors. Further analysis of Lsp1−/− mice may suggest ways to improve wound healing and/or treat fibrotic conditions of skin and other tissue.

Increased TGF-β-producing CD4+ T lymphocytes in postburn patients and their potential interaction with dermal fibroblasts in hypertrophic scarring

The development of hypertrophic scar involves a complex interplay between cells and cytokines. Although the mechanism underlying its pathogenesis is not well understood, a polarized T‐helper type 2 immune response has been reported, indicating a role for CD4+ T lymphocytes in hypertrophic scarring. Here, we report an increased frequency of CD4+/transforming growth factor‐β (TGF‐β)‐producing T cells in the peripheral blood and hypertrophic scar tissue of burn patients. These cells may play an indirect regulatory role in hypertrophic scar by affecting the functions of dermal fibroblasts. Our results show an increase in cell proliferation and collagen synthesis by dermal fibroblasts treated with medium derived from burn patient CD4+ T lymphocytes but not from the CD4+ T cells of normal subjects. Using confocal microscopy and immunoblotting, we found the level of α‐smooth muscle actin to be elevated in these treated dermal fibroblasts, which also showed an enhanced ability to contract collagen lattices. TGF‐β levels in medium conditioned by the culture of CD4+ T lymphocytes from burn patients were significantly higher than in the conditioned medium from CD4+ T lymphocytes of normal subjects. In addition, the application of a TGF‐β–neutralizing antibody significantly reduced the effect of burn patient CD4+ T lymphocyte medium on dermal fibroblast proliferation and collagen lattice contraction. Our study suggests that CD4+/TGF‐β–producing T lymphocytes may play an important role in postburn hypertrophic scarring.

North American survey of hydrotherapy in modern burn care

To investigate the role of hydrotherapy in the treatment of patients with burns, a survey was conducted of the use of hydrotherapy in Canada and the United States as part of an intensive investigation into the causes of Pseudomonas aeruginosa infections in burn injury. Results of the survey conducted indicate that hydrotherapy continues to be an important part of burn wound care in most (94.8%) burn centers in North America. Of the burn centers that use hydrotherapy, 81.4% continue to immerse patients, 82.8% perform hydrotherapy on all patients with burns regardless of total body surface area, and 86.9% continue with hydrotherapy throughout the entire phase of the patients hospitalization. Routine culturing of the hydrotherapy equipment is standard procedure in 49.7% of the units surveyed, and culturing of the water supply to the equipment on a regular basis is done in only 18.6% of those burn units regularly using hydrotherapy. Pseudomonas aeruginosa was identified as the most common, major cause of sepsis in 52.9% of the burn units surveyed, Staphylococcus aureus in 25.5%, and Candida albicans in 5.2%. This survey demonstrates the extensive use of hydrotherapy in North American burn units and the concern for serious infections in patients with burns from gram-negative organisms such as Pseudomonas species. With the increasing number of reports of Pseudomonas infections related to the use of hydrotherapy equipment, the importance for further investigation into burn wound care with and without hydrotherapy, infection rates, and cost analysis appears to be indicated.