Studies on the Role of Tumor Necrosis Factor in Adult Respiratory Distress Syndrome 1 - 3

R E. PARSONS, F. A. MOORE, E. E. MOORE, D. N. IKLE, P. M. HENSON, and G. S. WORTHEN

Introduction

Tumor necrosis factor (TNF), a cytokine produced by stimulated monocytes and macrophages, is rapidly becoming recognized as an important mediator of inflammation. Many of the characteristic features of septic shock once ascribed to endotoxin havebeen attributed to TNF (1,2), and TNF has been shown to have significant effects on inflammatory cells, particularly the neutrophils. Like endotoxin, TNF can prime neutrophils for an enhanced response to stimuli (3), and it enhances neutrophil adherence (4), phagocytosis (5), and superoxide production (6, 7). These features have led to the suggestion that TNF may playa role in multiple disease processes including sepsis and acute lung injury. The role of TNF in septic shock in animals has become increasingly welldefined. The intravascular administration of TNF reproduces the physiologic manifestations of shock seen with the infusion of endotoxin (8-10). The administration of antibodies to TNF prevents the mortality from endotoxic shock in mice, rabbits, and baboons (13-15). By contrast the association of TNF and sepsis in humans has been less certain. In humans, the physiologic response to a single-dose infusion of endotoxin is associated with a demonstrable rise in the plasma concentration of TNF (11, 12). The studies have been hampered, in part, by the rapid, transient nature of the release ofTNF in response to endotoxin. Michie and coworkers (11) found that TNF levels were maximal at 90 to 180 min following a single infusion of endotoxin into normal volunteers and by 4 h after the infusion the TNF was no longer measurable. Studies in patients with cancer suggest that the half-life of circulating TNF may actually be as short as 14-18 min (16). In humans the pattern of TNF release in response to a continuous endotoxin infusion or to repetitive infusions (both of which would mimic certain clinical situations) is not known, although there is evidence from studies in rabbits that af694

SUMMARY lUmor necrosis factor (TNF), rapidly becoming recognized as a mediator of Inflammation, may be Important In the pathogenesis of acute lung Injury. Its role In the development of the adult respiratory distress syndrome (ARDS) In humans, however, has been difficult to clarify. To determine If TNF could be Important early In the development of acute lung Injury from multiple causes, we enrolled 103 patients within 8 h of meeting the criteria for an at-risk Illness (sepsis, aspiration of gastric contents, severe pancreatitis, hypertransfuslon, abdominal trauma, chest trauma, multiple fractures) and obtained multiple frequent blood samples for TNF measurements. Using five methods of TNF analysis, we were unable to find an association between TNF and the development of ARDS. However, we found significant differences In TNF measurements depending on the methods of analysis used, which could, at least In part, account for the Inconsistencies In the published literature regarding the relationship between TNF and disease processes. AM REV RESPIR DIS 1992; 146:694-700

ter an initial exposure to endotoxin, further exposure does not increase TNF production/release (17). Despite the apparent brevity of the TNF response to a single dose of endotoxin, Waage and associates (18) demonstrated a clear relationship between TNF and mortality in meningococcemia in humans, and deGroote and colleagues (19) found measurable levels of TNF in 16070 of patients with septic shock in blood samples obtained a mean of 18.8 h after diagnosis. TNF has also been implicated in the acute lung injury associated with sepsis. The infusion of TNF into guinea pigs causes a significant increase in pulmonary permeability that wassimilar to that seen in response to the infusion of endotoxin (20). In cancer patients the infusion of TNF is followed by a decrease in the diffusion capacity for carbon monoxide and an increase in the alveolar to arterial oxygen difference (21). The relationship between TNF and the adult respiratory distress syndrome (ARDS) is also unclear. Although two recent studies demonstrated an association between TNF levels and the development of ARDS in septic patients (22,23) a third study comparing patients at risk for and with ARDS found increased levels of TNF on septic patients but no association between TNF levels and the development of ARDS (24). Possible explanations for the discrepancies between the studies include

the wide differences in the time of the initial TNF measurements relative to the onset of the disease, the variations in the intervals between TNF measurements, and the different assay systems used to measure TNF. Accordingly, we designeda prospective study that enrolled patients within 8 h of developing a risk illness for ARDS, measured TNF at frequent intervals, and used multiple methods to quantitate the TNF. Methods Patient Selection Seven groups of patients were defined as being at risk for the development of ARDS. These groups were selected based on the prospective studies of Fowler and coworkers (25) and Pepe and coworkers (26) that identified those patients who wereat significant risk

(Received in originaiform September 13,1991and in revised form March 5, 1992) 1 From the Departments of Medicine and Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Departments of Medicine and Surgery,Denver General Hospital, Department of Medicine, Surgery, and Pathology, University of Colorado School of Medicine,Denver,Colorado. 2 Supported by grant No. K08 HL0l849 and ARDS SCOR RFA - HL8701 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Polly E. Parsons, M.D., Department of Medicine #4000, Denver General Hospital, 777 Bannock Street, Denver, CO 80204.

695

TUMOR NECROSIS IN ARDS

for the development of the syndrome. The criteria for the seven at-risk categories were as follows: Sepsis. Evidence of a serious bacterial infection with two or more of the following: either (1) rectal or core temperature greater than 39° C or (2) peripheral white blood cell count greater than 12,000 cells/mm" or greater than 20070 immature neutrophils plus at least one of: (3) a positive blood culture of a commonly accepted pathogen, (4) a strongly suspected or proven source of systemic infection, (5) gross pus in an enclosed space, (6) unexplained systemic arterial hypotension (systolic blood pressure less than 80 mm Hg), (7) systemic vascular resistance less than 800 dyne x s x em", (8) unexplained metabolic acidosis. Severepancreatitis. Pancreatitis complicated by one or more of the following: white blood cell count> 16,000 cells/rum", lactic dehydrogenase> 350 u/ml, serum glutamic oxaloacetic transaminase > 250 u/ml, acute decrease in hematocrit of;; 10070, Pao2 < 55 mm Hg while breathing room air, serum calcium < 8.0 mg/dl. Hypertransfusion. Greater than 10 U of whole blood or packed red blood cells in less than 24 h. Witnessed aspiration of gastric contents. Abdominal trauma with an abdominal trauma index (27) greater than 15- determined at laparotomy. Chest trauma resulting in a flail chest or pulmonary contusion with a Pao, of < 70 mm Hg on > 40070 supplemental oxygen. Multiple fractures. Either (1) an unstable pelvic fracture requiring > 6 U of whole blood or packed red blood cells, (2) two major long bone fractures (long bones = femur, humerus, tibia), (3) pelvic fracture plus a major long bone fracture. ARDS. Diagnosed only if all of the following criteria were met: (1) acute respiratory failure requiring mechanical ventilation, (2) bilateral pulmonary infiltrates on chest radiograph, (3) pulmonary capillary wedge pressure < 18 mm Hg, (4) static pulmonary compliance < 50 mllcm H 20 , (5) arterial to alveolar partial pressure of oxygen ratio < 0.25. Within 8 h of fulfilling the criteria of one ofthe above categories, the patients were enrolled in the study. After informed consent was obtained from either the patient or a responsible family member, 10 cc of blood was drawn into EDTA from either an arterial line, central venous line, or via venipuncture of an antecubital vein. Plasma was separated from the blood by centrifuging at 1,000g for 10 min and stored at -70° C. The time that the first blood sample was drawn was designated T = o. Subsequent blood samples were obtained at T = 6 h, T = 12 h, T = 24 h, and T = 48 h. Twenty normal volunteers served as controls for this study. After informed consent was obtained, a single venous blood sample was obtained from these subjects. TNF Analysis Radioimmunoassay (RIA-Genzyme Corpo-

ration, Boston, MA). TNF was measured on all plasma samples with this assay, which is specific for hTNF-alpha. Freshly thawed plasma (100 ul) was incubated with rabbit antihTNF-alpha in the presence of bovine serum albumin buffer for 48 h at room temperature. 115 1hTNF-alpha was then added, and the samples were incubated for an additional 24 h. The second antibody, sheep anti-rabbit IgO, was added, and the samples vortexed and incubated an additional hour. The samples were then centrifuged at 1,500 x g at 4° C for 15 min. The supernatants weredecanted, and the assay test tubes were counted in a gamma counter. All specimens wererun in duplicate. With each assay, a standard curve was generated with known standards supplied with the assay, and nonspecific binding was accounted for with plasma controls. Cytotoxicity. TNF activity was assayed using the method described by Ruff and Gifford (28). L929 cells (American Tissue TypeCulture Collection, Rockville,MD) were seeded at a density of 5.5 x 104 cells per well in 96 well tissue culture plates (Costar, Cambridge, MA) in Dulbecco's Modified Essential Medium (Whittaker Bioproducts, Walkersville, MD) supplemented with 10070 fetal bovine serum, 0.25 mg/ml L-glutamine, 100u/ml penicillin, and 100ug/ml streptomycin. The cells were incubated at 37° C in 7.5070 CO 2 for 18-20 h, at which time the medium was replaced with medium containing 1 J.l.g/ ml actinomycin D (Sigma). One hundredmicroliter aliquots of serial dilutions of plasma samples and of human recombinant TNFalpha standards (Amgen, Thousand Oaks, CA) were added to wells in triplicate and incubated for 18to 20 h. The medium was then removed, and adherent cells stained with 100 ul 0.1070 crystal violet (Sigma Chemical Company, St. Louis, MO) in 1070 acetic acid for 15min. Stained cells werewashed with water, dried, and then the dye was solubilized with 100 ul 1070 SDS. Plates were read using an ELISA reader (Biotek) with a test wavelength of 590 mm. Immunoradiographicassay(IRMA-Medgenix Diagnostics, Brussels, Belgium). This assay is also reportedly specific for TNF-alpha according to the manufacturer. Plasma samples (200 ul) were added to assay tubes precoated with monoclonal antibodies reportedly selected for their ability to distinguish distinct epitopes of TNF-alpha. A signal antibody, anti-TNF-alpha Jl25 in phosphate buffer with bovine serum albumin and azide was added. Samples were incubated at room temperature for 16 to 20 h, the tubes decanted, washed once, and then counted in a gamma counter. All samples wereassayedin duplicate. A standard curve was generated using premeasured TNF-alpha standards supplied with the assay. Enzyme immunoassay (T-Cell Sciences, Inc.•Cambridge. MA). This is a sandwich immunoassay for TNF-alpha. A mouse monoclonal antibody to TNF-alpha was adsorbed onto polystyrene microtiter wells. Fifty milliliters of plasma were added to each well and

incubated for 2 h at 37° C. The wells were washed, and horseradish peroxidase conjugated with mouse monoclonal antibody to TNFalpha was added. After a 2-h incubation at 37° C, the wells were washed and colorimetric substrate was added. The optical density of all samples was read at 490 nm using an ELISA reader. All samples were run in duplicate and results quantitated by comparison with a standard curve generated with premeasured aliquots of TNF-alpha supplied with the assay. Enzyme immunoassay (Endogen, Inc.•Boston, MA). This is a dual antibody sandwich assay for TNF-alpha. Plasma samples were added to polystyrene wellscoated with mouse monoclonal anti-TNF, incubated for 2 h at 37° C, then washed once. Rabbit polyvalent anti-TNF, which reportedly binds to multiple epitopes of the TNF, was then added, the samples incubated for 1 h at 37° C, and then washed. Alkaline phosphatase labeled goat anti-rabbit IgG was added and the samples allowed to incubate for 1 h at 37° C, then washed. r-nitro-phenyl phosphate was added, and the samples wereread at 405 nm using an ELISA microplate reader. Samples were compared against a standard curve generated with TNF-alpha standards.

TNF Recovery To confirm that TNF was not being lost during the transport, processing, and freezing of blood samples, human recombinant TNF-a (600 pg/ml, Amgen, Thousand Oaks, CA) was added to blood immediately after it was obtained from two patients and one normal volunteer. Aliquots were centrifuged immediately and at 30, 60, 120, and 240 min to obtain plasma. The plasma samples were stored at -70° C until they were thawed and simultaneously assayed with two immunoreactivity assays (RIA - Genzyme, IRMA - Medgenix). TNF-a (600 pg/ml, Amgen) was also added to plasma samples from patients (n = 10)and normal subjects (n = 5) and assayed for TNF using the same two methods. Statistics To compare TNF measurements obtained in all patients over time, and in the subgroup of septic patients over time, a univariate repeated measures ANOVA was used with tests for subject, time, and group effects. The subset of patient samples analyzed at only two time points was assessed with a Wilcoxon signed rank sign test to determine differences between time points. A Kruskal-Wallis test was used to evaluate differences between groups at a single time point, and multiple Wilcoxon rank sum tests were then used to determine which specific groups were different. Unpaired t tests were used to compare the TNF recoveryfrom blood and plasma and to compare the samples analyzed with the TNF enzyme immunoassays. Simple linear regression and a paired t test were used to evaluate the correlation between two measured techniques. Upper limits of normal were computed as the mean plus two standard devi-

696

PARSONS, MOORE, MOORE, IKL~, HENSON, AND WORTHEN

velopment of ARDS was enrolled in this study. Twenty-eight of these patients developed ARDS and are referred to as "PRE ARDS." The other 75 patients are referred to as "AT RISK." The patient characteristics are shown in table 1. The only difference between the two patient groups was the APACHE II scores, which were greater in the ARDS group (p < 0.001). TNF levelswerequantitated in all pa- tient and normal subject plasma samples using the radioimmunoassay specific for TNF-a from Genzyme Corporation (RIA). As shown in figure 1, there were no differences in TNF levels or in TNF measurements over time in those patients at risk who did and did not develop ARDS. Furthermore, TNF levels in patients at T = 0 werenot greater than those found in normal subjects (mean ± 2 SD = 176.3 ± 54). The time point at which the patients developed ARDS varied, from having

TABLE 1 PATIENT CHARACTERISTICS

Sex (M/F) Age Apache II Diagnosis Sepsis Hypertransfusion Aspiration Pancreatitis Abdominal trauma Chest trauma Multiple fractures ARDS

At Risk (n = 75)

ARDS (n = 28)

57/18 44 ± 19 9.88 ± 6.4

20/8 52.2 ± 20.3* 15.8 ± 6.5*

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20

5

5

3

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17

2 1

4

7

5 2

* Mean ± SO.

ations in the normal sample. All tests of hypotheses were two tailed at the 0.05 level of significance.

Results

A total of 103patients at risk for the de-

300

250

200

Fig. 1. Serial TNF measurements (RIA-Genzyme) in patients at risk who did not develop ARDS ("ATRISK"), and who did develop ARDS ("PRE ARDS"). The upper limit of normal (230 pg/mlsolid line) represents the mean ± 2 SO plasma TNF level determined in 20 normal volunteers at a single time point.

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ARDS at the time of entry (T = 0) to developing it after the initial 48-h study period. This can be seen in figure 2. The number of patients who developedARDS at each time point is shown in figure 2A. As shown in figure 2B, there was no apparent pattern of TNF levels before or after the development 0 f ARDS. The patients with sepsis were identified and analyzed separately to see if the correlation between TNF levels and the subsequent development of ARDS in septic patients described by previous investigators (22, 23) was true for that subgroup of our patient population. As seen in figure 3, there were no differences in TNF levelsor in TNF measurements over time in those septic patients who did and did not develop ARDS. The TNF levels in the septic patients at T = 0 were not different than normals. Because we were unable to confirm the results of other investigators despite similarities in at least part of our patient populations (specifically septic patients), we were concerned that the different methods used to quantitate TNF could be contributing to the confusion. Accordingly, to confirm that the lack of association between TNF measurements and ARDS was not an artifact of methodology, patient samples were randomly selected and assayed for TNF using multiple methods. Plasma samples obtained at T = 0 and T = 24 from 23 patients (10 developed ARDS) and 9 normal subjects were assayed for TNF simultaneously with the same radioimmunoassay specific for human TNF-alpha (IRMA-Medgenix) used by Pinsky and colleagues (23) and Roten and coworkers (24) in their studies. Using this assay method, we also found that TNF levels in patients at risk for the development of ARDS were significantly greater than in normal subjects (figure 4, p < 0.001). However, there were no differences in TNF levels at time 0 and 24 h for either patient group and no differencesin TNF levelsbetween the two patient groups, indicating that there was no association between TNF levels and the subsequent development of ARDS (figure 4). Thirty of these same plasma samples were simultaneously assayed using the RIA (Genzyme). As can be seen in figure 5, there was poor correlation between the TNF levels measured with these two assays for human TNF-alpha. While the correlation coefficient between the two techniques is significant (r = 0.6, p < 0.001),the RIA (Genzyme) measurement is significantly higher than the IRMA (Medgenix) (difference 85.6 ± 17.5 pg/ml).

697

TUMOR NECROSIS IN ARDS

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TABLE 2 CYTOTOXICITY STUDIES

250

Fig. 3. Serial TNF levels (RIA-Genzyme) in the subset of patients who were septic (n - 29). The solid line represents the upper limit of normal (230 pglml) determined in 20 normal volunteers at a single time point. The data represent 19 "AT RISK" patients and 10 "PRE ARDS· patients. There were no differences between the patient groups or between either patient group and normals.

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200

PRE ARDS

2 4.53 2.13 4.51

± 0.41 Ulml* ± 0.531 U/ml* ± 0.72 Ulml*

± 0.68 Ulml*

• Level calculated assuming all cytotoxiCity secondary to TNF activity. The plasma and serum from six normal subjects were cytotoxic for L929 cells. If TNF was responsible for all the cytotoxicity, the TNF levels would be 2 ± 0.41 Ulml (plasma) and 4.53 ± 0.531 (serum). However, when the assays were repeated in the presence of anti-human antisera to TNF, the cytotoxicity persisted suggesting that something other than TNF was responsible.

150

AT RISK

100

Plasma Serum Plasma and antisera Serum and antisera

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700

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TNF was also quantitated with the L929 cell cytotoxicity assay. 1\venty-seven normal subjects were initially studied, and both serum and plasma wereassayed for TNF. Of these 27 subjects, the serum

of 21 was cytotoxic (correlating with a TNF level of 13.1 ± 3.2 U/ml), and the plasma of 13 was cytotoxic (correlating with a TNF level of 3.3 ± 1.9 U/ml). These values are similar to those recent-

ly reported by Levine and associates (29) for normal subjects. Goat anti-human antisera to TNF (generous gift of Dr. Richard J. Ulevitch, Scripps Clinic and Research Foundation, La Jolla, CA) sufficient to neutralize 25 U/ml TNF was added to the plasma and serum from six normal subjects, but there was no inhibition of cytotoxicity (table 2), suggesting that mediators other than TNF were being measured. Serial plasma samples (n = 26) from nine patients, four of whom developed ARDS, were found to have no cytotoxicity for L929 cells, suggesting that there was no TNF present. However, when known quantities of human TNF-alpha (Amgen) werethen added to five of these patient plasma samples, there was still no L929 cell cytotoxicity (data not shown), suggesting that either the TNF was rapidly inactivated by the plasma, or there was a factor(s) in the plasma that inhibited or interfered with the L929 cell killing. Similarly when TNF was measured in eight patient samples and six normal samples using the horseradish peroxidase ELISA assay (T Cell Science), the mean TNF levels were slightly but not significantly greater in the patient samples (8.4 ± 2.2 pg/ml) than the normal subjects (4.2 ± 1.5 pg/ml) (p = 0.16), but there were no differences between the two patient groups, AT RISK and PRE ARDS (data not shown). TNF was also measured in six normal and seven patient samples with the alkaline phosphatase ELISA assay (Endogen Inc.). The mean TNF level for the normal (70 ± 6.5 pg/ml) and patient (66 ± 3.6 pg/ml) samples were the same (P = 0.61).

TNF Recovery When samples of human recombinant TNF-alpha from different sources (Amgen, Medgenix, Genzyme) in KrebsRinger phosphate buffer with dextrose (KRPD) were measured with the different immunoreactive assays (RIA-Gen-

PARSONS, MOORE, MOORE, IKL~, HENSON, AND WORTHEN

698 TABLE 3 RECOVERY OF ADDED TNF*

Amgent Normal plasma (n 5) Patient plasma (n = 10)

=

RIA (Genzyme)

IRMA (Medgenix)

600 pg/ml 244 ± 17 pg/ml 302 ± 62 pg/ml

1,500 pg/ml 1,225 ± 192 pg/m 1,080 ± 45 pg/ml

* Human TNF-alpha (600 pg/ml-Amgen) was added to buffer, plasma from normal volunteers. and patient plasma and assayed with the RIA (Genzyme) and IRMA (Medgenix) method. Both assays detected the TNF, but the levels measured with the IRMA were consistently greater than those measured

by the RIA. t KRPD = Krebs-Ringer phosphate buffer with dextrose.

zyme, IRMA-Medgenix), the results varied as shown in table 3. Specifically,TNF, 600 pg/ml, from Amgen was measured as 600 pg/ml with the RIA (Genzyme) and 1,500pg/ml with the IRMA (Medgenix). Additionally, when TNF (Amgen) 600 pg/ml wasadded to whole blood, and the TNF recovered from the plasma over time was assessed, the levels measured by the different assays weredifferent. The amount of added TNF that could be recovered did decrease over time (figure 6) in all three subjects as measured by both assays. This decrease in measurable TNF was abrogated if the blood samples were kept on ice until the plasma was derived (data not shown). The recovery of TNF added to plasma samples from normal volunteers and patients is also shown in table 3. The recovery was not different for patients and normals (p = 0.065 for RIA, Genzyme, and p = 0.13 for IRMA, Medgenix), although the IRMA (Medgenix) results reflected an increased sensitivity to added TNF compared with the RIA (Genzyme). The recovery from plasma was consistently less than from KRPD buffer.

A

-

Discussion

In this study critically ill patients were studied within 8 h of being identified as at risk for the development of ARDS to determine if there wasany association between circulating levels of TNF and ARDS. Despite the early entry of patients into the study, the frequency of sampling, and the availability of multiple methods for the quantitation ofTNF, we were unable to demonstrate any relationship between TNF measurements and the development of ARDS. This in part confirms the results from Roten and associates (24), who evaluated patients with established ARDS and patients at risk and found no association between TNF measurements and ARDS. Our study also adds the observation that even when patients are studied within 8 h of being at risk for ARDS, no association between TNF measurements and the subsequent development of the syndrome could be found. This lack of association was true no matter how TNF was measured. This is in contrast to both the study of Roten and associates (24) and studies that have

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Fig. 6. TNF (600 pglml-Amgen) was added immediately to whole blood drawn from two patients and one normal volunteer, and serial plasma aliquots were obtained over time (0, 30, 60, 120, and 240 min). Initial plasma TNF levels were different as measured by the RIA (320-440 pglml, panel A) and the IRMA (1,200-1,550 pgiml, panel B). TNF measured by the two methods decreased over time in both the patients and the normal subject.

focused primarily on patients with sepsis. In a study of patients with septic shock, Marks and coworkers (22) found that 27 patients had measurable TNF (25/27 at the initial time point that was defined: "as early as possible after identification of hypotension and temperature abnormalities "), Of these 27 patients, 55070 developed ARDS, whereas only 26070 of the patients without circulating TNF at any time point developed the syndrome. deGroote and associates (19) studied 38 patients with presumed gram-negative bacteremia and found circulating TNF in 16070 ofthe patients' initial plasma samples (obtained 2 to 72 h after the diagnosis of presumed bacteremia). Thirteen of the 38 patients developed ARDS. Of note, none of those 13 patients had circulating TNF. Pinsky and colleagues evaluated 52 patients with undiagnosed hypotension (36 with sepsis or septic shock) and found that TNF levels were increased, but the development of multiple organ systemfailure and death were associated with a persistent elevation of TNF (> 150pg/ml) at 24 and 48 h after entry into their study rather than the initial circulating level of TNF. As mentioned previously, we found no differences in TNF levels measured over time between our patients at risk who did and did not develop ARDS. All three of the previously mentioned studies used different assays with different antibodies to TNF. Another major difference between our study and others is that the assaysweused gave conflicting data as to whether TNF levels in patients were greater than normal subjects. With the RIA (Genzyme), the alkaline phosphatase ELISA (Endogen), and the horseradish peroxidase ELISA (T Cell Sciences), there were no significant differences in TNF levels among patients and normals, whereas with the IRMA (Medgenix), the TNF levels in patients were significantly greater than in normal subjects. There are multiple potential explanations for the differences in results from the previous clinical studies and our study. The first consideration is the method for sample collection. It has been shown that some commercially available heparinized tubes are contaminated with endotoxin (24.68 ng/ml) and that blood drawn into these tubes had an initial mean TNF level of 97.5 ng/L, which increased to 3,431 ng/L after a 2-h incubation at 37° C (30). Similarly, blood collected into sterile glass tubes to which heparin had been added (which werealso

699

TUMOR NECROSIS IN ARDS

contaminated with endotoxin -1.28 ng/ml) had initial TNF levels of 115.6 ng/L, which increased to 997.7 ng/L after a 2-h incubation at 37° C. However, sterile polystyrene tubes with and without heparin and commercially available sterileglass tubes with EDTA(which were used for our study) were not found to be contaminated with endotoxin, and prolonged incubation did not increase TNF levelsin blood samples collected into those vials (30). Weconfirmed this by adding TNF to blood and obtaining aliquots of plasma over time. As discussed earlier (figure 6), the amount of TNF recovered decreased over time, suggesting there was no ongoing TNF production in our samples. All of the samples in our study were processed within less than 1 h (the majority within 30 min), so our inability to consistently find .greater TNF levels in patients than normals was not the result of TNF loss during sample preparation. The rate of decrease in measurable TNF is unlikely to account for the interassay variations in TNF measurements because it was the same for the two immunoreactive methods that we tested. Different antibodies to TNF have been used in the assays in clinical studies. It is possible that inherent differences in epitope specificity and affinity of these antibodies could vary in their recognition of TNF. Studies have shown that there are significant discrepancies between TNF levels determined by cytotoxicity and immunoreactivity (31, 32). Specifically, as suggested by Meager and associates (33), there is no wayto be certain that a specific antibody is exclusively recognizing biologically active TNF and not inactive, denatured, aggregated, or fragmented TNF molecules. The varying specificity of anti-TNF antibodies may well account for some of the differences between published studies and within our study (34). As we demonstrated, the quantification of a standard amount of TNF could vary from a measured level of 400 pg/ml to 1,500 pg/ml depending on the method used. Furthermore, the measurements of TNF in normal subjects ranged from minimal relative to those in patients to the same as those in patients. This strongly suggests that different antibodies recognize different epitopes of TNF that likely represent different inactive forms of TNF. If we assume that the high TNF levels found in normal subjects with the RIA (Oenzyme) represent primarily inactive or less active forms of TNF, it ap-

pears that these forms may be generated in vivo over a prolonged period of time, because they did not develop when TNF was added to whole blood, and plasma was derived 4 h later. Furthermore, the consistently high TNF levelsfound in patients relative to normal subjects with the IRMA (Medgenix) could suggest that assay is a more specific measurement for a form of TNF that is present in critically ill patients and not present in normal subjects. A circulating inhibitor to TNF-alpha has been previously described (35), and the results from our cytotoxicity assays suggest that circulating inhibitors could also account for some of the differences between clinical studies. As discussed in the Results section, we found that when known amounts of TNF were added to the plasma from patients, there was no increase in cytotoxicity, suggesting that inhibitors of TNF-induced cytotoxicity could be present. We did not find similar inhibitory activity in the plasma from normal subjects. In contrast, when we added TNF to normal and patient plasma and assayed for immunoreactivity, there was some inhibition by plasma, but it was the same for patient and normal plasma. If inhibitors of TNF develop during some acute illnesses, in at least some patients the timing of the sample collection for the measurement of TNF during the course of a disease could be critical, and differences in study protocol could significantly alter the results from clinical series. We have no a priori reason to favor any particular assay system. At face value the IRMA (Medgenix) appears to have some superior features: The sensitivity is high, TNF levels in normal subjects are low, and TNF levels are increased in patient samples relative to normal subjects. Whether this reflects the real presence of active TNF in these critically ill patients awaits an understanding of both the fate of circulating TNF and the recognition of the various epitopes by the available antibodies. Further compounding the interpretation of studies that investigate the role of TNF in human disease states is the supposition that circulating levelsof TNF accurately reflect tissue levels of TNF (36), which may not actually be the case. Stimulated alveolar macrophages produce more TNF than peripheral blood monocytes (37, 38), suggesting that tissue TNF concentrations could be substantially greater than circulating TNF concentrations. Furthermore, although

we have hypothesized that a major effect of TNF could be the priming of circulating neutrophils, local TNF effects may also be important. There is abundant evidence that TNF induces a variety of alterations in the endothelium, including upregulation of adhesion molecules and release of IL-8, which could lead to an increase in the adherence of neutrophils to the endothelial cell surface (39, 40). Because adherent neutrophils are known to secrete more of their constituents in response to stimuli (including TNF) (41, 42), the tissue level concentration of TNF may be far more relevant than circulating levels of TNF in the pathogenesis of organ-specific disease states. Acknowledgment The authors thank May Gillespie, Michael Owens, and Paul Noble, M.D., for their excellent technical support and the patient care staff of Denver General Hospital. They also gratefully acknowledge William F. Kastner, M.S., for his assistance with the statistical analysis.

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