Suzan deSerres

Allogeneic fibroblasts used to grow cultured epidermal autografts persist in vivo and sensitize the graft recipient for accelerated second-set rejection

INTRODUCTION Cultured epidermal autografts (CEAs) have been used for wound coverage in patients with massive burns and other skin defects. However, CEAs often display late breakdown, which may be immunologically mediated and initiated by persistent foreign fibroblasts used as a feeder layer to optimize keratinocyte growth. This study investigates whether these fibroblasts, previously shown to persist in vitro, survive after grafting and induce host sensitization to alloantigen. METHODS CEAs from CBA donors (H-2k) were grown on allogeneic NIH 3T3 (H-2q) or syngeneic LTK (H-2k) fibroblasts, which were removed by trypsinization 7 days later. CBA mice (n = 85) were flank-grafted with NIH allografts (positive control), CEA/3T3s, CEA/LTKs, or CBA autografts (negative control). Hosts were challenged with second set NIH tail allografts 3 weeks later. Median graft survival was compared between groups by Wilcoxon rank and chi 2 analysis. Additional CBA mice (n = 15) received CEAs that were biopsied 0, 4, and 8 days after grafting. The presence of allogeneic fibroblasts was determined by Western immunoblotting, using KL295, a monoclonal antibody that recognizes H-2q (but not H-2k) class II histocompatibility antigens. RESULTS Allogeneic fibroblasts persisted after grafting but decreased over time, as determined by alloantigen expression on Western immunoblots. Accelerated tail graft rejection occurred in hosts primed by NIH allografts (9 days, p < 0.05), as well as by CEAs growth with an allogeneic (10 days, p < 0.05) but not a syngeneic feeder layer (12 days, NS). Mice receiving flank autografts rejected second set tail allografts at 12 days. CONCLUSIONS Immunogenic fibroblasts used to grow CEAs survive in vivo and sensitize the graft recipient for accelerated second-set rejection. These persistent cells may initiate an inflammatory response that may result in late graft breakdown and limit the utility of CEAs grown with a foreign fibroblast feeder layer.

Antigenic cross-reactivity between media supplements for cultured keratinocyte grafts

Patients who were grafted with cultured human keratinocytes have been shown to make antibodies to the xenogeneic proteins, which supplement the culture media. An immune response to these antigens may lead to late graft loss or accelerated rejection of subsequent cultured keratinocyte grafts. Methods to eliminate this potential problem include growing cells in a serum-free medium with bovine pituitary extract as a serum substitute. To determine whether bovine pituitary extract would be subject to an immune response similar to that which is evoked by fetal bovine serum, we investigated their antigenic cross-reactivity. When compared by sodium dodecylsulfate-polyacrylamide gel electrophoresis, considerable similarity was identified between the two supplements. Immunostaining with antibodies to both bovine pituitary extract and fetal bovine serum confirmed antigenic cross-reactivity. Inhibitory enzyme-linked immunosorbent assay studies demonstrated specific cross-reactivity that ranged from 60% to 68%. When tested for antibody cross-reactivity to bovine pituitary extract, serum samples from five patients who were receiving human-keratinocyte grafts that were grown in fetal bovine serum-supplemented media and that exhibited a positive antibody titer to fetal bovine serum showed cross-reactivity that ranged from 30% to 141%. Bovine pituitary extract contains proteins that cross-react with fetal bovine serum. Human keratinocytes that are cultured in media that is supplemented with bovine pituitary extract would therefore be subject to an immune response similar to that which is evoked by human keratinocytes that have been cultured in fetal bovine serum.

Antibody response to xenogeneic proteins in burned patients receiving cultured keratinocyte grafts.

Cultured keratinocytes (CK) have been used to resurface large burn wounds with some success. These CK are grown in the presence of fetal bovine serum (FBS) and mouse fibroblasts (MF). Serum from ten patients who received CK grafts as part of burn wound coverage was studied by ELISA technique for antibody to these xenogeneic antigens. All patients had some amount of antibody to FBS but no detectable antibody to MF. The amount of antibody to FBS varied between patients and with respect to time after graft application. Generally, the levels of antibody to FBS were moderate by 30 days, declined, and then rose slowly by 5 to 6 months. The majority of antibody to FBS was against bovine serum albumin (BSA), demonstrated by Western blot technique. The presence of such antibody to FBS might produce clinical problems of graft rejection, anaphylaxis, and serum sickness in patients receiving CK grown in FBS supplemental medium. Further investigation will need to determine the likelihood and potential severity of such clinical problems.

Early, complete burn wound excision partially restores cytotoxic T lymphocyte function.

BACKGROUND Cytotoxic lymphocytes (CTLs) are an important component of immune function, involved in antigen recognition and resistance to viral infection. Burn injury suppresses cell-mediated immunity, induces allograft tolerance, and increases the risk of viral infection, but the mechanisms are not well understood. This study analyzes the effect of burn size and burn wound excision on CTL activity. METHODS Anesthetized CBA mice (n = 12) received a 0%, 20%, or 40% body surface area contact burn. Additional mice (n = 16) received a 40% burn that was totally, partially, or not excised 72 hours after burn. Excised areas were covered with normal, syngeneic skin. Two weeks later harvested splenocytes were cocultured with allogeneic stimulators. CTL activity was determined by a 51Cr release assay, in which CTL effectors were tested on allogeneic, radiolabeled targets. Dilution curves of CTL activity were compared by ANOVA: RESULTS Both 20% and 40% burns significantly inhibited CTL activity (p < 0.05). Total but not partial excision of a 40% burn restored CTL activity (p < 0.01). Both total and partial wound excision also improved survival (p < 0.05). CONCLUSIONS Burn injury inhibits CTL activity in a size-dependent manner, and total wound excision significantly improves both CTL function and survival after injury. This study suggests a mechanism for the immunosuppressive effects of burn injury and provides an immunologic rationale for early, complete burn wound excision.

Early but not late Burn wound excision partially restores viral-specific T lymphocyte cytotoxicity

OBJECTIVE Early burn wound excision restores immunocompetence and improves patient survival, but the exact mechanisms have not yet been defined. Burn injury impairs cytotoxic T lymphocyte (CTL) activity as a function of burn size, increasing the risk of infection. The purpose of this study was to determine if early wound excision improved viral-specific CTL function. METHODS Anesthetized C57BL/6 mice (n = 20) received 0%, 20%, or 40% total body surface area full-thickness contact burns and were inoculated 3 days later with intraperitoneal lymphocytic choriomeningitis virus. Eight days after infection, or 11 days after burn, CTL effectors (E) were harvested and tested against infected, radiolabeled L-Dh targets (T) in a 51Cr-release assay, at varied E:T ratios. Dilution curves of CTL activity were compared by analysis of variance. In the second experiment, mice (n = 18) underwent a 30% burn that was totally excised and grafted on postburn days (PBDs) 0, 3, and 7. Control groups included sham burn and no excision of a 30% burn. In the third experiment, mice (n = 22) received a 30% burn that was partially, completely, or not excised on PBD 3. Control groups included sham burn with and without excision. All groups were infected with intraperitoneal lymphocytic choriomeningitis virus on PBD 3. Viral-specific CTL activity was determined on PBD 11. RESULTS Both 20% and 40% burn injury impaired viral-specific CTL function. Wound excision on PBDs 0 and 3, but not on PBD 7, partially restored CTL function. Total excision of the 30% burn improved CTL activity to a greater extent than did partial excision. CONCLUSION Burn injury inhibits viral-specific CTL activity. Early, complete wound excision augments CTL function. Improved CTL activity after burn may reduce the risk of infection, providing an immunologic rationale for expeditious wound excision.

Gene expression and cytokine and enzyme activation in the liver after a burn injury.

The liver plays a critical role in the inflammatory response to injury; however, the mechanisms by which the liver is affected and how it influences the rest of the immune system are not well understood. Partial hepatectomy is a direct injury to the liver, whereas a burn is an indirect injury to liver, but both injuries appear to produce damage to the liver. In this study, we used a mouse model of 25% total body surface area and 40% total body surface area full-thickness burns to investigate the mechanism of liver damage and response to burn injury by measuring levels of c-Jun messenger (m)RNA, NFkappaB nuclear protein, interleukin-6, transaminases, and liver tissue histology over time. c-Jun and NFkappaB are 2 transcription factors that are induced by partial hepatectomy and related to hepatocyte injury and growth. In both groups of mice with burns, expression of c-Jun mRNA and NFkappaB nuclear protein was activated within 30 minutes after the burn injury, followed by increased levels of interleukin-6 and, finally, elevated enzyme levels. Liver injuries were similar in both groups despite the magnitude of the burns. We believe that these gene products are initiated in the hepatocyte injury after a burn and that they precede other inflammatory responses such as cytokine release, plasma transaminase levels, and histologic changes.

Xenogeneic mouse fibroblasts persist in human cultured epidermal grafts: a possible mechanism of graft loss.

Recent reports suggest that long-term graft take of cultured epidermal autografts (CEAs) is less than 50% when late graft loss is considered. The characteristics of late CEA loss suggest that it may occur as a result of an immunologic reaction to persistent xenogeneic cells and/or proteins used to grow CEA. In this study we examined whether immunologically reactive, mouse 3T3 fibroblasts used as feeder layers can persist in primary, secondary, and tertiary human CEA. We cocultured keratinocytes from 11 separate burn patients with growth-arrested 3T3 fibroblasts. After removing visible 3T3 fibroblasts from CEA with trypsinization, we allowed CEA to reach confluence. We then harvested CEA either as primary, secondary, or tertiary cultures. We detected mouse fibroblasts using fluorescence activated cell sorting (FACS) with a monoclonal antibody specific for mouse major histocompatibility (MHC) antigens. We detected mouse MHC class II antigens by performing Western immunoblotting with another mouse MHC-specific monoclonal antibody. By FACS we identified mouse fibroblasts in 100, 75, and 62.5% of primary, secondary, and tertiary passage CEAs, respectively. Similarly by immunoblotting we found mouse MHC class II antigen in 100, 80, and 66.7% of primary, secondary, and tertiary CEAs. These results demonstrate that xenogeneic fibroblast feeder layers capable of generating immunogenic transplantation antigens persist in CEAs. The persistence of these cells and their antigen expression may contribute to CEA loss.

Persistence of fetal bovine serum proteins in human keratinocytes.

Cultured human keratinocytes are used for skin grafts, but their success is limited by late graft loss. Development of antibody to fetal bovine serum (FBS) protein used in culture media for in vitro keratinocyte growth has been identified. The persistence of FBS antigen in skin grafts is important in the induction of the immune response and the susceptibility of the keratinocytes to immune-mediated injury. The magnitude and longevity of FBS protein persistence on human keratinocytes was studied. Secondary passage human keratinocytes were grown in media supplemented with 5% FBS. The media was changed to one supplemented with pooled human AB serum, and the amount of FBS protein incorporated in the tissue was measured over the following 8 days by an ELISA reaction directed against FBS antigen. Incorporated FBS antigen decreased for the first 3 days to 31% of maximum. There was no further significant decrease for 5 days. Keratinocytes grown in alternative serum supplements (NuSerum [Collaborative Research Inc., Bedford, Mass.] and Serum Plus [Hazelton Research Products Inc., Lenexa, Kan.]), which contain reduced amounts of FBS, offered no significant reduction in FBS protein incorporation. This duration of antigen persistence would make human keratinocytes susceptible to cell destruction by immune response to FBS and may contribute to delayed loss of human keratinocyte grafts.

Transforming growth factor-β1 and splenocyte apoptotic cell death after burn injuries

Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that mediates apoptotic cell death in human lymphocytes in vitro. To better understand the mechanism through which TGF-beta1 exerts its apoptotic effect, we investigated the role of TGF-beta1 in the relationship between burn injury and cell death of splenocytes in a mouse model of either 0%, 25%, or 40% full-thickness burns. Mice were killed and spleens were harvested at 15 and 30 minutes and at 1, 2, 4, 8, 12, and 24 hours after the burn. The spleens were divided and used for both histologic analyses with H-E stain and TUNEL stain and for total messenger RNA isolation and reverse transcriptase-polymerase chain reaction amplification. Amplified polymerase chain reaction products were analyzed for signal strength by electrophoresis. TGF-beta1 RNA expression was highest at 2 hours after the burn injuries in the 40% full-thickness burns and at 4 hours after the burn injuries in the 25% full-thickness burns. The relative increase in TGF-beta1 RNA was 3 times greater with the larger burn than with the smaller burn. In histologic analysis, splenocyte apoptotic cell death was observed at 4 to 24 hours after the burn in the 40% full-thickness burns but at only 4 to 12 hours in the 25% full-thickness burns. TGF-beta1 RNA peak expression was observed at different times after the burn in 25% and 40% full-thickness burns. Histologic analysis showed apoptotic cell death in proportion with respective messenger RNA expressions. This suggests that TGF-beta1 may be associated with apoptosis of splenocytes in vivo and that the effect of TGF-beta1 after a burn injury may be important in the immune system.

Impact of burn injury on hepatic TGF-β1 expression and plasma TGF-β1 levels

Background: The liver plays a critical regulatory role in the acute inflammatory response to injury, although the mechanisms of this regulation are not well understood, transforming growth factor-β 1 (TGF-β 1 ) is induced after burn injury and may contribute to an inhibitory or fatal effect on hepatocytes. We investigated the association over time between plasma concentration of TGF-β 1 , expression of TGF-β 1 m-RNA in liver tissue, and histologic analysis of liver apoptosis after burn injury. Methods: Male BALB/c mice were anesthetized and randomized to receive 0% (sham), moderate (approximately 25%) (M), or large (approximately 50%) (L) body surface area full-thickness contact burn, followed by resuscitation and analgesia. Animals were killed over a time course from 15 minutes to 24 hours after burn injury, and liver tissue and peripheral blood were collected. Plasma levels of TGF-β 1 (nanograms per milliliter) were measured by enzyme-linked immunosorbent assay. TGF-β 1 m-RNA was extracted from liver and measured by reverse transcription-polymerase chain reaction. Histology of liver apoptosis was examined after fixation and staining with TdT-mediated dUTP nick-end labeling (TUNEL) method. Results: The plasma concentration of TGF-β 1 in burn group L was significantly increased at 4 hours after burn when compared with sham and M burn groups. This rise in plasma TGF-β 1 was preceded by an increase in hepatic TGF-β 1 m-RNA expression at 30 minutes, 1, 2, and 4 hours after burn in the L group. Histologic analysis found greater hepatocyte death in the L group than in the M group at 8 hours after burn. Conclusion: The levels of induced TGF-β 1 and TGF-β 1 m-RNA after L burn injury are higher and peak earlier than after M burn injury. Elevated TGF-β 1 may be associated with cell death in hepatocytes. The TGF-β 1 rise may be associated with hepatocyte injury and systemic response to massive burn.