Tumor Necrosis Factor Levels in Serum and Bronchoalveolar Lavage Fluid of Patients with the Adult Respiratory Distress Syndrome1- 3
THOMAS M. HYERS, SALLY M. TRICOMI, PATRICIA A. DETTENMEIER, and ALPHA A. FOWLER
Introduction
In gram-negative bacterial sepsis, tumor necrosis factor (TNF) is thought to be a major mediator of cellular injury after its release from mononuclear cells following exposure to endotoxin (1). Studies in normal individuals indicate that a small bolus of intravenous endotoxin results in a short-lived release and clearance of TNF in the serum over a period of approximately 4 h (2). The appearance of TNF in the serum is followed by somewhat longer lived increases in heart rate, temperature, white blood cell count, and adrenocorticotropic hormone (ACTH) and epinephrine release. It is not clear from human studies how the continuous or intermittent release of endotoxin into the circulation affects blood TNF levels. In pathologic human states relative to the adult respiratory distress syndrome (ARDS), TNF has been reported to be elevated in plasma of patients with septic shock (3), in bronchial secretions of patients with ARDS (4), and in limited bronchoalveolar lavage (BAL) samples of patients with ARDS (5). The studies of plasma samples of patients in septic shock showed that TNF was elevated transiently in patients with either gramnegative or gram-positive infections. The highest plasma elevations of TNF occurred in the first 12h after septic shock wasidentified (3). In the two small studies of patients with ARDS, TNF appeared to be elevated in lavage fluids and bronchial secretions but not in plasma. We studied patients with ARDS and individuals at high risk for ARDS. Half the patients with ARDS had sepsis as the cause of their ARDS, and about a third of the patients at risk also had sepsis. We measured levels of tumor necrosis factor immunologically and functionally in BAL fluid and serum in these patients. Our goal was to determine if elevated levels were associated with the onset of ARDS or clinical sepsis or with subsequent mortality. 268
SUMMARY 1\.Imornecrosis factor (TNF) was measurad antigenlcally and functionally In serum and bronchoalveolar lavage fluid (BAL) of patients with ARDS and those at high risk for ARDS. Of 22 patients with ARDS, 14 had sepsis or serious Infection as the major clinical pradlsposltlon, and 10 of 20 hlgh·rlsk patients had sepsis or serious infection. Mean levels of TNF In serum of patients with ARDS and high risk showed a trand toward elevation but wera not significantly higher than mean serum levaIs In normal SUbJects. Mean levels of TNF In BAL of ARDS patients (242 ± 126 pg/ml) wera significantly higher than in normal subjects (9 ± 5 pg/ml), p < 0.05. Antigenic levels of TNF wera undetectsble in approximately half the patients with ARDS or the high. risk state. Levels of TNF in BAL appearad to be highest In the first day of ARDS. Thera appeared to be no ralatlonshlp between levels of TNF in serum or BAL and subsequent mortality. However, serum levels of TNF wera significantly higher In septic patients than In nonseptlc patients, wheraas this dlfferance was not apparant In BAL. These rasults show that functional and antigenic elevations of TNF ara prasent In BAL and perhaps In serum of patients with ARDS or with the hlgh.rlsk state. AM REV RESPIR DIS 1991; 144:268-271
Methods The patients were selected from the intensive care units and wards of the St. Louis University Hospital and the Medical College of Virginia of Virginia Commonwealth University. All studies were performed after obtaining informed consent, and all studies were approved by the respective institutional review boards of the two institutions.
Definition of ARDS and High Risk The high-risk state for ARDS was defined as one or more of the following predispositions: (1) sepsis confirmed by culture of a known pathogen from the patient's blood; (2) sepsis syndrome defined as one from each of the following groups: Group a, temperature > 38.5° C, white blood cell count> 12 with at least 10070 band forms, known or suspected focus of infection; Group b, blood pressure < 100 mm Hg or requirement for vasopressors for > 2 h, urine output < 30 ml/h, systemic vascular resistance < 800 dyn/cm", or blood lactate> 2 mM; (3) trauma requiring multiple blood transfusions; (4) witnessed aspiration of gastric contents; (5) other, including pneumonia, smoke inhalation, and postsurgical shock. The adult respiratory distress syndrome was defined as bilateral and widespread infiltrates on chest X-ray, an arterial Pa02 to fraction of inspired oxygen (FI02) ratio < 150off positive end-expiratory pressure (PEEP) or < 200 on PEEP, and a pulmonary artery occlusion pressure < 18 mm Hg. Each patient with ARDS had to have at least one of the highrisk predispositions.
Normal control subjects werenonsmoking individuals in generally good health. These volunteers were recruited from the personnel at both participatory institutions. See table 1 for a summary of the study groups.
Performance of Bronchoalveolar Lavage BAL was performed through an endotracheal tube by wedging a 5.2-mm fiberoptic bronchoscope into a subsegmental bronchus. Lavage was performed using 50-ml aliquots of warmed normal saline introduced by a syringe through the bronchoscopic aspiration port. Saline (250 to 300 ml) was infused sequentially, and the return was obtained through the same syringe. All returned volume was then pooled and measured and immediately chilledfor performance of cellcounts and other assays. The material was taken rapidly to the laboratory where a cell count was performed and a differential stain via cytocen-
(Received in originalform August 20, 1990and in revisedform February 28, 1991) 1 From the Department of Internal Medicine, St. Louis University School of Medicine, Missouri, and the Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. 2 Supported by Grants No. HL30572-07 and HL07050-15 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Thomas M. Hyers, M.D., UniversityHospital, Divisionof Pulmonology, 3635 Vista at Grand Boulevard, P. O. Box 15250, St. Louis, MO 63110-0250.
TNF IN ARDS AND SEPSIS
269 TABLE 1
BRONCHOALVEOLAR LAVAGE SUBJECT CHARACTERISTICS
Group
n
Predispositions
Sex
Age
Mortality Rate
ARDS
22
Sepsis = 9 Aspiration = 5 Sepsis + aspiration = 2 Sepsis syndrome = 2 Trauma = 3 Pneumonia = 1
10 F 12 M
44 ± 4
6/22
Sepsis = 5 Aspiration = 8 Sepsis + aspiration = 2 Sepsis syndrome = 2 Pneumonia = 1 Smoke inhalation = 1 Postsurgical shock = 1
8F 12 M
55 ± 5
13 F 9M
29 ± 1
Risk
20
Normal
22
A 600 of sample)/A6oo of cell blank. Sample values were read from the standard curve (0.13 to 16U/well). Units of activity were calibrated to the National Biological Standards Board (World Health Organization) interim TNF standard code 87/650 kindly provided by Dr. Craig Reynolds of the National Cancer Institute, Frederick, MD.
Statistical Analysis Differences between ARDS, risk, and normal TNF levelswere analyzed by a Kruskal-Wallis nonparametric analysis of variance followed by a Dunn's test for differences between groups. When TNF levels in only two groups were compared, a two-tailed Mann-Whitney U test was employed. Agreement between immunologic and functional assays was assessed by Spearman's nonparametric correlation (7).
7/20
Results trifuge was prepared from the whole-lavage fluid. The pooled fluid was then spun at 4 0 C at 400 x g for 15 min for the cell pellet. This supernatant was centrifuged at 80,000 x g for 30 min at 4 0 C to remove the surfactant-rich fraction. The resultant supernatant was concentrated lO-fold on a 5,000 molecular weight cutoff filter (Amicon, Danvers, MA) under nitrogen. The concentrated supernatant was then divided into 2oo-J.11 aliquots and rapidly frozen at -70 0 C. An aliquot was thawed at room temperature for each assay. Lavage samples from patients with ARDS were studied after storage for an average of 1 yr. Lavage samples from high-risk patients and normal subjects were stored for slightly longer periods on the average. There was no evidence by comparing older samples with more recent samples that length of storage time influenced TNF levels or bioactivity. Serum was obtained by drawing whole blood through standard venipuncture technique or through a central line. The serum was decanted from the clotted blood after centrifugation and was frozen in aliquots in the same way as the BAL fluid.
Immunologic Assays Antigenic levels of tumor necrosis factor in serum were measured by a radioimmunoassay (RIA; Genzyme Corporation, Boston, MA) specific for human TNF-a with a detection range of 157to 5,000 pg/rnl, TNF levels in BAL fluid were measured by an ELISA (Genzyme) that specifically quantifies human TNF-a in the range of 12to 800 pg/ml. Spikes
of TNF standard were added to random samples to determine recovery and to rule out interference of inhibitors with an assay.
L929 Bioassay The biologic activity of TNF in BAL fluid was determined by a modification of the method of Flick and Gifford (6). Murine L929 cells (ATCC CCL-I) were grown in 75-cm 2 culture flasks in modified Eagle's medium (MEM; Sigma Chemical Co., St. Louis, MO) with 10070 heat-inactivated fetal bovine serum (Hazelton, Lenexa, KS) and plated when at less than 80070 confluence in 96-well plates at 2 x lQ4 cells per well in the same medium. Plates were incubated overnight and medium decanted and replaced with 100J.11 per well of the same medium supplemented with 2 J.1g/ ml of actinomycin D (Sigma). Dilutions of standard rhu-TNF (Genzyme)or samples were made in the medium with no actinomycin D and filtered through a 0.2-J.1m filter. Standard, sample, or medium alone (100 J.11) was added to each well in triplicate, with 10 J.11 Triton X-loo added to one triplicate as a kill blank. Plates were reincubated for 18to 20 hat 370 C under 5070 CO 2 , and then medium was decanted and plates fixed and stained in 0.2070 crystal violet (Sigma) in 100070 methanol for 20 min. Plates were then washed extensively in deionized water and dried. Absorbance at 600 nm was read on a Bio-TekEL309 microplate reader (Bio-Tek Instruments Inc., Winooski, VT). A kill blank was subtracted from each value and the percentage cytotoxicity calculated as (A 6oo of cell blank [medium only] TABLE 3
TABLE 2
TUMOR NECROSIS FACTOR LEVELS IN BRONCHOALVEOLAR LAVAGE (BY ELISA)
TUMOR NECROSIS FACTOR LEVELS IN SERUM {BY RADIOIMMUNOASSAy}· Diagnosis
n
pglml ± SEM
Range (pg/ml)
ARDS Risk Normal
13 10 10
128 ± 36 102 ± 48 16 ± 16
0-311 0-448 0-164
• These groups are not significantly different.
Diagnosis ARDS* Risk Normal •p
n
pg/ml ± SEM
Range (pg/ml)
22 20 22
242 ± 126 2,817 ± 2,653 9±5
0-2,542 0-53,206 0-63
< 0.05 (ARCS versus normal).
Sepsis and aspiration of gastric contents tended to be the predominant predispositions in the group with ARDS and in the high-risk group. Deaths generally occurred within the first 2 wk and were often related to sepsis and multiple-organ failure. The mortality rate for patients with ARDS (270/0) was lower than has been reported in recent series and probably reflects the selection of only moderately ill patients who would be candidates for BAL. Tumor necrosis factor levelsdetectable by RIA were found in approximately half the ARDS sera and half the sera of patients at risk for ARDS but in only one of 10 sera of normal individuals (table 2). TNF levels in sera of 16 patients who lived (134 ± 35) were not significantly different from those in seven patients who died (76 ± 50). However, serum levels ofTNF in 12patients with sepsis (181 ± 42) were significantly higher than those in 11 patients who had other causes of risk or ARDS (46 ± 33) (p < 0.05). Tumor necrosis factor levels measured by ELISA were significantly elevated in BAL fluids of ARDS patients compared with those of normal control subjects but not compared to those of patients at risk for ARDS (table 3). Eighteen septic patients did not have a significantly higher level than did 24 patients with other causes of either ARDS or the high-risk state. Thirteen patients who died did not have significantly higher TNF levels in lavage fluid than did 29 patients who survived (figure 1). The 17patients in whom aspiration of gastric contents was a cause of ARDS or the risk state had a significantly higher level of TNF in the BAL fluid than did the 25 patients who had not aspirated gastric contents (p < 0.01). Concurrent serum samples were not al-
270
HYERS, TRICOMI, DETTENMEIER, AND FOWLER
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YES
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YES
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NO
YES
NO
DIED
SEPSIS
than 40 pg/ml of TNF by ELISA showed no killing in the L929 assay. The results of random spiking of samples indicated that inhibitors werenot interfering with the assay. When 500 pg/ml spikes of TNF standard were added to six serum samples, an average of 92070 recovery was obtained. When 80 pg/ml spikes of TNF standard were added to five different BAL fluids in the ELISA assay, an average of 107070 of TNF was recovered.
ways available for each patient who underwent lavage. However, in a subgroup of 14 patients who had both BAL and serum TNF levels measured at the same time, the correlation coefficient (Spearman's nonparametric) was -0.26 (not significant). The correlation between TNF levels and macrophage numbers in BAL was poor. The Spearman's nonparametric correlation coefficient was - 0.01 in the ARDS patients and 0.31 in the risk patients. Neither correlation was visually impressive or significant. We also analyzed the TNF levels in BAL as a function of total protein. Analyzing the data this way did not change the significant differences in TNF levelsbetween ARDS patients and normal subjects and between aspiration and nonaspiration patients. Agreement between pg/ml of TNF by ELISA and Vlml by L929 bioassay was assessedin BAL fluids of 18patients with ARDS or at risk for the syndrome (figure 2). A Spearman's nonparametric correlation of 0.65 was obtained (P < 0.01).Eight normal BAL fluids with less
Discussion
These studies show that TNF is frequently found in serum and BAL fluid of patients with ARDS and at high risk for the syndrome. However, many patients with ARDS or the high-risk state did not have detectable levels of TNF, in agreement with previous findings in normal subjects in whom TNF release was not sustained after a single bolus of endotoxin (2). The failure to find TNF elevations in some patients may be explained by_a time effect. For instance, TNF in
•
200
Ol
150
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il
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100
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Fig. 1. Distribution of TNF levels by ELISA in patients with and without aspiration, sepsis, and fatal outcome. The y axis is expressed as a logarithmic scale because of the very large range. Mean pg/ml of TNF ± SEM are as follows: aspiration, 3,354 ± 3,119; noaspiration, 186 ± 112; sepsis, 199 ± 140; no sepsis, 2,420 ± 2,209; died, 4,296 ± 4,072; lived, 201 ± 93. Only aspiration versus no aspiration reached statistical significance by Spearman's nonparametric correlation (p < 0.05).
50
• •• •
•
o +->--tI-------+------t---------1 o 1000 2000 3000 pg/ml TNF by ELISA
Fig. 2. Correlation between functional activity (Ulml of TNF by L929 bioassay, vertical axis) and immunologic activity (pglml of TNF by ELISA, horizontal axis) in 18 patients with ARDS or at high risk (p < 0.05). Standard used in both assays is 20 U/ng.
BAL fluid of patients with ARDS averaged 552 ± 349 pg/ml (n = 8) on Day 1 of the syndrome, 101 ± 69 pg/ml (n = 6) on Day 2, and 38 ± 24 pg/ml (n = 8) on Days 3 to 7. There appeared to be no relationship between blood levelsof TNF and subsequent death in this study. This relationship appeared to hold in previous studies of meningococcal sepsis, childhood infectious purpura, acquired immune deficiency syndrome, and Kawasaki disease (8-11) but has not been confirmed in other studies (12-15). However, the rapid clearance of TNF from blood makes it difficult to draw conclusions about cause-and-effect relationships. It does appear, however, that serum levels of TNF can be persistently elevated in patients with severe, chronic congestive heart failure (16). Finally, it must be emphasized that ARDS is a complicated syndrome whose pathogenesis is multifactorial (17, 18). Isolated studies of blood and bronchoalveolar fluid at one point in time cannot be used alone to unravel the pathogenic sequence. The biologic assay (L929 cell assay) did not always detect the same amount of activity as the immunologic assays, but this finding is in agreement with other studies (19) and is probably due in part to interactions of TNF with other cytokines in the functional assay. However, it is clear that significant levels of functional TNF are present in serum and BAL fluid of patients with sepsis and ARDS. The elevation of TNF in BAL of patients with ARDS appears to be most prominent on Day 1 of the syndrome. References 1. Tracey KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234:470-4. 2. Michie H, Manogue KR, Spriggs DA, et al. Detection of circulating levels of tumor necrosis factor after endotoxin administration. N Engl J Med 1988; 318:1481-6. 3. Marks JD, Marks CB, Luce JM, et al. Plasma tumor necrosis factor in patients with septic shock. Am Rev Respir Dis 1990; 141:94-7. 4. Millar AB, Singer M, Meager A, Foley NM, Johnson NM, Rook GAW. Tumor necrosis factor in bronchopulmonary secretions of patients with adult respiratory distress syndrome. Lancet 1989; 2:712-4. 5. Roberts DJ, Davies JM, Evans CC, Bell M, Mostafa SM. Tumor necrosis factor and adult respiratory distress syndrome. Lancet 1989;2:1043-4. 6. Flick DA t Gifford GE. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods 1984; 68:167-75. 7. Zar JH. Biostatistical analysis, 2nd ed. Englewood Cliffs, NJ: Prentice-Hall, 1984. 8. Waage A, Halstensen A, Espevik I. Associa-
271
TNF IN ARDS AND SEPSIS
tion between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1987; 1:355-7. 9. Giradin E, Grau GE, Dayer J-M, Roux-LombardP, LambertP-H, J-5studygroup. Thmornecrosis factor and interleukin-1 in the serum of children with severeinfectious purpura. N Engl J Med 1988; 319:397-400. 10. Lahdevirta J, Maury CPJ, Teppo A-M, Repo H. Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immune deficiency syndrome. Am J Med 1988; 85:289-91. 11. Furukawa S, Matsubara T, Jujoh K, et al. Peripheral blood monocytes!macrophages and serum tumor necrosis factor in Kawasaki disease. Clin Immunol Immunopathol 1988; 48:247-51.
12. Silva CL, Foss NT. Thmor necrosis factor in leprosy patients. J Infect Dis 1989; 159:787-90. 13. Scuderi P, Sterling KE, Lam KS, et al. Raised serum levels of tumor necrosis factor in parasitic infections. Lancet 1986; 2:1364-5. 14. SaxneT, PalladrinoMAJr, HeinegardD, Talal N, Wollheim FA. Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 1988; 31:1041-5. 15. Maury CP, Teppo A-M. Tumor necrosis factor in the serum of patients with systemic lupus erythematosis. Arthritis Rheum 1989; 32:146-50. 16. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levelsof tumor necrosis factor in severechronic heart failure. N Engl J Med
1990; 323:266-41. 17. Stephenson AH, Lonigro AJ, Hyers TM, Webster RO, Fowler AA. Increased concentrations of leukotrienes in bronchoalveolar lavage fluid of patients with ARDS or at risk for ARDS. Am Rev Respir Dis 1988; 138:714-9. 18. Siler TM, Swierkosz JE, Hyers TM, Fowler AA, Webster RO. Immunoreactive interleukin-1 bronchoalveolar lavage fluid of high-risk patients with the adult respiratory distress syndrome. Exp Lung Res 1989; 15:881-94. 19. Meager A, Leung H, Wooley J. Assays for tumor necrosis factor and related cytokines. J Immunol Methods 1989; 116:1-179.
THOMAS M. HYERS, SALLY M. TRICOMI, PATRICIA A. DETTENMEIER, and ALPHA A. FOWLER
Introduction
In gram-negative bacterial sepsis, tumor necrosis factor (TNF) is thought to be a major mediator of cellular injury after its release from mononuclear cells following exposure to endotoxin (1). Studies in normal individuals indicate that a small bolus of intravenous endotoxin results in a short-lived release and clearance of TNF in the serum over a period of approximately 4 h (2). The appearance of TNF in the serum is followed by somewhat longer lived increases in heart rate, temperature, white blood cell count, and adrenocorticotropic hormone (ACTH) and epinephrine release. It is not clear from human studies how the continuous or intermittent release of endotoxin into the circulation affects blood TNF levels. In pathologic human states relative to the adult respiratory distress syndrome (ARDS), TNF has been reported to be elevated in plasma of patients with septic shock (3), in bronchial secretions of patients with ARDS (4), and in limited bronchoalveolar lavage (BAL) samples of patients with ARDS (5). The studies of plasma samples of patients in septic shock showed that TNF was elevated transiently in patients with either gramnegative or gram-positive infections. The highest plasma elevations of TNF occurred in the first 12h after septic shock wasidentified (3). In the two small studies of patients with ARDS, TNF appeared to be elevated in lavage fluids and bronchial secretions but not in plasma. We studied patients with ARDS and individuals at high risk for ARDS. Half the patients with ARDS had sepsis as the cause of their ARDS, and about a third of the patients at risk also had sepsis. We measured levels of tumor necrosis factor immunologically and functionally in BAL fluid and serum in these patients. Our goal was to determine if elevated levels were associated with the onset of ARDS or clinical sepsis or with subsequent mortality. 268
SUMMARY 1\.Imornecrosis factor (TNF) was measurad antigenlcally and functionally In serum and bronchoalveolar lavage fluid (BAL) of patients with ARDS and those at high risk for ARDS. Of 22 patients with ARDS, 14 had sepsis or serious Infection as the major clinical pradlsposltlon, and 10 of 20 hlgh·rlsk patients had sepsis or serious infection. Mean levels of TNF In serum of patients with ARDS and high risk showed a trand toward elevation but wera not significantly higher than mean serum levaIs In normal SUbJects. Mean levels of TNF In BAL of ARDS patients (242 ± 126 pg/ml) wera significantly higher than in normal subjects (9 ± 5 pg/ml), p < 0.05. Antigenic levels of TNF wera undetectsble in approximately half the patients with ARDS or the high. risk state. Levels of TNF in BAL appearad to be highest In the first day of ARDS. Thera appeared to be no ralatlonshlp between levels of TNF in serum or BAL and subsequent mortality. However, serum levels of TNF wera significantly higher In septic patients than In nonseptlc patients, wheraas this dlfferance was not apparant In BAL. These rasults show that functional and antigenic elevations of TNF ara prasent In BAL and perhaps In serum of patients with ARDS or with the hlgh.rlsk state. AM REV RESPIR DIS 1991; 144:268-271
Methods The patients were selected from the intensive care units and wards of the St. Louis University Hospital and the Medical College of Virginia of Virginia Commonwealth University. All studies were performed after obtaining informed consent, and all studies were approved by the respective institutional review boards of the two institutions.
Definition of ARDS and High Risk The high-risk state for ARDS was defined as one or more of the following predispositions: (1) sepsis confirmed by culture of a known pathogen from the patient's blood; (2) sepsis syndrome defined as one from each of the following groups: Group a, temperature > 38.5° C, white blood cell count> 12 with at least 10070 band forms, known or suspected focus of infection; Group b, blood pressure < 100 mm Hg or requirement for vasopressors for > 2 h, urine output < 30 ml/h, systemic vascular resistance < 800 dyn/cm", or blood lactate> 2 mM; (3) trauma requiring multiple blood transfusions; (4) witnessed aspiration of gastric contents; (5) other, including pneumonia, smoke inhalation, and postsurgical shock. The adult respiratory distress syndrome was defined as bilateral and widespread infiltrates on chest X-ray, an arterial Pa02 to fraction of inspired oxygen (FI02) ratio < 150off positive end-expiratory pressure (PEEP) or < 200 on PEEP, and a pulmonary artery occlusion pressure < 18 mm Hg. Each patient with ARDS had to have at least one of the highrisk predispositions.
Normal control subjects werenonsmoking individuals in generally good health. These volunteers were recruited from the personnel at both participatory institutions. See table 1 for a summary of the study groups.
Performance of Bronchoalveolar Lavage BAL was performed through an endotracheal tube by wedging a 5.2-mm fiberoptic bronchoscope into a subsegmental bronchus. Lavage was performed using 50-ml aliquots of warmed normal saline introduced by a syringe through the bronchoscopic aspiration port. Saline (250 to 300 ml) was infused sequentially, and the return was obtained through the same syringe. All returned volume was then pooled and measured and immediately chilledfor performance of cellcounts and other assays. The material was taken rapidly to the laboratory where a cell count was performed and a differential stain via cytocen-
(Received in originalform August 20, 1990and in revisedform February 28, 1991) 1 From the Department of Internal Medicine, St. Louis University School of Medicine, Missouri, and the Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. 2 Supported by Grants No. HL30572-07 and HL07050-15 from the National Institutes of Health. 3 Correspondence and requests for reprints should be addressed to Thomas M. Hyers, M.D., UniversityHospital, Divisionof Pulmonology, 3635 Vista at Grand Boulevard, P. O. Box 15250, St. Louis, MO 63110-0250.
TNF IN ARDS AND SEPSIS
269 TABLE 1
BRONCHOALVEOLAR LAVAGE SUBJECT CHARACTERISTICS
Group
n
Predispositions
Sex
Age
Mortality Rate
ARDS
22
Sepsis = 9 Aspiration = 5 Sepsis + aspiration = 2 Sepsis syndrome = 2 Trauma = 3 Pneumonia = 1
10 F 12 M
44 ± 4
6/22
Sepsis = 5 Aspiration = 8 Sepsis + aspiration = 2 Sepsis syndrome = 2 Pneumonia = 1 Smoke inhalation = 1 Postsurgical shock = 1
8F 12 M
55 ± 5
13 F 9M
29 ± 1
Risk
20
Normal
22
A 600 of sample)/A6oo of cell blank. Sample values were read from the standard curve (0.13 to 16U/well). Units of activity were calibrated to the National Biological Standards Board (World Health Organization) interim TNF standard code 87/650 kindly provided by Dr. Craig Reynolds of the National Cancer Institute, Frederick, MD.
Statistical Analysis Differences between ARDS, risk, and normal TNF levelswere analyzed by a Kruskal-Wallis nonparametric analysis of variance followed by a Dunn's test for differences between groups. When TNF levels in only two groups were compared, a two-tailed Mann-Whitney U test was employed. Agreement between immunologic and functional assays was assessed by Spearman's nonparametric correlation (7).
7/20
Results trifuge was prepared from the whole-lavage fluid. The pooled fluid was then spun at 4 0 C at 400 x g for 15 min for the cell pellet. This supernatant was centrifuged at 80,000 x g for 30 min at 4 0 C to remove the surfactant-rich fraction. The resultant supernatant was concentrated lO-fold on a 5,000 molecular weight cutoff filter (Amicon, Danvers, MA) under nitrogen. The concentrated supernatant was then divided into 2oo-J.11 aliquots and rapidly frozen at -70 0 C. An aliquot was thawed at room temperature for each assay. Lavage samples from patients with ARDS were studied after storage for an average of 1 yr. Lavage samples from high-risk patients and normal subjects were stored for slightly longer periods on the average. There was no evidence by comparing older samples with more recent samples that length of storage time influenced TNF levels or bioactivity. Serum was obtained by drawing whole blood through standard venipuncture technique or through a central line. The serum was decanted from the clotted blood after centrifugation and was frozen in aliquots in the same way as the BAL fluid.
Immunologic Assays Antigenic levels of tumor necrosis factor in serum were measured by a radioimmunoassay (RIA; Genzyme Corporation, Boston, MA) specific for human TNF-a with a detection range of 157to 5,000 pg/rnl, TNF levels in BAL fluid were measured by an ELISA (Genzyme) that specifically quantifies human TNF-a in the range of 12to 800 pg/ml. Spikes
of TNF standard were added to random samples to determine recovery and to rule out interference of inhibitors with an assay.
L929 Bioassay The biologic activity of TNF in BAL fluid was determined by a modification of the method of Flick and Gifford (6). Murine L929 cells (ATCC CCL-I) were grown in 75-cm 2 culture flasks in modified Eagle's medium (MEM; Sigma Chemical Co., St. Louis, MO) with 10070 heat-inactivated fetal bovine serum (Hazelton, Lenexa, KS) and plated when at less than 80070 confluence in 96-well plates at 2 x lQ4 cells per well in the same medium. Plates were incubated overnight and medium decanted and replaced with 100J.11 per well of the same medium supplemented with 2 J.1g/ ml of actinomycin D (Sigma). Dilutions of standard rhu-TNF (Genzyme)or samples were made in the medium with no actinomycin D and filtered through a 0.2-J.1m filter. Standard, sample, or medium alone (100 J.11) was added to each well in triplicate, with 10 J.11 Triton X-loo added to one triplicate as a kill blank. Plates were reincubated for 18to 20 hat 370 C under 5070 CO 2 , and then medium was decanted and plates fixed and stained in 0.2070 crystal violet (Sigma) in 100070 methanol for 20 min. Plates were then washed extensively in deionized water and dried. Absorbance at 600 nm was read on a Bio-TekEL309 microplate reader (Bio-Tek Instruments Inc., Winooski, VT). A kill blank was subtracted from each value and the percentage cytotoxicity calculated as (A 6oo of cell blank [medium only] TABLE 3
TABLE 2
TUMOR NECROSIS FACTOR LEVELS IN BRONCHOALVEOLAR LAVAGE (BY ELISA)
TUMOR NECROSIS FACTOR LEVELS IN SERUM {BY RADIOIMMUNOASSAy}· Diagnosis
n
pglml ± SEM
Range (pg/ml)
ARDS Risk Normal
13 10 10
128 ± 36 102 ± 48 16 ± 16
0-311 0-448 0-164
• These groups are not significantly different.
Diagnosis ARDS* Risk Normal •p
n
pg/ml ± SEM
Range (pg/ml)
22 20 22
242 ± 126 2,817 ± 2,653 9±5
0-2,542 0-53,206 0-63
< 0.05 (ARCS versus normal).
Sepsis and aspiration of gastric contents tended to be the predominant predispositions in the group with ARDS and in the high-risk group. Deaths generally occurred within the first 2 wk and were often related to sepsis and multiple-organ failure. The mortality rate for patients with ARDS (270/0) was lower than has been reported in recent series and probably reflects the selection of only moderately ill patients who would be candidates for BAL. Tumor necrosis factor levelsdetectable by RIA were found in approximately half the ARDS sera and half the sera of patients at risk for ARDS but in only one of 10 sera of normal individuals (table 2). TNF levels in sera of 16 patients who lived (134 ± 35) were not significantly different from those in seven patients who died (76 ± 50). However, serum levels ofTNF in 12patients with sepsis (181 ± 42) were significantly higher than those in 11 patients who had other causes of risk or ARDS (46 ± 33) (p < 0.05). Tumor necrosis factor levels measured by ELISA were significantly elevated in BAL fluids of ARDS patients compared with those of normal control subjects but not compared to those of patients at risk for ARDS (table 3). Eighteen septic patients did not have a significantly higher level than did 24 patients with other causes of either ARDS or the high-risk state. Thirteen patients who died did not have significantly higher TNF levels in lavage fluid than did 29 patients who survived (figure 1). The 17patients in whom aspiration of gastric contents was a cause of ARDS or the risk state had a significantly higher level of TNF in the BAL fluid than did the 25 patients who had not aspirated gastric contents (p < 0.01). Concurrent serum samples were not al-
270
HYERS, TRICOMI, DETTENMEIER, AND FOWLER
100000
10000
. ..
1000
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than 40 pg/ml of TNF by ELISA showed no killing in the L929 assay. The results of random spiking of samples indicated that inhibitors werenot interfering with the assay. When 500 pg/ml spikes of TNF standard were added to six serum samples, an average of 92070 recovery was obtained. When 80 pg/ml spikes of TNF standard were added to five different BAL fluids in the ELISA assay, an average of 107070 of TNF was recovered.
ways available for each patient who underwent lavage. However, in a subgroup of 14 patients who had both BAL and serum TNF levels measured at the same time, the correlation coefficient (Spearman's nonparametric) was -0.26 (not significant). The correlation between TNF levels and macrophage numbers in BAL was poor. The Spearman's nonparametric correlation coefficient was - 0.01 in the ARDS patients and 0.31 in the risk patients. Neither correlation was visually impressive or significant. We also analyzed the TNF levels in BAL as a function of total protein. Analyzing the data this way did not change the significant differences in TNF levelsbetween ARDS patients and normal subjects and between aspiration and nonaspiration patients. Agreement between pg/ml of TNF by ELISA and Vlml by L929 bioassay was assessedin BAL fluids of 18patients with ARDS or at risk for the syndrome (figure 2). A Spearman's nonparametric correlation of 0.65 was obtained (P < 0.01).Eight normal BAL fluids with less
Discussion
These studies show that TNF is frequently found in serum and BAL fluid of patients with ARDS and at high risk for the syndrome. However, many patients with ARDS or the high-risk state did not have detectable levels of TNF, in agreement with previous findings in normal subjects in whom TNF release was not sustained after a single bolus of endotoxin (2). The failure to find TNF elevations in some patients may be explained by_a time effect. For instance, TNF in
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Fig. 1. Distribution of TNF levels by ELISA in patients with and without aspiration, sepsis, and fatal outcome. The y axis is expressed as a logarithmic scale because of the very large range. Mean pg/ml of TNF ± SEM are as follows: aspiration, 3,354 ± 3,119; noaspiration, 186 ± 112; sepsis, 199 ± 140; no sepsis, 2,420 ± 2,209; died, 4,296 ± 4,072; lived, 201 ± 93. Only aspiration versus no aspiration reached statistical significance by Spearman's nonparametric correlation (p < 0.05).
50
• •• •
•
o +->--tI-------+------t---------1 o 1000 2000 3000 pg/ml TNF by ELISA
Fig. 2. Correlation between functional activity (Ulml of TNF by L929 bioassay, vertical axis) and immunologic activity (pglml of TNF by ELISA, horizontal axis) in 18 patients with ARDS or at high risk (p < 0.05). Standard used in both assays is 20 U/ng.
BAL fluid of patients with ARDS averaged 552 ± 349 pg/ml (n = 8) on Day 1 of the syndrome, 101 ± 69 pg/ml (n = 6) on Day 2, and 38 ± 24 pg/ml (n = 8) on Days 3 to 7. There appeared to be no relationship between blood levelsof TNF and subsequent death in this study. This relationship appeared to hold in previous studies of meningococcal sepsis, childhood infectious purpura, acquired immune deficiency syndrome, and Kawasaki disease (8-11) but has not been confirmed in other studies (12-15). However, the rapid clearance of TNF from blood makes it difficult to draw conclusions about cause-and-effect relationships. It does appear, however, that serum levels of TNF can be persistently elevated in patients with severe, chronic congestive heart failure (16). Finally, it must be emphasized that ARDS is a complicated syndrome whose pathogenesis is multifactorial (17, 18). Isolated studies of blood and bronchoalveolar fluid at one point in time cannot be used alone to unravel the pathogenic sequence. The biologic assay (L929 cell assay) did not always detect the same amount of activity as the immunologic assays, but this finding is in agreement with other studies (19) and is probably due in part to interactions of TNF with other cytokines in the functional assay. However, it is clear that significant levels of functional TNF are present in serum and BAL fluid of patients with sepsis and ARDS. The elevation of TNF in BAL of patients with ARDS appears to be most prominent on Day 1 of the syndrome. References 1. Tracey KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234:470-4. 2. Michie H, Manogue KR, Spriggs DA, et al. Detection of circulating levels of tumor necrosis factor after endotoxin administration. N Engl J Med 1988; 318:1481-6. 3. Marks JD, Marks CB, Luce JM, et al. Plasma tumor necrosis factor in patients with septic shock. Am Rev Respir Dis 1990; 141:94-7. 4. Millar AB, Singer M, Meager A, Foley NM, Johnson NM, Rook GAW. Tumor necrosis factor in bronchopulmonary secretions of patients with adult respiratory distress syndrome. Lancet 1989; 2:712-4. 5. Roberts DJ, Davies JM, Evans CC, Bell M, Mostafa SM. Tumor necrosis factor and adult respiratory distress syndrome. Lancet 1989;2:1043-4. 6. Flick DA t Gifford GE. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods 1984; 68:167-75. 7. Zar JH. Biostatistical analysis, 2nd ed. Englewood Cliffs, NJ: Prentice-Hall, 1984. 8. Waage A, Halstensen A, Espevik I. Associa-
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TNF IN ARDS AND SEPSIS
tion between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1987; 1:355-7. 9. Giradin E, Grau GE, Dayer J-M, Roux-LombardP, LambertP-H, J-5studygroup. Thmornecrosis factor and interleukin-1 in the serum of children with severeinfectious purpura. N Engl J Med 1988; 319:397-400. 10. Lahdevirta J, Maury CPJ, Teppo A-M, Repo H. Elevated levels of circulating cachectin/tumor necrosis factor in patients with acquired immune deficiency syndrome. Am J Med 1988; 85:289-91. 11. Furukawa S, Matsubara T, Jujoh K, et al. Peripheral blood monocytes!macrophages and serum tumor necrosis factor in Kawasaki disease. Clin Immunol Immunopathol 1988; 48:247-51.
12. Silva CL, Foss NT. Thmor necrosis factor in leprosy patients. J Infect Dis 1989; 159:787-90. 13. Scuderi P, Sterling KE, Lam KS, et al. Raised serum levels of tumor necrosis factor in parasitic infections. Lancet 1986; 2:1364-5. 14. SaxneT, PalladrinoMAJr, HeinegardD, Talal N, Wollheim FA. Detection of tumor necrosis factor alpha but not tumor necrosis factor beta in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 1988; 31:1041-5. 15. Maury CP, Teppo A-M. Tumor necrosis factor in the serum of patients with systemic lupus erythematosis. Arthritis Rheum 1989; 32:146-50. 16. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levelsof tumor necrosis factor in severechronic heart failure. N Engl J Med
1990; 323:266-41. 17. Stephenson AH, Lonigro AJ, Hyers TM, Webster RO, Fowler AA. Increased concentrations of leukotrienes in bronchoalveolar lavage fluid of patients with ARDS or at risk for ARDS. Am Rev Respir Dis 1988; 138:714-9. 18. Siler TM, Swierkosz JE, Hyers TM, Fowler AA, Webster RO. Immunoreactive interleukin-1 bronchoalveolar lavage fluid of high-risk patients with the adult respiratory distress syndrome. Exp Lung Res 1989; 15:881-94. 19. Meager A, Leung H, Wooley J. Assays for tumor necrosis factor and related cytokines. J Immunol Methods 1989; 116:1-179.
