Tumor necrosis factorealfa in nonhealing venous leg ulcers Carlos A. Charles, MD,a Paolo Romanelli, MD,a Zuleika Bonilla Martinez, MD,a Fangchao Ma, MD, PhD,a Brenda Roberts, BS,a and Robert S. Kirsner, MD, PhDa,b Miami, Florida Background: Venous leg ulcers are responsible for more than half of all lower extremity ulcerations, affecting more than one million Americans annually. Studies have demonstrated alterations in levels of proinflammatory cytokines in patients with chronic wounds, including tumor necrosis factorealfa (TNFa), which may be implicated in wound chronicity. Objective: To test the hypothesis that recalcitrant venous leg ulcers have increased local tissue TNFa as compared to normal skin. Methods: Five patients with nonhealing healing chronic venous leg ulcers were recruited. Two 4-mm punch biopsy specimens were obtained: one from the wound margin and one from noninvolved, nonesun exposed normal skin on the flexor aspect of the forearm. Tissue samples were processed using fixed with formalin stained by immunohistochemistry for TNFa. Qualitative and quantitative comparisons were made for the presence of TNFa receptor in all tissue samples, specifically comparing the presence of TNFa in nonhealing venous leg ulcer samples versus normal skin. Results: The overall staining score for nonhealing venous leg ulcers was significantly higher compared to respective normal skin samples (P = .01). In addition, immunostaining for TNFa was significantly less in the two nonhealing venous leg ulcers that were present for the shortest duration compared to the other ulcers of longer duration (P = .048). Limitations: The small sample size may mitigate the clinical implications of findings. Conclusions: Increased levels of TNFa in nonhealing venous leg ulcers, especially those of longer duration, implies that excessive inflammation may be causal in wound chronicity and suggests potential therapeutic alternatives. ( J Am Acad Dermatol 2009;60:951-5.)
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enous leg ulcers are responsible for more than half of all lower extremity ulcerations, affecting more than one million Americans annually.1 The associated direct cost of ulcer treatments for a patient amounts to approximately $30,000 per year in the United States, and in some industrialized countries, this number may represent From the Departments of Dermatology and Cutaneous Surgerya and Epidemiology and Public Health,b University of Miami Miller School of Medicine, Miami. Funding sources: None. Conflicts of interest: None declared. Reprints not available from the authors. Correspondence to: Robert S. Kirsner, MD, PhD, Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, PO Box 016250 (R250), Miami, FL 33136. E-mail:
[email protected]. Published online April 3, 2009. 0190-9622/$36.00 ª 2008 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2008.09.012
approximately 1% of the total health care budget.2 The standard of care—multilayered compression bandages—is cumbersome, slow to demonstrate healing, and effective in only one half to two-thirds of patients.3-5 Significant interest has been focused on understanding the population of patients who fail to heal with standard therapy. Studies have shown that local alterations in levels of proinflammatory cytokines, including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alfa (TNFa) within chronic wound fluid may be implicated in wound chronicity.6 The purpose of this pilot study was to test the hypothesis that recalcitrant venous leg ulcers have increased local cutaneous levels of TNFa as compared to normal skin as per immunohistochemical analysis. Elucidating this hypothesis may provide further insight into the pathophysiology of recalcitrant venous leg ulcers and guidance for the development of therapeutic modalities for these wounds. 951
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Table I. Patient demographics Subject
Age
Gender
Ulcer location
Ankle brachial index
Ulcer duration (wks)
Ulcer size (cm2)
1 2 3 4 5
64 56 33 68 62
Male Male Male Male Female
Left medial malleolus Right medial malleolus Left medial malleolus Left medial malleolus Left lateral malleolus
0.99 1.1 0.98 1.1 1.0
6 40 144 28 144
6.75 46.8 162 25 80
All patients had active ulcers (C6 of the CEAP classification system [www.phlebology.org]) and deep perforator and superficial vein involvement by ultrasound.
METHODS After institutional review board approval and obtaining informed consent, five patients with nonhealing healing chronic venous leg ulcers were recruited. Clinical factors that have been identified as independent prognostic factors for wound healing were used to stratify the wounds as nonhealing as described previously by Margolis et al.7 Venous ulcers that were present for longer than 6 months and/or had an area greater than 5 cm2 were characterized as nonhealing. Venous etiology of the leg ulcers was determined clinically and confirmed by vascular Doppler radiologic studies, if needed. Clinical parameters used to determine venous etiology of leg ulcers were the presence of a lower leg ulcer and at least two of the following: venous dermatitis, atrophie blanche, varicosities, hyperpigmentation, or lipodermatosclerosis. All patients had the presence of arterial disease excluded, initially by an ankle brachial index. Patients with active cellulitis, wound infection, and/or osteomyelitis were excluded from the study, as were patients treated with topical or systemic steroids. Active ulcer infection was defined by the presence of pain, warmth, erythema, malodorous exudates, and/or a previous untreated, culture-proven infection in the past 6 months. Any patient that had undergone treatment in the previous 6 weeks with a bioengineered skin equivalent or topical growth factor was excluded, as were any patients that had undergone previous treatment with anti-TNF therapy. At inclusion, two 4-mm punch biopsy specimens were obtained: one from the wound margin and one from noninvolved, nonesun exposed normal skin on the flexor aspect of the forearm. Tissue samples were processed using fixed with formalin and processed according to standard histologic processing protocol. All patients were subsequently treated with compression bandages using the standard of care treatment for venous leg ulcers. All biopsy samples were stained by immunohistochemistry for TNFa (Gene Tex Inc, San Antonio, TX). Qualitative and quantitative comparisons were made for the presence of TNFa receptor in all tissue
Table II. Overall staining score per tissue sample nonhealing versus healing venous leg ulcers Subject
1 2 3 4 5
Nonhealing ulcer
Normal skin
2 6 6 2 6
0 1 0 0 0
Overall staining score for nonhealing venous leg ulcers was significantly higher compared to respective normal skin samples (t test for paired data; P = .01).
samples, specifically comparing the presence of TNFa in nonhealing venous leg ulcer samples versus normal skin. The intensity of intracytoplasmatic TNFa immunostaining was graded semiquantitatively from 0 (no staining) to 3 (maximal intensity of staining) per high-powered field. The number of positive cells was counted and scored semiquantitatively from 0 to 4 (0 = 0; 1 = 1-30; 2 = 31-60; 3 = 61-90; and 4 = [90 stained cells in representative high-powered field). An overall score per venous leg ulcer was calculated by multiplying the value for intensity of immunostaining by the score of positive cells. Depending on the outcome of the tests for normality and equal variances, parametric one-way t tests or the Wilcoxon rank sum test were used to determine differences between overall TNFa staining between nonhealing leg ulcers and normal skin. P \.05 was considered statistically significant.
RESULTS A total of five patients were enrolled. All patients had active ulcers (C6 of the CEAP classification) and all had deep perforator and superficial involvement by ultrasound (Table I). Biopsy samples were obtained from each patient’s nonhealing leg ulcer and normal skin. Demographic information is presented for patients with nonhealing venous leg ulcers (Table I). Overall staining score for nonhealing venous leg ulcers was significantly higher compared to respective normal skin samples
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Table III. Overall staining score per tissue sample with respective ulcer duration Subject
Nonhealing ulcer (staining score)
Ulcer duration (wks)
2 6 6 2 6
6 40 144 28 144
1 2 3 4 5
Immunostaining for tumor necrosis factor-alfa was significantly less in the two nonhealing venous leg ulcers that were present for the shortest duration (subjects 1 and 2) compared to the other ulcers of longer duration (Wilcoxon rank sum test, one-sided; P = .048).
Fig 1. Immunostaining for tumor necrosis factorealfa in normal skin. Note that there is no significant immunostaining. (Original magnification: 340.)
(P = .01; Table II). In addition, immunostaining for TNFa was significantly less in the two nonhealing venous leg ulcers that were present the shortest duration compared to the other ulcers of longer duration (P = .048; Table III). Representative tissue immunostaining between nonhealing venous leg ulcers and normal skin are illustrated in Figs 1 and 2.
DISCUSSION Inflammation is critical to the wound healing process. However, when that inflammatory process does not progress, leading to a chronic inflammatory state, wound healing is impaired. Persistently elevated levels of proinflammatory cytokines adversely affect the growth and viability of cell types necessary for appropriate formation of a healthy extracellular matrix.8 Studies comparing wound fluid from healing and nonhealing wounds have found that nonhealing wounds have low levels of mitogenic activity, high concentrations of proinflammatory cytokines, such as TNFa and IL-1,6 and high levels of proteases,9 low levels of protease inhibitors,10 growth factors, and competent fibroblasts.11 In this pilot study, we have shown the presence of increased levels of TNFa within recalcitrant venous leg ulcers by immunohistochemistry and increased staining with ulcers of longer duration. This is compared to normal forearm skin. While this is presumably similar to other normal areas, it is possible that a regional variation of TNFa may exist. TNFa is an important proinflammatory cytokine that has been implicated in the pathophysiology of many cutaneous diseases. TNFa is synthesized as a 26-kDa membrane-bound precursor that is then proteolytically cleaved at the cell surface, giving rise to the mature secreted 17-kDa polypeptide that is biologically active.12,13 TNFa has disparate physiologic cellular effects and can regulate cellular activation, proliferation, or apoptotic cell death depending on its concentration and/or target cell
Fig 2. A, Immunostaining for tumor necrosis factorealfa (TNFa) in a nonhealing venous leg ulcer. Note the diffuse light and dark brown dermal staining for TNFa within the inflammatory infiltrate. B, At a higher magnification, note the intense diffuse dark brown and black dermal staining for TNFa within the inflammatory infiltrate. (Original magnification: A, 340; B, 3100.)
type. Additionally, various postreceptor signaling processes may also determine the physiologic effect of TNFa.14 TNFa stimulates the proliferation of dermal fibroblasts in vitro.15 Alternatively, TNFa inhibits cell proliferation and induces cell adhesion molecules in keratinocytes.16 TNFa can stimulate functional activation of endothelial cells and impair endothelial cell growth and even their induce apoptosis in certain conditions.17
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The role of TNFa in wound healing is not completely understood; however, the relationship between TNFa and venous leg ulcers has been studied. TNFa has been detected in intracapillary monocytes in venous ulcer biopsies.18 Studies focusing on the relationship between TNFa and healing of venous leg ulcers found significantly higher levels of immunoreactive TNFa in samples of nonhealing versus healing venous leg ulcers by enzyme-linked immunosorbent assay (ELISA).19 Interestingly enough, that same study did not find significant differences in the levels of bioactive TNFa in the wound fluid of healing versus nonhealing venous leg ulcers. These findings suggest that other mechanisms may be responsible for altering the bioactivity of TNFa in venous leg ulcers, and that TNFa alone may not be the only inflammatory mediator of healing. Others have found increased systemic levels of TNFa in nonhealing venous leg ulcers and observed that reductions in serum cytokine levels TNFa parallel healing of venous leg ulcers in patients undergoing compression therapy.20 This suggests but remains to be studied that reductions in systemic levels of TNFa in venous leg ulcers may lead improved healing. Furthermore, Wallace et al, recently found that carriage of the TNFA-308A allele was a significant risk factor for the development of venous leg ulceration, and this polymorphism has been shown to increase TNFa protein levels in some studies.21 The study of TNFa and venous leg ulcers is important from a clinical standpoint, because antiTNF agents have become an important therapeutic modality for many conditions. While recent data has found anti-TNF agents beneficial in the treatment of ulcers secondary to inflammatory conditions, such as pyoderma gangrenosum,22,23 to our knowledge, these agents have yet to have been employed in the treatment of venous leg ulcers. However, one recent study did demonstrate that the application of the anti-TNF agent etanercept to chronic wound fluid and fibroblasts reduced the cytotoxic effect of the wound fluid on the fibroblasts by approximately 30% and neutralized TNFa binding by up to 80% measured by ELISA. These findings suggest that direct application of an antiTNF agent to chronic wounds may reduce the inflammatory activity of TNFa and improve healing.24 In addition, a recent study demonstrated that systemic administration of an anti-TNFa agent in an acutely wounded rodent model of obesity and diabetes led to significantly improved healing rates compared to nontreated subjects, with significantly more rapid and complete neoepidermal coverage of the impaired wound tissue in the face of a diabetic phenotype.25
Herein, we demonstrated increased levels of TNFa in nonhealing venous leg ulcers compared to normal skin with immunohistochemistry. We also showed that venous ulcers of longer durations displayed more TNFa than ulcers of shorter durations. Despite our small sample size, a relationship between local levels of TNFa and nonhealing venous leg ulcers is suggested. Future studies comparing nonhealing versus healing leg ulcers would be critical to further elucidate the findings of this study, as would studies employing a larger sample size. REFERENCES 1. Valencia IC, Falabella A, Kirsner RS, Eaglstein WH. Chronic venous insufficiency and venous leg ulceration. J Am Acad Dermatol 2001;44:401-21. 2. Ruckley CV. Socio-economic impact of chronic venous insufficiency and leg ulcers. Angiology 1997;48:67-9. 3. Falanga V, Margolis D, Alvarez O, Auletta M, Maggiacomo F, Altman M, et al. Rapid healing of venous ulcers and lack of clinical rejection with an allogeneic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch Dermatol 1998;134:293-300. 4. Phillips TJ, Machado F, Trout R, Porter J, Olin J, Falanga V. Prognostic indicators in venous ulcers. J Am Acad Dermatol 2000;43:627-30. 5. Phillips TJ. Successful methods of treating venous ulcers: the tried and true, plus the novel and new. Postgrad Med J 1999; 105:159-79. 6. Trengove NJ, Stacey MC, MacAuley S, Bennett N, Gibson J, Burslem F, et al. Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen 1999;7:442-52. 7. Margolis DJ, Berlin JA, Strom BL. Which venous leg ulcers will heal with limb compression bandages? Am J Med 2000;109: 15-9. 8. Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N. Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration. FASEB J 2002;16: 963-74. 9. Wysocki AB, Staiano-Coico L, Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol 1993;101:64-8. 10. Chen SM, Ward SI, Olutoye OO, Diegelmann RF, Kelman Cohen I. Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Repair Regen 1997;5:23-32. 11. Tarnuzzer RW, Schultz GS. Biochemical analysis of acute and chronic wound environments. Wound Repair Regen 1996;4: 321-5. 12. Jones E, Stuart D, Walker N. Structure of tumour necrosis factor. Nature 1989;338:225-8. 13. Jue DM, Sherry B, Luedke C, Manogue KR, Cerami A. Processing of newly synthesized cachetin/tumor necrosis factor in endotoxin-stimulated macrophages. Biochemistry 1990;29: 8371-7. 14. Schu¨tze S, Scheurich P, Schlu¨ter C, Ucer U, Pfizenmaier K, Kro¨nke M. Tumor necrosis factor-induced changes of gene expression in U937 cells. Differentiation-dependent plasticity of the responsive state. J Immunol 1988;140:3000-5. 15. Vilcek J, Palombella V, Henrikson-DeStefano D, Swenson C, Feinman R, Hirai M, et al. Fibroblast enhancing activity of
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