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Human beta defensin is absent in burn blister fluid
Burns 26 (2000) 724±726
www.elsevier.com/locate/burns
Human beta defensin is absent in burn blister ¯uid M.R. Ortega a, T. Ganz b, S.M. Milner a,* a
Institute for Plastic and Reconstructive Surgery, Southern Illinois University School of Medicine, Spring®eld, IL 62794, USA b Department of Medicine, UCLA School of Medicine, CHS 37-055, Los Angeles, CA 90095, USA Accepted 3 April 2000
Abstract Defensins are a family of cationic antimicrobial peptides that participate in innate host defence. Human beta defensin-2 (HBD-2) is produced by human keratinocytes, and has a potent bactericidal activity against a wide spectrum of microorganisms. We have recently shown that expression of HBD-2 is present in normal skin and lost in the full-thickness burn wound. Defensins have been found in the blister ¯uid of chronic wounds. Our study was designed to examine blister ¯uid from partialthickness burns for defensin content. Fluid from ®ve patients was collected from partial-thickness burn blisters, and then analysed by sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with a monoclonal antibody and rabbit polyclonal antibody to HBD-2. The assay was validated against a Western blot assay for HBD-2 in samples of bronchoalveolar lavage ¯uid from patients with in¯ammatory lung disease. No HBD-2 was detectable in any of the burn blister ¯uids analysed. HBD-2 is lost in the full-thickness burn wound, and we have now demonstrated its absence in burn blister ¯uid. This ®nding represents evidence of a host defence defect within the burn wound and suggests a possible therapeutic role for antimicrobial peptides in the management of burn wounds. 7 2000 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Burns; Sepsis; Defensins; Blister ¯uid; Antimicrobial peptides
1. Introduction The concept of human skin as an ``immunological organ'' is now widely accepted [1]. Antimicrobial peptides, which are emerging as an important means of host defence, have been found in the cells and body ¯uids of a wide variety of multicellular animals [2]. Defensins, a particular class of antimicrobial peptides, have six cysteine residues, which form intramolecular disulphide bonds, are rich in arginine and are only 29±47 amino acids in length. There are three major structural sub-families of defensins, alpha, beta and insect defensins, whose ability to kill microorganisms is based on their capacity to form pores in microbial membranes [3]. Epithelial surfaces are protected by antimicrobial * Corresponding author.
peptides that form part of an innate immune mechanism. The mucosal surfaces of mammals are protected by defensins produced by epithelial lining cells and specialised epithelial cells, such as Paneth cells [4]. In human skin, Harder et al. demonstrated the presence of HBD-2 [5]. The peptide has potent bactericidal activity against a wide spectrum of gram positive, gram negative and fungal organisms, which are commonly implicated in burn sepsis [6]. The burn wound is an ideal substrate for bacterial growth and provides a portal for microbial invasion. In a partial thickness burn, blister ¯uid is liberated freely across the surface of the wound. It has been shown that alpha defensins are present in blisters of chronic wounds and pemphigoid [7]. In view of our previous studies showing decreased HBD-2 expression in burned skin [8], we wished to examine whether HBD-2 would be present in the burn blister.
0305-4179/00/$20.00 7 2000 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 0 5 2 - 8
M.R. Ortega et al. / Burns 26 (2000) 724±726
725
Table 1 Characterisation of patients, burn area and sites of blister ¯uid collectiona TBSA (%) Patient
Age (yr)
Sex
Past medical history
A
69
F
B C
17 80
M F
D E
63 81
M M
Hypertension Myocardial infarction Coronary artery bypass Brain aneurysm clipping None Myocardial infarction Congestive heart failure Asthma Abdominal hysterectomy Kidney stones None
a
PT
FT
Burn type
Blister source
±
Grease
Right lower leg
17.5 10.0
4.5 10.0
Flame Flame
Abdomen Right forearm
18.0 16.0
± ±
Flame Flame
Right upper arm Right thigh
4
Total body surface area (TBSA) burn percentage is expressed in terms of total partial thickness (PT)/full-thickness (FT) burns.
2. Methods and results After informed consent was obtained, blister ¯uid was aspirated from intact burn blisters of ®ve patients, admitted to our Regional Burn Centre, within 8 h of the thermal injuries. Aspiration with an 18-gauge needle was performed prior to formation of any gel-like coagulation within the blisters. Each specimen was centrifuged to remove cells, and then stored at ÿ708C. We analysed blister ¯uid from three males and two females with age ranges from 17 to 81 years and total body surface area burns ranging from 4±22%. Blister ¯uid was collected from various sites as outlined in Table 1. The blister ¯uid samples were analysed by sandwich ELISA with a monoclonal antibody to HBD-2 as the anchoring antibody, rabbit polyclonal antibody to HBD-2 as the detecting antibody, and development with goat-anti rabbit IgG peroxidase conjugate and Ophenylenediamine OPD/hydrogen peroxide. Recombinant HBD-2 produced in baculovirus-infected insect cells was used as a standard. The assay was validated against a Western blot assay for HBD-2 in samples of bronchoalveolar lavage ¯uid from patients with in¯ammatory lung disease. The sensitivity of the assay was 0.125 ng/ml. No HBD-2 was detectable in any of the burn blister ¯uids analysed. 3. Discussion Burn wound sepsis is due to a failure of local immunity of the burn wound. Factors such as a loss of the skin's physical barrier and generalised post-burn immuno-suppression contribute to susceptibility of the burn wound to microbial invasion. Partial thickness burns develop blistering of skin. Blister ¯uid is comprised of plasma proteins, ions and nutrient substances
that leak out of the microcirculation, with the addition of substances generated by injured cells or in¯ammatory cells. Thus the ¯uid has a composition similar to plasma but with notable increases in complement, leukotrienes and prostaglandins. Despite the presence of these host defence substances, the ¯uid was reported not to exert an antibacterial eect in vitro [9]. However, systemically administered antibiotics diuse into burn blister ¯uid with resultant bactericidal activity [9,10]. Elevated levels of alpha defensins and lysozyme have been detected in bullous pemphigoid blister ¯uid but no analysis of burn blister ¯uid for defensins has been previously attempted [7]. This study demonstrates that HBD-2 concentrations in burn blister ¯uid are below the level detectable by our highly sensitive assay, and at least a thousand-fold below the concentrations that would exert antimicrobial activity. Thus cutaneous burns breach not only the physical antimicrobial barrier but disrupt chemical antimicrobial defences as well. The disruption of the chemical antimicrobial shield may be yet another factor predisposing these patients to infection. It also suggests that application of antimicrobial peptides to the burn wound may have a therapeutic part to play in the prevention and treatment of invasive burn sepsis.
References [1] Allgower M, Schoenberger GA, Sparkes BG. Burning the largest immune organ. Burns 1995;21(Suppl. 1):1±30. [2] Ganz T. Defensins and host defense. Science 1999;286:420±1. [3] Ganz T, Lehrer RI. Defensins. Pharm Ther 1995;66:191±205. [4] Diamond G, Bevins CL. b-defensins: endogenous antibiotics of the innate host defense response. Clin Immunol Immunopath 1998;88(3):221±5. [5] Harder J, Bartels J, Christophers E, Schroder J-M. A peptide antibiotic from human skin. Nature 1997;387:861.
726
M.R. Ortega et al. / Burns 26 (2000) 724±726
[6] Lawyer C, Pai S, Watabe M, Borgia P, Mashimo T, Eagleton L, et al. Antimicrobial activity of a 13 amino acid tryptophanrich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides. FEBS Lett 1996;390:95±8. [7] Frohm M, Gunne H, Bergman AC, et al. Biochemical and antibacterial analysis of human wound and blister ¯uid. Eur J Biochem 1996;237:86±92.
[8] Milner SM, Ortega MR. Reduced antimicrobial peptide expression in human burn wounds. Burns 1999;25:411±3. [9] Heggers JP, Ko F, Robson MC, Heggers R, Craft KE. Evaluation of burn blister ¯uid. Plast Reconstr Surg 1980;65(6):798±804. [10] Nishizaki A, Aoyama H, Koh T, Izawa Y. Transfer of cefmenoxime to burn blister ¯uids. Jpn J Antibiot 1984;37:295±302.
www.elsevier.com/locate/burns
Human beta defensin is absent in burn blister ¯uid M.R. Ortega a, T. Ganz b, S.M. Milner a,* a
Institute for Plastic and Reconstructive Surgery, Southern Illinois University School of Medicine, Spring®eld, IL 62794, USA b Department of Medicine, UCLA School of Medicine, CHS 37-055, Los Angeles, CA 90095, USA Accepted 3 April 2000
Abstract Defensins are a family of cationic antimicrobial peptides that participate in innate host defence. Human beta defensin-2 (HBD-2) is produced by human keratinocytes, and has a potent bactericidal activity against a wide spectrum of microorganisms. We have recently shown that expression of HBD-2 is present in normal skin and lost in the full-thickness burn wound. Defensins have been found in the blister ¯uid of chronic wounds. Our study was designed to examine blister ¯uid from partialthickness burns for defensin content. Fluid from ®ve patients was collected from partial-thickness burn blisters, and then analysed by sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with a monoclonal antibody and rabbit polyclonal antibody to HBD-2. The assay was validated against a Western blot assay for HBD-2 in samples of bronchoalveolar lavage ¯uid from patients with in¯ammatory lung disease. No HBD-2 was detectable in any of the burn blister ¯uids analysed. HBD-2 is lost in the full-thickness burn wound, and we have now demonstrated its absence in burn blister ¯uid. This ®nding represents evidence of a host defence defect within the burn wound and suggests a possible therapeutic role for antimicrobial peptides in the management of burn wounds. 7 2000 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: Burns; Sepsis; Defensins; Blister ¯uid; Antimicrobial peptides
1. Introduction The concept of human skin as an ``immunological organ'' is now widely accepted [1]. Antimicrobial peptides, which are emerging as an important means of host defence, have been found in the cells and body ¯uids of a wide variety of multicellular animals [2]. Defensins, a particular class of antimicrobial peptides, have six cysteine residues, which form intramolecular disulphide bonds, are rich in arginine and are only 29±47 amino acids in length. There are three major structural sub-families of defensins, alpha, beta and insect defensins, whose ability to kill microorganisms is based on their capacity to form pores in microbial membranes [3]. Epithelial surfaces are protected by antimicrobial * Corresponding author.
peptides that form part of an innate immune mechanism. The mucosal surfaces of mammals are protected by defensins produced by epithelial lining cells and specialised epithelial cells, such as Paneth cells [4]. In human skin, Harder et al. demonstrated the presence of HBD-2 [5]. The peptide has potent bactericidal activity against a wide spectrum of gram positive, gram negative and fungal organisms, which are commonly implicated in burn sepsis [6]. The burn wound is an ideal substrate for bacterial growth and provides a portal for microbial invasion. In a partial thickness burn, blister ¯uid is liberated freely across the surface of the wound. It has been shown that alpha defensins are present in blisters of chronic wounds and pemphigoid [7]. In view of our previous studies showing decreased HBD-2 expression in burned skin [8], we wished to examine whether HBD-2 would be present in the burn blister.
0305-4179/00/$20.00 7 2000 Elsevier Science Ltd and ISBI. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 0 0 ) 0 0 0 5 2 - 8
M.R. Ortega et al. / Burns 26 (2000) 724±726
725
Table 1 Characterisation of patients, burn area and sites of blister ¯uid collectiona TBSA (%) Patient
Age (yr)
Sex
Past medical history
A
69
F
B C
17 80
M F
D E
63 81
M M
Hypertension Myocardial infarction Coronary artery bypass Brain aneurysm clipping None Myocardial infarction Congestive heart failure Asthma Abdominal hysterectomy Kidney stones None
a
PT
FT
Burn type
Blister source
±
Grease
Right lower leg
17.5 10.0
4.5 10.0
Flame Flame
Abdomen Right forearm
18.0 16.0
± ±
Flame Flame
Right upper arm Right thigh
4
Total body surface area (TBSA) burn percentage is expressed in terms of total partial thickness (PT)/full-thickness (FT) burns.
2. Methods and results After informed consent was obtained, blister ¯uid was aspirated from intact burn blisters of ®ve patients, admitted to our Regional Burn Centre, within 8 h of the thermal injuries. Aspiration with an 18-gauge needle was performed prior to formation of any gel-like coagulation within the blisters. Each specimen was centrifuged to remove cells, and then stored at ÿ708C. We analysed blister ¯uid from three males and two females with age ranges from 17 to 81 years and total body surface area burns ranging from 4±22%. Blister ¯uid was collected from various sites as outlined in Table 1. The blister ¯uid samples were analysed by sandwich ELISA with a monoclonal antibody to HBD-2 as the anchoring antibody, rabbit polyclonal antibody to HBD-2 as the detecting antibody, and development with goat-anti rabbit IgG peroxidase conjugate and Ophenylenediamine OPD/hydrogen peroxide. Recombinant HBD-2 produced in baculovirus-infected insect cells was used as a standard. The assay was validated against a Western blot assay for HBD-2 in samples of bronchoalveolar lavage ¯uid from patients with in¯ammatory lung disease. The sensitivity of the assay was 0.125 ng/ml. No HBD-2 was detectable in any of the burn blister ¯uids analysed. 3. Discussion Burn wound sepsis is due to a failure of local immunity of the burn wound. Factors such as a loss of the skin's physical barrier and generalised post-burn immuno-suppression contribute to susceptibility of the burn wound to microbial invasion. Partial thickness burns develop blistering of skin. Blister ¯uid is comprised of plasma proteins, ions and nutrient substances
that leak out of the microcirculation, with the addition of substances generated by injured cells or in¯ammatory cells. Thus the ¯uid has a composition similar to plasma but with notable increases in complement, leukotrienes and prostaglandins. Despite the presence of these host defence substances, the ¯uid was reported not to exert an antibacterial eect in vitro [9]. However, systemically administered antibiotics diuse into burn blister ¯uid with resultant bactericidal activity [9,10]. Elevated levels of alpha defensins and lysozyme have been detected in bullous pemphigoid blister ¯uid but no analysis of burn blister ¯uid for defensins has been previously attempted [7]. This study demonstrates that HBD-2 concentrations in burn blister ¯uid are below the level detectable by our highly sensitive assay, and at least a thousand-fold below the concentrations that would exert antimicrobial activity. Thus cutaneous burns breach not only the physical antimicrobial barrier but disrupt chemical antimicrobial defences as well. The disruption of the chemical antimicrobial shield may be yet another factor predisposing these patients to infection. It also suggests that application of antimicrobial peptides to the burn wound may have a therapeutic part to play in the prevention and treatment of invasive burn sepsis.
References [1] Allgower M, Schoenberger GA, Sparkes BG. Burning the largest immune organ. Burns 1995;21(Suppl. 1):1±30. [2] Ganz T. Defensins and host defense. Science 1999;286:420±1. [3] Ganz T, Lehrer RI. Defensins. Pharm Ther 1995;66:191±205. [4] Diamond G, Bevins CL. b-defensins: endogenous antibiotics of the innate host defense response. Clin Immunol Immunopath 1998;88(3):221±5. [5] Harder J, Bartels J, Christophers E, Schroder J-M. A peptide antibiotic from human skin. Nature 1997;387:861.
726
M.R. Ortega et al. / Burns 26 (2000) 724±726
[6] Lawyer C, Pai S, Watabe M, Borgia P, Mashimo T, Eagleton L, et al. Antimicrobial activity of a 13 amino acid tryptophanrich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides. FEBS Lett 1996;390:95±8. [7] Frohm M, Gunne H, Bergman AC, et al. Biochemical and antibacterial analysis of human wound and blister ¯uid. Eur J Biochem 1996;237:86±92.
[8] Milner SM, Ortega MR. Reduced antimicrobial peptide expression in human burn wounds. Burns 1999;25:411±3. [9] Heggers JP, Ko F, Robson MC, Heggers R, Craft KE. Evaluation of burn blister ¯uid. Plast Reconstr Surg 1980;65(6):798±804. [10] Nishizaki A, Aoyama H, Koh T, Izawa Y. Transfer of cefmenoxime to burn blister ¯uids. Jpn J Antibiot 1984;37:295±302.