MATERIALS AND METHODS

Patients

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. 1979}). Arterial Doppler pressures were performed to assess the presence of arterial disease (^{Yao et al. 1968}). 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.

Collection of wound fluid

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. 1996}). 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- 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.

TNF- bioassay

Bioactive TNF- 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 1991}). Recombinant human TNF- (TNF-H, Genzyme, Boston, MA) was used as the standard for the quantitation of TNF- in the test samples. Serial dilutions of recombinant human TNF- 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 1.43 10⁸ U per mg) were used to construct the standard curve. Test wound fluid samples were filter-sterilized after thawing (0.22 m membrane, Millex^R-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 2 10⁵ cells per ml and 100 l 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 l per well of 5 mg MTT per ml phosphate-buffered saline) (^{Mosmann 1983}) 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. 1989}) after 2 h at 37°C, and the optical densities read at 570 nm.

TNF- immunoassays

Immunoreactive TNF- in wound fluid was determined by DuoseT^TM (Genzyme) and EASIA^TM (Medgenix, Fleurus, Belgium) ELISA for human TNF-. Assays were performed according to the manufacturer's instructions. The same preparation of recombinant human TNF- 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.

Immunoassays to detect soluble TNF-receptors

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- or TNF- (lymphotoxin ).

Statistical analysis

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- was examined, regression analysis was performed using the statistical software package S-PLUS version 3.2 (StatSci, Seattle, WA).

RESULTS

Patient dataWound 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 noninsulin-dependent 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 of the 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%.

Immunoreactive TNF- are higher in the nonhealing phase

The levels of immunoreactive TNF- 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- significantly decreased, by 70%, in the transition from the nonhealing phase to the healing phase in both ELISA (Table 1). The two ELISA assigned consistently different absolute levels of TNF-, despite the use of a common reference standard, with the Medgenix assay assigning approximately 3-fold more TNF- than the Genzyme assay for the same sample (Table 1).

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.

Figure 1.

Positive nonlinear association between values of TNF- detected by Medgenix EASIA^TM and Genzyme DuoseT^TM ELISA in 40 wound fluid samples, but large differences in absolute values of TNF- assigned by each. The pattern of TNF- content of the wound fluid samples is similar within either ELISA, but values cannot be compared between assays.

Full figure and legend (10K)

Bioactive TNF- are not significantly different between the healing phases

The bioassay results followed the same trend as the ELISA results, but the difference between the nonhealing and healing phases was not significant (Table 2). 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- 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 (F_1,34 = 7.6, p < 0.01), indicating that healing status is a factor that significantly influences the relationship between ELISA and bioactive TNF- levels (Figure 2). Figure 2 demonstrates that for a given amount of immunoreactive TNF-, the bioactivity is lower in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers.

Figure 2.

Relationship between ELISA and bioactive TNF- is different for nonhealing and healing wound fluid samples. When healing status is included as a factor in regression analysis, two parallel lines representing the nonhealing and healing samples explain the variation in the data significantly better than a single line (F_1,34 = 7.6, p < 0.01). The bioactivity of a given amount of immunoreactive TNF- was approximately 100 U per ml less in wound fluid from nonhealing ulcers than in wound fluid from healing ulcers.

Full figure and legend (12K)

Soluble p75 TNF receptor levels are higher in the nonhealing phase

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 3). Like TNF- 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) (Figure 3), but there was no association between the levels of either soluble receptor and immunoreactive or bioactive TNF- levels (data not shown).

Figure 3.

Significant linear correlation between soluble p75 and soluble p55 TNF receptor levels in wound fluid samples. The relationship between the two types of soluble TNF receptor measured by ELISA in 34 wound fluid samples is shown. r = 0.78; p < 0.025.

Full figure and legend (10K)

Molar ratio of soluble TNF receptor levels to immunoreactive TNF- is not high enough for substantial inhibition of TNF bioactivity

Molar ratios of sTNF-R levels to immunoreactive TNF- 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- (^{Loetscher et al. 1991;Van Zee et al. 1992}). These estimations can help predict whether levels of sTNF-R measured in vivo are sufficient to block immunoreactive TNF- levels found in vivo. The cytotoxic effect of TNF- 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 300-fold excess of soluble p75 receptors (^{Van Zee et al. 1992}). 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-, and 1.1–14.6 (median 6.5) for soluble p55/TNF- (using the Medgenix TNF- data). Such calculations depend critically upon the absolute amount of TNF- detected by ELISA, so the ratios would be approximately 3-fold higher if the Genzyme values were used.

DISCUSSION

In wound fluid samples from chronic venous ulcers at nonhealing and healing phases, a significantly higher concentration of immunoreactive TNF- was detected in the nonhealing wound fluid samples by both ELISA. The median amount of TNF- 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- ELISA, the quantitative differences in TNF- 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- (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- ELISA kits has been documented by^{Ledur et al. (1995)}. Despite the differences in the absolute values of TNF- assigned, the relative values between samples were consistent using either assay.

When we examined the correlation between immunoreactive TNF- and bioactive TNF- we found a significant linear relationship. Clinical studies have often demonstrated high levels of immunoreactive TNF- and sTNF-R coexisting with extremely low values of bioactive TNF- as measured by the murine L929 and WEHI 164 clone 13 assays (^{Jackson et al. 1995;Linderholm et al. 1996}). 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 1991}). Using this assay we showed substantial levels of bioactive TNF- in both the nonhealing and healing phases.

In contrast to the levels of immunoreactive TNF- decreasing significantly with healing, the levels of bioactive TNF- were not significantly different between the nonhealing and healing phases, although the median values did follow the same trend. When the relationship between immunoreactive and bioactive TNF- was examined, it was found that the observed variation in the data was significantly better explained when healing status was assigned as a separate factor. That is, the linear relationship between immunoreactivity and bioactivity was different for each phase of healing. The bioactivity of immunoreactive TNF- in wound fluid from nonhealing ulcers was 100 U per ml less than the bioactivity of the same amount of immunoreactive TNF- in wound fluid from healing ulcers. The implication of this finding is that some variable associated with healing status is able to modify the bioactivity of the TNF- present in the wound fluid.

The levels of the soluble p75 receptor were significantly elevated in wound fluid from nonhealing ulcers compared with healing ulcers (p < 0.025), and it is tempting to speculate that this was responsible for modulating the activity of the TNF. Both types of sTNF-R have been shown to be able to inhibit the cytotoxic activity of TNF-in vitro at high molar excesses of sTNF-R (^{Loetscher et al. 1991;Higuchi and Aggarwal 1992;Spinas et al. 1992;Van Zee et al. 1992;Mohler et al. 1993}), with Van Zee et al. estimating that a 30-fold excess of soluble p55, or a 300-fold excess of soluble p75, is required to neutralize the cytotoxicity of 1500 pg recombinant human TNF- per ml. Because the median value of the molar excess of soluble p75/immunoreactive TNF- in nonhealing wound fluid was only 12.4, and the median level of TNF- in nonhealing wound fluid was 2428.5 pg per ml (Medgenix), it is unlikely that these levels of soluble receptor were responsible for a substantial decrease in bioactivity. In addition, there was no association between immunoreactive TNF- levels and the levels of either of the sTNF-R; however, calculations such as molar ratios are difficult to compare between laboratories and assays, and it cannot be ruled out that the combined effect of p55 and p75 soluble receptors had some inhibitory activity.

At low values of the molar excess of sTNF-R, the effects of the soluble receptors can augment the bioactivity of TNF-in vitro by stabilizing the structure of the trimeric TNF- molecule (^{Aderka et al. 1992}). These authors found that at the concentrations of TNF- present in these wound fluid samples, TNF- deteriorates very rapidly at 37°C (e.g., by 70% in 48 h) unless stabilized by sTNF-R. Consequently, within the 48 h time-frame of the KYM-1D4 bioassay it is possible that the measured cytotoxicity was a combination of the free TNF- present in the wound fluid at the beginning of the incubation period, plus receptor-stabilized TNF- that was released during the incubation period. This confounding phenomenon may, in part, explain the lack of an obvious relationship between bioactivity and sTNF-R levels.

If the molar excess of soluble p75 receptors was not sufficient to down-modulate the bioactivity of the TNF- in nonhealing wound fluid on its own, other factors may also be responsible. It has been shown that neutrophil-derived proteolytic enzymes (cathepsin-G, elastase) can inactivate TNF- (^{Scuderi et al. 1991}) and large numbers of neutrophils are found in the dermis at the base and edge of venous ulcers (^{Stacey et al. 1995}) in which elastase activity has been detected (^{Mirshahi et al. 1995}).

In this study, the levels of the p55 and p75 soluble receptors showed a strong linear correlation (p < 0.001), which is a feature of some, but not all, in vivo scenarios. The same association has been found in plasma of HIV-1-seropositive humans (^{Aukrust et al. 1994}) and in a sample of critically ill patients (^{Van Zee et al. 1992}), but not in plasma from humans with acute experimental endotoxinaemia (^{Spinas et al. 1992}). Interestingly, the levels of neither soluble p55 nor soluble p75 receptors were correlated with immunoreactive TNF levels in our wound fluid samples. In HIV-1-seropositive humans the plasma TNF- levels were associated with both soluble p55 and soluble p75 levels (^{Aukrust et al. 1994}), and in acute experimental endotoxinemia in humans TNF- levels were correlated with p75 but not p55 (^{Spinas et al. 1992}). Such differences may be induced by factors at the sites of TNF- production/activity, e.g., the proteolytic enzyme profile. It appears that a common protease (or group of proteases) may be responsible for some of the cleavage of the transmembrane TNF receptors in this chronic venous ulcer environment, and that this is not the same protease(s) involved in the cleavage of transmembrane TNF-. Collaborative work in our laboratory has shown that there are elevated levels of matrix metalloproteinases in nonhealing versus healing wound fluid.² The major enzyme catalysing the release of TNF- from cells (TNF- convertase) is believed to be a nonmatrix metalloproteinase (^{Black et al. 1996}).

Although a linear association was demonstrated between soluble p75 and p55 receptor levels, the significant decline in soluble p75 levels but not p55 levels with healing suggests that additional regulatory mechanisms for TNF receptor cleavage also exist. The cleavage of membrane p75, but not p55, is reported to be dependent on phosphorylation of the cytoplasmic domain (^{Brakebusch et al. 1992;Crowe et al. 1993}). In addition, the proteolytic enzymes present may favor the cleavage of p75 over p55. Elastase from activated neutrophils has been reported to mediate shedding of the p75 receptor but not the p55 receptor (^{Porteu et al. 1991}).

In conclusion, this study enhances our knowledge of the role of TNF- in the healing of chronic wounds, and also demonstrates that local factors may influence the way that TNF activity is regulated. Total TNF- was significantly elevated in the nonhealing wound versus the healing wound, but the accompanying bioactivity was not. Whereas there is apparently a greater stimulus for TNF- release in the nonhealing phase, there appear to be other mechanisms operating that can modulate TNF- activity. The soluble TNF receptors do not appear to play the sole role in limiting excessive TNF- activity in this chronic wound environment. The molar levels were low compared with the levels of TNF- present, and were not related to the amount of TNF- present. Whether additional factors such as neutrophil elastase or cathepsin-G were responsible for downregulating excessive TNF- activity can only be speculated from this study. This study suggests that excessive TNF- activity may not be the key factor in the impaired healing of chronic venous ulcers, because healing was initiated without a significant decline in the level of bioactive TNF-. Further work to examine the forms of TNF- in wound fluid by electrophoresis and immunoblotting, and to determine the levels of key proteolytic enzymes, is necessary to assess the relative importance of receptor binding and proteolytic cleavage in the regulation of TNF- bioactivity in the chronic wound environment.

Notes

¹ Trengove N, Bielefeldt-Ohmann H, Stacey M: Cytokine profile of wound fluid from chronic leg ulcers. Wound Rep Reg 2:228, 1994 (abstr.)

² Tarnuzzer R, Schultz G, Macauley S, Trengove N, Stacey M: Protease levels in fluids collected from chronic venous/arterial leg ulcers before and during healing. Wound Rep Reg 4:A142, 1996 (abstr.)

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Acknowledgments

This work was supported by a grant from the Raine Foundation, University of Western Australia. We thank Jeremy Wallace for performing the regression analysis and for his general statistical advice.