Title: Levels of Tumor Necrosis Factor-α (TNF-α) and Soluble TNF Receptors in Chronic Venous Leg Ulcers – Correlations to Healing Status
Abstract: This study tested the hypothesis that excessive tumor necrosis factor-α (TNF-α) levels in chronic venous leg ulcers are associated with impaired healing. TNF-α was measured by two enzyme-linked immunosorbent assays and a bioassay (KYM-1D4) in paired wound fluid samples collected during the nonhealing and healing phases from 21 human patients with venous leg ulcers. Soluble TNF receptor levels (p55 and p75) were also measured. The levels of immunoreactive TNF-α were significantly higher in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers (p < 0.005), whereas the levels of bioactive TNF-α were not. Statistical analysis confirmed that TNF-α bioactivity relative to the amount of immunoreactive TNF-α was downregulated in wound fluid from nonhealing ulcers compared with healing ulcers. The levels of soluble p55 and p75 receptors in wound fluid showed a significant linear correlation (p < 0.001), suggesting a partially coordinated or common regulatory mechanism for the cleavage of transmembrane TNF receptors in chronic venous ulcers in vivo. Although the levels of soluble p75 receptors were significantly higher in nonhealing wound fluid compared with healing wound fluid (p < 0.025), these levels were theoretically inadequate to substantially neutralize the bioactivity of the accompanying TNF-α levels on their own. The bioactivity accompanying the elevated levels of immunoreactive TNF-α in wound fluid from nonhealing ulcers may have been further down-modulated by an additional mechanism. Because healing was initiated without a significant decline in the level of bioactive TNF-α, TNF-α-mediated events may not be the key events contributing to the impaired healing seen in chronic venous ulcers. This study tested the hypothesis that excessive tumor necrosis factor-α (TNF-α) levels in chronic venous leg ulcers are associated with impaired healing. TNF-α was measured by two enzyme-linked immunosorbent assays and a bioassay (KYM-1D4) in paired wound fluid samples collected during the nonhealing and healing phases from 21 human patients with venous leg ulcers. Soluble TNF receptor levels (p55 and p75) were also measured. The levels of immunoreactive TNF-α were significantly higher in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers (p < 0.005), whereas the levels of bioactive TNF-α were not. Statistical analysis confirmed that TNF-α bioactivity relative to the amount of immunoreactive TNF-α was downregulated in wound fluid from nonhealing ulcers compared with healing ulcers. The levels of soluble p55 and p75 receptors in wound fluid showed a significant linear correlation (p < 0.001), suggesting a partially coordinated or common regulatory mechanism for the cleavage of transmembrane TNF receptors in chronic venous ulcers in vivo. Although the levels of soluble p75 receptors were significantly higher in nonhealing wound fluid compared with healing wound fluid (p < 0.025), these levels were theoretically inadequate to substantially neutralize the bioactivity of the accompanying TNF-α levels on their own. The bioactivity accompanying the elevated levels of immunoreactive TNF-α in wound fluid from nonhealing ulcers may have been further down-modulated by an additional mechanism. Because healing was initiated without a significant decline in the level of bioactive TNF-α, TNF-α-mediated events may not be the key events contributing to the impaired healing seen in chronic venous ulcers. The factors associated with venous insufficiency that initiate ulceration and that impair wound healing are still being debated. It has been established that one effect of venous insufficiency is impaired tissue oxygen perfusion (Mani et al., 1989Mani R. White J. Barrett D. Weaver P. Tissue oxygenation, venous ulcers and fibrin cuffs.J R Soc Med. 1989; 82: 345-346PubMed Google Scholar, Falanga et al., 1991Falanga V. McKenzie A. Eaglestein W. Heterogeneity in oxygen diffusion around venous ulcers.J Dermatol Surg Oncol. 1991; 17: 336-339Crossref PubMed Scopus (16) Google Scholar) that can influence cell proliferation (Balin et al., 1984Balin A. Fisher A. Carter D. Oxygen modulates growth of human cells at physiological partial pressures.J Exp Med. 1984; 160: 152-166Crossref PubMed Scopus (93) Google Scholar, Siddiqui et al., 1996Siddiqui A. Galiano R. Connors D. Gruskin E. Wu L. Mustoe T. Differential effects of oxygen on human dermal fibroblasts.Wound Rep Reg. 1996; 4: 211-218Crossref PubMed Scopus (53) Google Scholar) and matrix deposition (Herrick et al., 1996Herrick S. Ireland G. Simon D. McCollum C. Ferguson M. Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions.J Invest Dermatol. 1996; 106: 187-193Crossref PubMed Scopus (74) Google Scholar). Others have suggested that a key event in the pathogenesis of chronic venous ulcers is inflammation generated by activated leukocytes trapped in the microcirculation (Thomas et al., 1988Thomas P. Nash G. Dormandy J. White cell accumulation in dependent legs of patients with venous hypertension: a possible mechanism for trophic changes in the skin.Br Med J. 1988; 296: 1693-1695Crossref PubMed Scopus (272) Google Scholar). During normal wound healing, an initial transient inflammatory response is an essential part of the tissue repair process. Work in our department shows that persistent inflammation is a feature of nonhealing venous ulcers, and that its resolution correlates with healing. Levels of the pro-inflammatory cytokines interleukin-1, interleukin-6, and TNF-α, and a clinical marker of inflammation, C- reactive protein, have all been shown to decrease in wound fluid from healing chronic venous ulcers compared with nonhealing ulcers (Trengove et al., 1996Trengove N. Langton S. Stacey M. Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers.Wound Rep Reg. 1996; 4: 234-239Crossref PubMed Scopus (167) Google Scholar) 1Trengove N, Bielefeldt-Ohmann H, Stacey M, Cytokone profile of wound fluid from chronic leg ulcers. Wound Rep Reg 2:228, 1994(abstr.)1Trengove N, Bielefeldt-Ohmann H, Stacey M, Cytokone profile of wound fluid from chronic leg ulcers. Wound Rep Reg 2:228, 1994(abstr.). We hypothesize that prolonged inflammation impairs the healing of chronic venous ulcers via the adverse action of cytokines that affect the growth or viability of cell types found in healing skin, and that affect the integrity of the extracellular matrix. Tumor necrosis factor- α (TNF-α) is an important proinflammatory cytokine that has been detected in intracapillary monocytes in venous ulcer biopsies (Mirshahi et al., 1995Mirshahi S. Soria J. Mirshahi M. et al.Expression of elastase and fibrin in venous leg ulcer biopsies: a pilot study of pentoxifylline vesus placebo.J Cardiovasc Pharmacol. 1995; 25: S101-S105Crossref PubMed Scopus (18) Google Scholar). TNF-α is synthesized as a 26 kDa membrane-bound precursor and is proteolytically cleaved at the cell surface to yield the mature secreted 17 kDa polypeptide (Jue et al., 1990Jue D. Sherry B. Luedke C. Manogue K. Cerami A. Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages.Biochem. 1990; 29: 8371-8377Crossref PubMed Scopus (82) Google Scholar) that is biologically active as a homo-trimer (Jones et al., 1989Jones E. Stuart D. Walker N. Structure oftumour necrosis factor.Nature. 1989; 338: 225-228Crossref PubMed Scopus (471) Google Scholar). TNF-α can mediate functional activation, proliferation, or apoptotic death of cells depending on its concentration and the target cell type. The diversity of responses induced by TNF-α may also be due to subtle complexities of postreceptor signaling processes (Schutze et al., 1988Schutze S. Scheurich P. Schluter C. Ucer U. Pfizenmaier K. Kronke M. Tumor necrosis factor-induced changes of gene expression in U937 cells: Differentiation-dependent plasticity of the responsive state.J Immunol. 1988; 140: 3000-3005PubMed Google Scholar). In vitro, TNF-α stimulates the proliferation of dermal fibroblasts (Vilcek et al., 1986Vilcek J. Palombella V. Henriksen-DeStefano D. Swenson C. Feinman R. Hirai M. Tsujimoto M. Fibroblast growth enhancing activity of tumor necrosis factor and its relationship to other polypeptide growth factors.J Exp Med. 1986; 163: 632-643Crossref PubMed Scopus (487) Google Scholar), but inhibits cell proliferation and induces cell adhesion molecules in keratinocytes (Detmar and Orfanos, 1990Detmar M. Orfanos C. Tumor necrosis factor-alpha inhibits cell proliferation and induces class II antigens and cell adhesion molecules in cultured normal human keratinocytes in vitro.Arch Dermatol Res. 1990; 282: 238-245Crossref PubMed Scopus (55) Google Scholar). Whereas TNF-α is a potent functional activator of endothelial cells, it impairs their growth in vitro (Frater-Schröder et al., 1987Frater-Schröder M. Risau W. Hallmann R. Gautschi P. Bohlen P. Tumor necrosis factor type α, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo.Proc Natl Acad Sci USA. 1987; 84: 5277-5281Crossref PubMed Scopus (589) Google Scholar) and under certain conditions can induce endothelial cell apoptosis (Polunovsky et al., 1994Polunovsky V. Wendt C. Ingbar D. Peterson M. Bitterman P. Induction of endothelial cell apoptosis by TNF alpha: modulation by inhibitors ofprotein synthesis.Exp Cell Res. 1994; 214: 584-594Crossref PubMed Scopus (266) Google Scholar). There are several mechanisms that may serve to regulate the bioactivity of TNF-α in vivo. The effects of TNF-α are mediated by two types of cell surface receptors, the p55 and p75 TNF receptors. Both receptor subtypes may be proteolytically cleaved to produce soluble receptor proteins, which may compete with membrane-bound TNF receptors for TNF-α binding. There is evidence that at high concentrations of soluble TNF receptors (sTNF-R) in vitro these receptors can inhibit the bioactivity of TNF-α (Higuchi and Aggarwal, 1992Higuchi M. Aggarwal B. Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of p60 and p80 receptors.Biochem Biophys Res Commun. 1992; 182: 638-643Crossref PubMed Scopus (34) Google Scholar). The functional role of sTNF-R in vivo is still being elucidated. Reports of high levels of TNF-α, soluble p55, and soluble p75 measured by enzyme-linked immunosorbent assay (ELISA) in combination with barely detectable levels of bioactive TNF-α in human plasma, have been proposed as indirect evidence for soluble receptors reducing the bioactivity of TNF-α in vivo (Jackson et al., 1995Jackson A. Alexandrov A. Prescott S. James K. Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients after bacillus Calmette-Guerin immunotherapy.Cancer Immunol. 1995; 40: 119-124Google Scholar, Linderholm et al., 1996Linderholm M. Ahlm C. Settergren B. Waage A. Tarnvik A. Elevated levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, and IL-10 in patients with hemorrhagic fever with renal syndrome.J Infect Dis. 1996; 173: 38-43Crossref PubMed Scopus (166) Google Scholar). In other studies, circulating soluble receptor levels in critically ill patients and in experimental endotoxinaemia have not been sufficient to block the accompanying TNF activity entirely (Spinas et al., 1992Spinas G. Keller U. Brockhaus M. Release of soluble receptors for tumor necrosis factor (TNF) in relation to circulating TNF during experimental endotoxinemia.J Clin Invest. 1992; 90: 533-536Crossref PubMed Scopus (216) Google Scholar, Van Zee et al., 1992Van Zee K. Kohno T. Fischer E. Rock C. Moldawer L. Lowry S. Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor a in vitro and in vivo.Proc Natl Acad Sci USA. 1992; 89: 4845-4849Crossref PubMed Scopus (749) Google Scholar). A sTNF-R fusion protein has been shown to protect mice from lethal endotoxin challenge (Mohler et al., 1993Mohler K. Torrance D. Smith C. et al.Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists.J Immunol. 1993; 151: 1548-1561PubMed Google Scholar). Conversely, it is suggested that at low concentrations of sTNF-R, the reversible association of the soluble receptors with TNF-α can slow the spontaneous degradation of the labile trimeric form of the TNF- a molecule and increase its bioavailability (Aderka et al., 1992Aderka D. Englemann H. Maor Y. Brakebusch C. Walach D. Stabilization ofthe bioactivity of tumor necrosis factor by its soluble receptors.J Exp Med. 1992; 175: 323-329Crossref PubMed Scopus (742) Google Scholar). The aim of this study was to examine the role of TNF-α in chronic venous leg ulcers, and in particular to determine whether the activity of TNF-α is likely to contribute to delayed healing. To investigate this we tested the hypothesis that the levels ofTNF-α in the extracellular fluid collected from venous ulcers would decrease as the ulcers transform from a nonhealing to a healing state. Bioactive levels of TNF-α were compared with immunoreactive levels (two different ELISA) in the nonhealing phase and after 2 wk bed rest when the ulcers showed early clinical signs of healing. The levels of soluble p55 and p75 receptors were also determined by ELISA to establish if they have a role in regulating the bioactivity of TNF-α in the chronic wound environment. Wound fluid was collected from chronic ulcers on the lower leg of 21 patients with venous disease. Patients with ulcers on the foot were excluded from this study. The presence of venous disease was determined by clinical history, examination, and investigations that included venous refilling time by photoplethysmography (Abramowitz et al., 1979Abramowitz H. Queral L. Finn W. Nora P. Peterson L. Bergan J. Yao J. The use of photoplethysmography in the assessment of venous insufficiency: a comparison to venous pressure measurements.Surg. 1979; 86: 434-441PubMed Google Scholar). Arterial Doppler pressures were performed to assess the presence of arterial disease (Yao et al., 1968Yao S. Hobbs J. Irvine W. Pulse examination by ultrasonic method.Br Med J. 1968; 4: 555-557Crossref PubMed Scopus (37) Google Scholar). A diagnosis of venous disease was confirmed by a venous refilling time less than 25 s and arterial disease was determined by an ankle/brachial Doppler arterial ratio of less than 0.9. A panel of blood tests was also performed to rule out systemic contributing factors to ulceration, such as diabetes. In all cases the patients' ulcers had failed to respond to outpatient treatment (compression therapy), showing no reduction in ulcer size over more than 3 mo or a continued increase in ulcer size. Patients were admitted to hospital for bed rest, six-hourly saline solution compresses, and eventual skin grafting. There were no additional interventions with respect to other potential causes of delayed wound healing. Wound fluid was collected from the patients' ulcers within 24 h of admission to hospital (nonhealing phase) and after 2 wk of regular saline dressings and bed rest (healing phase). Wound fluid was collected from each patient in a standardized manner as previously described (Trengove et al., 1996Trengove N. Langton S. Stacey M. Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers.Wound Rep Reg. 1996; 4: 234-239Crossref PubMed Scopus (167) Google Scholar). After aspiration the fluid was transferred into Greiner Vacuette serum collection tubes (Interpath, Melbourne, Australia). These tubes were kept on ice for less than 1 h prior to centrifugation of the wound fluid and storage in multiple aliquots at —80°C. Measurements of TNF-α and soluble TNF receptor levels were performed on aliquots thawed for the first time. The edge of each ulcer was traced onto a transparent plastic sheet and the area determined by planimetry. The ulcer size measurements were performed on admission to hospital and just prior to the second wound fluid collection. Bioactive TNF-α was assayed colorimetrically by its cytotoxic effect on the human rhabdomyosarcoma cell line, KYM-1D4 (a kind gift of Dr. Jay Steer, University Department of Pharmacology, University of Western Australia), essentially as previously described (Meager, 1991Meager A. A cytotoxicity assay for tumour necrosis factor using a human rhabdomyosarcoma cell line.J Immunol Meth. 1991; 144: 141-143Crossref PubMed Scopus (33) Google Scholar). Recombinant human TNF-α (TNF-H, Genzyme, Boston, MA) was used as the standard for the quantitation of TNF-α in the test samples. Serial dilutions of recombinant human TNF-α in RPMI-1640 medium (Gibco, Grand Island, NY) plus 5% fetal calf serum (CSL, Melbourne, Australia) from 143 to 0.143 U per ml (specific activity 1.43 X 108 U per mg) were used to construct the standard curve. Test wound fluid samples were filter-sterilized after thawing (0.22 μm membrane, MillexR-GV13, Millipore, MA) and diluted 15-fold in the same medium. One hundred microliter aliquots of test and standard samples were assayed in triplicate in 96 well tissue culture plates (Falcon, Franklin Lakes, NJ). The KYM-1D4 cells were resuspended in culture medium at 2 X 105 cells per ml and 100 μl added to each test and standard well before incubation of the plates at 37°C, 5% CO2. Cell survival was estimated with MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO) (10 ml per well of 5 mg MTT per ml phosphate-buffered saline) (Mosmann, 1983Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J Immunol Meth. 1983; 65: 55-63Crossref PubMed Scopus (43038) Google Scholar) after 48 h. The blue formazan product of MTT conversion was eluted with 20% sodium dodecyl sulfate (ultrapure grade, USB, Cleveland, OH) in 50% dimethylformamide pH 4.7 (Hansen et al., 1989Hansen M. Nielsen S. Berg K. Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.J Immunol Meth. 1989; 119: 203-210Crossref PubMed Scopus (3231) Google Scholar) after 2 h at 37°C, and the optical densities read at 570 nm. Immunoreactive TNF-α in wound fluid was determined by DuoseTTM (Genzyme) and EASIATM (Medgenix, Fleurus, Belgium) ELISA for human TNF-α. Assays were performed according to the manufacturer's instructions. The same preparation of recombinant human TNF-α that was used in the bioassay was used as the reference standard for both ELISA (TNF-H, Genzyme). Briefly, all test samples were diluted 10-fold for each ELISA. Standards or test samples in duplicate were incubated in microtitre wells coated with one (Genzyme) or several (Medgenix) murine monoclonal antibodies and detected with horseradish peroxidase-conjugated rabbit polyclonal antibodies. After color development of the tetramethylbenzidine substrate the optical densities were measured at 450 nm. The levels of sTNF-R p55 and p75 were measured in wound fluid using Biotrak ELISA (Amersham, U.K.). Assays were performed according to the manufacturer's instructions with all test samples being diluted 50-fold for accurate measurement. Standards and test samples were assayed in duplicate. Both assays incorporate a specific murine monoclonal antibody for each sTNF-R as a coating antibody, together with horseradish peroxidase-conjugated polyclonal anti-sTNF-R antibodies. According to the manufacturers, these ELISA detect both free receptor and receptor bound to TNF, as the assays are relatively insensitive to added TNF-α or TNF- P (lymphotoxin α). Wilcoxon's sign rank test (two-tailed) was used to compare the values for the nonhealing and healing phases. The linear association between pairs of assays was tested using Pearson's correlation coefficient (r). When the effect of healing status on the relationship between immunoreactive and bioactive TNF-α was examined, regression analysis was performed using the statistical software package S-PLUS version 3.2 (StatSci, Seattle, WA). Wound fluid was collected from 21 patients with chronic leg ulcers in both the nonhealing and the healing phases. There were 13 male and eight female patients, with a median age of 78 y (range 31—91 y). In nine patients the ulcers were due to venous disease alone, and another nine had combined venous and arterial disease. Two patients with venous disease had noninsulin-dependent diabetes, and one patient had venous and arterial disease and noninsulindependent diabetes. These patients did not receive additional treatment for the arterial component of their disease or any alteration in their diabetic medications after admission to hospital. The median initial size ofthe ulcers was 46 cm2 with an interquartile range of 20—80 cm2. The median reduction in the size of the ulcers after 2 wk bed rest was 8%, with an interquartile range of 2% to 23%. The levels of immunoreactive TNF-α in the wound fluid samples were very high compared with normal plasma levels, with a median of 1999.5 pg per ml and a range of 8.1 to 10970 pg per ml in the Medgenix ELISA (normal plasma range 1—20 pg per ml, Medgenix product information). The amount of TNF-α significantly decreased, by ≈70%, in the transition from the nonhealing phase to the healing phase in both ELISA (Table I). The two ELISA assigned consistently different absolute levels of TNF-α, despite the use of a common reference standard, with the Medgenix assay assigning approximately 3-fold more TNF-α than the Genzyme assay for the same sample (Table I).Table ILevels of immunoreactive TNF-a measured in wound fluid from nonhealing and healing ulcers with two different ELISA (pg per ml)Nonhealing median (interquartile range)Healing median (interquartile range)SignificancelevelMedgenix2428.5895.2(1535.0-3549.2)(549.4-2523.8)p < 0.002Genzyme694.7190.8p < 0.003(402.2-1295.0)(100-848.3) Open table in a new tab Figure 1 illustrates the strong association between the two sets of ELISA results, and demonstrates that comparisons made between samples are consistent within either assay, but that values cannot be compared between assays. The bioassay results followed the same trend as the ELISA results, but the difference between the nonhealing and healing phases was not significant (Table II). The relationship between the ELISA (Medgenix) and bioassay results was examined using simple linear regression, and then by adding healing status as a factor in multiple regression, fitting separate lines for the nonhealing and healing groups. A significant linear correlation was found between the ELISA and bioassay results (p < 0.001) with 71% (r2 = 0.71) of the observed variation in bioactive TNF-α levels being explained by the ELISA levels. There was a significant improvement in the amount of variation explained when separate parallel lines were fitted for each healing status (F1,34 = 7.6, p < 0.01), indicating that healing status is a factor that significantly influences the relationship between ELISA and bioactive TNF-α levels (Fig 2). Figure 2 demonstrates that for a given amount of immunoreactive TNF-α, the bioactivity is lower in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers.Table IILevels of bioactive TNF-α in wound fluid from nonhealing and healing ulcers (U per ml)Nonhealing median(interquartile range)Healing median(interquartile range)Significance level156.1 (32.6-353.8)49 (15.3-221.2)NSa Open table in a new tab There were significantly greater levels of soluble p75 receptors in wound fluid from nonhealing ulcers than from healing ulcers, but the levels of soluble p55 receptors remained at similar levels in both healing phases (Table III). Like TNF-α levels, the levels of both types of soluble receptors were much higher than those found in normal plasma, with median values of 24423.5 pg per ml and 14044 pg per ml, respectively, for the p75 and p55 soluble receptors (normal plasma ranges; soluble p75, 1003-3170 pg per ml; soluble p55, 748.7-1966 pg per ml). A significant linear association was shown between soluble p75 and soluble p55 levels (r = 0.78, p < 0.001) (Fig 3), but there was no association between the levels of either soluble receptor and immunoreactive or bioactive TNF-α levels (data not shown).Table IIILevels of p75 and p55 soluble TNF receptors in wound fluid from nonhealing and healing ulcers (pg per ml)Nonhealing median (interquartile range)Healing median (interquartile range)SignificancelevelSoluble p75b2689720616(21433.2-1264.5)(15874.2-5929.2)p < 0.025Soluble p5514858.513930.5(11693.5-17599)(8874-15396.8)NSaNS, not significant.a NS, not significant. Open table in a new tab Molar ratios of sTNF-R levels to immunoreactive TNF- a levels have been calculated by some authors to determine the excess of soluble receptor required to inhibit the in vitro cytotoxicity of a given amount of recombinant TNF-α (Loetscher et al., 1991Loetscher H. Gentz R. Zulauf M. et al.Recombinant 55-kDa tumor necrosis factor (TNF) receptor.J Biol Chem. 1991; 266: 18324-18329Abstract Full Text PDF PubMed Google Scholar, Van Zee et al., 1992Van Zee K. Kohno T. Fischer E. Rock C. Moldawer L. Lowry S. Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor a in vitro and in vivo.Proc Natl Acad Sci USA. 1992; 89: 4845-4849Crossref PubMed Scopus (749) Google Scholar). These estimations can help predict whether levels of sTNF-R measured in vivo are sufficient to block immunoreactive TNF-α levels found in vivo. The cytotoxic effect of TNF-α on WEHI 164 clone 13 murine cells is reported to be inhibited by 50% in the presence of a 30-fold molar excess of soluble p55 receptors, or a 300fold excess of soluble p75 receptors (Van Zee et al., 1992Van Zee K. Kohno T. Fischer E. Rock C. Moldawer L. Lowry S. Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor a in vitro and in vivo.Proc Natl Acad Sci USA. 1992; 89: 4845-4849Crossref PubMed Scopus (749) Google Scholar). If the molar ratios are determined for the data in this experiment using the same calculations as Van Zee et al (assumed same molecular weight for all molecules, not a true molar ratio), the range of ratios in the nonhealing phase is 2.4-35.1 (median 12.4) for soluble p75/TNF-a, and 1.1-14.6 (median 6.5) for soluble p55/TNF-a (using the Medgenix TNF-α data). Such calculations depend critically upon the absolute amount of TNF-α detected by ELISA, so the ratios would be approximately 3fold higher if the Genzyme values were used. In wound fluid samples from chronic venous ulcers at nonhealing and healing phases, a significantly higher concentration of immunoreactive TNF-α was detected in the nonhealing wound fluid samples by both ELISA. The median amount of TNF-α in the nonhealing wound fluid was extremely high (Medgenix, 2428 pg per ml). Whereas there was a very strong positive association between the two TNF-α ELISA, the quantitative differences in TNF-α concentrations between the assays probably reflect differences in the epitopes detected by the antibodies in the kits. The Medgenix ELISA may detect more forms of TNF-α (e.g., proteolytically cleaved or complexed) than the Genzyme ELISA, because it uses a cocktail of monoclonal coating antibodies rather than a single monoclonal antibody. Such variation between commercial TNF-α ELISA kits has been documented by Ledur et al., 1995Ledur A. Fitting C. David B. Hamberger C. Cavaillon J.-M. Variable estimates of cytokine levels produced by commercial ELISA kits: results using international cytokine standards.J Immunol Meth. 1995; 186: 171-179Crossref PubMed Scopus (117) Google Scholar. Despite the differences in the absolute values of TNF-α assigned, the relative values between samples were consistent using either assay. When we examined the correlation between immunoreactive TNF-α and bioactive TNF-α we found a significant linear relationship. Clinical studies have often demonstrated high levels ofimmunoreactive TNF-α and sTNF-R coexisting with extremely low values of bioactive TNF-α as measured by the murine L929 and WEHI 164 clone 13 assays (Jackson et al., 1995Jackson A. Alexandrov A. Prescott S. James K. Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients after bacillus Calmette-Guerin immunotherapy.Cancer Immunol. 1995; 40: 119-124Google Scholar, Linderholm et al., 1996Linderholm M. Ahlm C. Settergren B. Waage A. Tarnvik A. Elevated levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, and IL-10 in patients with hemorrhagic fever with renal syndrome.J Infect Dis. 1996; 173: 38-43Crossref PubMed Scopus (166) Google Scholar). The KYM-1D4 bioassay used here offers advantages over the murine cell lines, in that it is a highly sensitive assay and species preference is eliminated (Meager, 1991Meager A. A cytotoxicity assay for tumour necrosis factor using a human rhabdomyosarcoma cell line.J Immunol Meth. 1991; 144: 141-143Crossref PubMed Scopus (33) Google Scholar) Using this assay we showed substantial levels of bioactive TNF- α in both the nonhealing and healing phases. In contrast to the levels of immunoreactive TNF-α decreasing significantly with healing, the levels of bioactive TNF-α were not significantly different between the nonhealing and healing phases, although the median values did fol