JOURNAL
OF SURGICAL
RESEARCH
(1991)
60,362-367
Increased PMN CD1 1 b/CD1 8 Expression following Post-traumatic ARDS H. HANK Division
of Surgical
Presented
Research,
at the Annual
Rhode Meeting
Island
SIMMS, Hospital,
of the Association
M.D., AND R. D’AMICO, Brown
University
School
for Academic
Surgery,
B.S.
of Medicine, Houston,
Providence, Texas,
Rhode
November
Island
14-17,
02903 1990
part by the surface expression of the receptor CDllb/ CD18 (CR3) [g-lo]. In this study, the components of this hypothesis were dissecting by assessing the state of activation of circulating pulmonary artery PMN before and after the clinical recognition of ARDS. Second, we performed longitudinal studies on patients at risk for the development of ARDS to determine if changes in PMN function might precede the development of ARDS. To investigate the ability of PMN to adhere to pulmonary artery endothelial cells and subsequently release reactive oxygen species, we measured the number of CDllb/CDlE) receptors per PMN, the production of MTT-Formazan, intracellular H,O, production, and superoxide anion production by circulating pulmonary artery PMN. Our results suggest that post-traumatic ARDS is associated both with an increased number of CDllb/CD18 receptors on the PMN cell surface and an upregulation of the oxidative burst. Second, both the numbers of CDllb/CD18 receptors and MTT-Formazan production increased in six of seven patients prior to the clinical recognition of ARDS. These results suggest that alterations in circulating artery PMN may provide markers for the incipient development of ARDS in the critically ill traumatized patient.
We investigated the role of polymorphonuclear leukocytes (PMN) in the pathogenesis of post-traumatic adult respiratory distress syndrome (ARDS). Two groups of patients were studied: Group I (n = 29) represents trauma patients studied within 24 hr of admission to the SICU, and Group II (n = 10) represents a subset of Group I patients who subsequently developed ARDS. Circulating pulmonary artery PMN were then assayed for CDllb/CD18 expression, MTT-Formazan production, intracellular H,Oz production, and superoxide anion release. PMN from Group II patients were upregulated with regard to all of the PMN functions assayed within 24 hr of the diagnosis of ARDS being made. Subsequently, longitudinal assays were performed on 17 patients at risk for the development of ARDS. In 6 of 7 patients prior to the clinical recognition of ARDS, CDllb/CD18 expression and MTT-Formazan production increased significantly over baseline. These results suggest that: (i) ARDS coincides with increased CDllb/CDlS expression on PMN cell surfaces, (ii) PMN oxidative metabolism increases at the onset of ARDS, and (iii) changes in circulating pulmonary artery PMN may provide markers for the development of ARDS in the traumatized patient. o 1991 Academic PWS, I~C.
INTRODUCTION
The adult respiratory distress syndrome (ARDS) remains a common and potentially lethal problem in modern ICU patient care. While a number of disease processeshave been associated with ARDS, the exact etiology of the syndrome remains unknown [l-5]. Among several potential theories, there is increasing evidence that activated polymorphonuclear neutrophils (PMN) may play a role in the pathogenesis of ARDS [6-71. Specifically, adherence of PMN to pulmonary artery endothelial cells followed by release of reactive oxygen species and subsequent endothelial cell basement membrane damage may be the trigger that induces a leaky capillary state in the pulmonary circulation [8]. PMN adherence to endothelial cells is mediated in large 0022-4804/91$1.50 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
362 Inc. reserved.
MATERIALS
AND
METHODS
Reagents and Buffers Hanks’ balanced salt solution with and without Ca2+ and Mg2+ ( HBSS2+, HBSS2-) and RPM1 1640 containing L-glutamine were obtained from GIBCO Laboratories (Grand Island, NY). Reagents were obtained from the following sources: Lymphocyte separation medium, Litton Bionetics, Kensington, Maryland; C5a, MTTFormazan, ferricytochrome c, FMLP, superoxide dismutase, Sigma Chemical Co., St. Louis, Missouri; Dextran T500, Pharmacia Fine Chemicals, Uppsala, Sweden; and 2-ethylhexylphthalate and dibutyl phthalate
SIMMS
were purchased New York.
AND
D’AMICO:
from Eastman-Kodak
INCREASED
PMN
Co., Rochester,
Study Population Trauma patients admitted to the Surgical Intensive Care Unit at the Rhode Island Hospital were considered for study. Group I (n = 29) was composed of trauma patients studied on admission to SICU. The mean ISS score of these patients was 27.2 + 2.3. Group II (n = 10) represented a subset of Group I patients who subsequently developed ARDS. The mean ISS score of these patients was 30.6 f 2.9. ARDS was defined as the presence of all of the following: hypoexemia (P,O, sat < 90%) requiring F,O, > 0.5 with PEEP 2 5, Q,/Q, 2 15%, decreased static and dynamic compliance, PCWP < 15, and bilateral interstitial infiltrates seen in chest X-ray. The mean length of stay until the development of ARDS was 5.2 + 0.8 days and all Group II patients were studied within 24 hr of the diagnosis of ARDS. At the time the diagnosis of ARDS was made, invasive bacterial infection had not yet been documented in any of these patients. A second series of trauma patients (n = 17) was studied prospectively to determine if alterations in circulating pulmonary artery PMN function would precede the clinical development of ARDS. Of these 17 patients, 7 developed ARDS and the mean ISS score of these patients was 29.8 * 3.0. The mean length of stay until the development of ARDS in these patients was 4.9 + 0.8 days. Patients were studied every other day until discharge from the ICU, removal of the Swan-Ganz catheter, or the development of ARDS. Phagocyte
Preparation
Correct placement of the Swan-Ganz catheter was confirmed by chest X-ray and appropriate pulmonary artery pressure wave forms. Whole blood (15 cc) was then withdrawn from the PA port of the Swan-Ganz catheter. PMN were separated from venous blood samples by Ficoll-Hypaque density centrifugation and dextran sedimentation [12]. The erythrocytes were removed by two hydroponic saline lysis steps. The remaining PMN were washed two additional times and resuspended in RPM1 or HBSS2- depending upon the particular assay (vide infra). The Ficoll-Hypaque media was found to be free of endotoxin using a chromogenic substrate in the Limulus amebocyte lysate assay. The limit of endotoxin detection in this assay was 10 pg/ml (Kit No. QCL-1000, MA-Bioproducts, Walkerville, MD). Monoclonal
Antibodies
Anti-Macl, which recognizes the CDllb/CD18 surface receptor, was provided by Dr. Michael Frank (LCI, NIAID, NIH). Anti-Mac1 was radiolabeled using the Io-
FOLLOWING
POST-TRAUMATIC
dobead iodination procedure of the Mab was 1.25 &i/pg. Binding
ARDS
[13]. The specific
363 activity
of Iz51Anti-Mac1
For “‘1 anti-Mac1 binding assays, PMN at 20 X 106/ ml were prepared in HBSS2-. One hundred microliters of cells was added to 12 X 75-mm polypropylene tubes, followed by the addition of 1251anti-Macl. In parallel, 1251anti-Mac1 was added in the presence of 50- to lOOfold excess of unlabeled anti-Macl. The cells were incubated for 60 min at 0°C and then pelleted by centrifuging through an oil mixture consisting of one part bis-2-(ethylhexyl)phthalate to three parts dibutyl phthalate. Bound anti-Mac1 was determined from the level of radioactivity in the cell pellets and specific binding was calculated by subtracting 1251anti-Mac1 bound in the presence of 50- to loo-fold excess unlabeled anti-Macl. PMN MTT-Formazan
Production
The utilization of oxygen by PMN was measured using the production of MTT-Formazan [ 14-161. PMN at concentrations ranging from 1 to 8 X 106/ml were adhered for 60 min at 37°C in flat-bottom 96-well microtiter plates. MTT in DMSO was then added to the PMN and the OD at 550 mm was recorded after 60 min at room temperature. The MTT-Formazan produced was calculated based upon known standards and the results were recorded as nanomoles MTT-Formazan produced/ PMN/GO min. PMN Oxidative Response The production of intracellular H,O, by both baseline and stimulated PMN was assessedby the formation of intracellular 2’,7’-dichlorofluorescein [ 171. PMN (1 X lo7 ml) in calcium- and magnesium-free GHBSS were loaded by incubation with 2.5 PM 2’,7’-dichlorofluorescein diacetate (DCF-DA; Eastman-Kodak) for 15 min at 37°C then washed, and resuspended in 1 ml GHBSS. Aliquots of 100 ~1 of PMN were then incubated with buffer or FMLP (final concentration 5 X 10e7M) for 30 min at 37°C. The reaction was stopped by addition of 200 ~1 ice-cold PBS, and intracellular fluorescence was determined by flow cytometry. Results are expressed as the fold increase in fluorescence over Group I PMN in buffer alone. Superoxide Anion Production The amount of superoxide anion generated was assayed by measurement of the reduction of ferricytochrome c [18]. Assays were performed in 96-well flatbottom plates (Costar, Cambridge, MA). One hundred microliters of cytochrome c, with and without C5a at 5 X lo-‘M, was added to each chamber and 5 ~1 of superoxide dismutase (60,000 U/ml) was added to control chambers. After the addition of 100 ~1 of PMN at 5
364
JOURNAL
PATIENT
PATIENT
OF
SURGICAL
RESEARCH:
GROUP
VOL.
50, NO.
4, APRIL
1991
CDllb/CD18 expression increased from 3.32 f 0.58 X lo3 to 8.62 f 1.08 X lo3 in Groups I and II, respectively (P < 0.001 comparing Group I vs Group II.) The nanomoles MTT-Formazan/PMN increased from 1.42 f 0.31 to 3.04 + 0.26 in Groups I and II, respectively (data shown for PMN at 8 X 106; P < 0.001 comparing Group I vs Group II). Finally, both intracellular H,O, and superoxide anion production increased by both baseline and stimulated PMN. Fluorescence obtained from Group I PMN in buffer (baseline H,O, production-mean channel fluorescence 68.98) was arbitrarily assigned a value of 1.0. PMN from Group II patients in buffer demonstrated a 2.5-fold increase in fluorescence over Group I PMN in buffer (P < 0.001 comparing Group I vs Group II in buffer). While FMLP induced an increase in fluorescence for both groups of patients, a 2.45-fold and a 4.92fold increase in fluorescence over baseline was seen in Groups I and II, respectively (P = 0.028 comparing Group I vs Group II for H,O, production with FMLP). Superoxide anion production (nM/PMN/hr) from baseline PMN was 0.52 + 0.06 and 0.89 k 0.09 for Groups I and II, respectively. Superoxide anion production from
GROUP
FIG. 1. (Top) PMN at 20 X 106/ml were incubated with “‘1 antiMac1 in the presence of unlabeled anti-Macl. Results represent the number of CDllb/CDlS per PMN in patient Groups I and II. The number of CDllb/CD18 per PMN increased significantly between patient groups. *P < 0.001 comparing Group I vs Group II. (Bottom) PMN at 8 x 106/ml were incubated with MTT in DMSO for 60 min and the nM MTT-Formazan produced/PMN/hr was recorded. The nM MTT Formazan produced/PMN/hr increased significantly between patient groups. *P < 0.001 comparing Group I vs Group II. 2
1
X 105/ml, the plate was incubated for 60 min at 37°C in a 5% CO, incubator. Optical density was assayed at 550 mm on a dual beam microplate reader (Model MR600; Dynatech Laboratories). Production of superoxide anion was determined using the molar extinction coefficient of cytochrome c (6.3 with a light path of 3 mm).
PATIENT
GROUP
PATIENT
GROUP
Statistical Analysis Experiments conducted between Group I and Group II patients were analyzed by the unpaired t test. Differences over time in the group of patients studied longitudinally were analyzed using the one-way ANOVA with the Newman-Keuls test. A P value < 0.05 was considered statistically significant. RESULTS
ARDS Is Associated with PMN
Upregulation
The results of experiments designed to assessthe state of activation of pulmonary artery PMN at the time the diagnosis of ARDS was made are shown in Figs. 1 and 2.
2
1
FIG. 2. (Top) PMN at 1 X lO’/ml were incubated with 2.5 pM 2’,7’-dichlorofluorescein diacetate and the production of intracellular H,O, was measured. Comparing Group I vs Group II, PMN incubated in buffer or with FMLP demonstrated increased intracellular H,O, production. (*P < 0.001 comparing Group I vs Group II for PMN in buffer; +P = 0.028 comparing Group I vs Group II for PMN incubated with FMLP). (Bottom) PMN at 5 x 105/ml were incubated with or without C5a in the presence of ferricytochrome c and superoxide anion production was measured. Comparing Group I vs Group II, PMN incubated in buffer or with C5a demonstrated increased superoxide anion production. (*P < 0.001 comparing Group I vs Group II for bufferor C5a-stimulated PMN).
SIMMS
AND
D’AMICO:
INCREASED
PMN
FOLLOWING
POST-TRAUMATIC
TABLE
365
ARDS
1
Increased CDllb/CDlS Expression and MTT-Formazan Production Precedes the Development of ARDS Control
I
I ::‘I
0
.
’ 1
”
2
”
3
.
” 4
’ 5
I 6
I 7
DAYS
FIG. 3. The seven patients who developed ARDS are each represented by a separate symbol over Days l-7. CDllb/CDlS expression increased on six of seven patients prior to the clinical recognition of ARDS (all patients except that patient represented by the solid triangles). The mean length of time until the onset of ARDS is demonstrated by the vertical bar on Day 4.8. CDllb/DClS expression in patients who did not develop ARDS is shown by the open triangles. *P < 0.01 comparing patients with ARDS-open triangles vs the six patients who did develop ARDS.
C5a (final concentration 5 x lo-’ M) stimulated PMN was 1.08 +- 0.09 and 2.10 +- 0.04 for Groups I and II, respectively (P < 0.01 comparing Group I vs Group II for superoxide anion production). Longitudinal
(x103)
3
ARDS-Day
3.5 + 0.35
5.7 f 0.46*
8.3 + 0.61
1.45 r?r: 0.07
2.5 f 0.18.l
3.8 f 0.24t
6
Note. Control, patients who did not develop ARDS. * P < 0.003 comparing control vs ARDS-Day 3; ARDS-Day 3 vs ARDS-Day 6. t P = 0.02 comparing ARDS-Day 3 vs control; P < 0.001 comparing ARDS-Day 3 vs ARDS-Day 6.
prior to the development of ARDS. Significant differences between patients with or without ARDS with respect to these two PMN functions appeared by Hospital Day 3 and these data are summarized in Table 1. Intracellular H,O, production and superoxide anion production increased significantly in only two of seven patients prior to the clinical recognition of ARDS (data not shown).
Studies
The results of experiments designed to determine if changes in circulating pulmonary artery PMN would precede the clinical diagnosis of ARDS are shown in Figs. 3 and 4. CDllb/CD18 expression increased significantly over baseline in six of seven patients 24-48 hr prior to the clinical recognition of ARDS. Concomitantly, MTT-Formazan production increased significantly over baseline in six of seven patients 24-48 hr
F
CDllb/CD18 MTT-Formazan production (nM/PMN/hr)
ARDS-Day
I 11~1~~~u~u~4~r~~~’ 0 1 2 3 4
I 5
6
7
8
DAYS
FIG. 4. The seven patients who developed ARDS are each represented by a separate symbol over Days l-7. The nM MTT-Formazan/ PMN increased in six of seven patients prior to the clinical recognition of ARDS (all patients except that patient represented by the solid triangles). The mean length of time until the onset of ARDS is demonstrated by the vertical bar on Day 4.8. MTT-Formazan production in patients who did not develop ARDS is shown by the open triangles. *P < 0.01 comparing patients without ARDS-open triangles vs the six patients who did develop ARDS.
DISCUSSION
Polymorphonuclear neutrophils are a unique component of the immune system largely because of their capacity to markedly increase oxidative metabolism and intracellular bacteriacidal activity. There is increasing evidence, however, that active oxidative metabolism may be deleterious. Although the presence of PMN is not absolutely required for the development of ARDS [19-201, recent experimental evidence has implicated neutrophils as an important element in the pathogenesis of ARDS. Increased concentrations of PMN have been found in the bronchoalveolar lavage fluid of patients with ARDS [21-221. Furthermore, increased concentrations of neutrophil collagenase and myeloperoxidase activity have been found to correlate with lung permeability changes in patients with ARDS [23]. Neutrophil elastase has been shown to augment acute edematous lung injury [24] and neutrophil leukotriene B, precedes the development of pulmonary failure and has been implicated as a potential marker in the development of ARDS [25]. Although the exact etiology of ARDS remains unclear, it is apparent that a pulmonary permeability defect is present. This permeability defect is not limited to the lungs and in fact ARDS may represent a particular manifestation of a generalized permeability defect [26]. While complement activation has been shown to play a role in the subsequent upregulation of PMN function,
366
JOURNAL
OF
SURGICAL
RESEARCH:
complement activation per se does not appear to be a sufficient stimulus to cause ARDS [27-301. Upregulation of PMN function has been seen during early periods of sepsis and is manifested by increased phagocytosis and elevated intracellular pH [31]. Various authors have demonstrated that PMN are capable of binding to pulmonary endothelial cells [32] primarily via ICAM-1, a surface receptor expressed by pulmonary endothelial cells. Subsequent release of reactive oxygen species in close proximity to endothelial cells may then damage these cells and induce a leaky capillary state. We have attempted to analyze the components of this hypothesis by measuring the PMN surface receptor expression of CDllb/CD18 (CR3) which is the primary adhesion molecule on the PMN cell surface and various aspects of the PMN oxidative burst. Our results suggest that post-traumatic ARDS is associated with an increased number of CDllb/CD18 on the PMN cell surface. While this result does not prove that enhanced PMN adhesiveness to endothelial cells is occurring during ARDS, it does provide experimental evidence that upregulation of pulmonary artery PMN with regard to potential adhesiveness has occurred. Furthermore, there is a marked upregulation of various components of the PMN oxidative burst following posttraumatic ARDS. These changes were seen with both intracellular (H202) and extracellular (superoxide anion) oxidative products. Kew et al. have proposed that elevated C-reactive protein found in patients with ARDS might help downregulate PMN migration and be a marker for patients with ARDS [33]. Their results, however, looked at C-reactive protein levels after the diagnosis of ARDS had been made. While C-reactive protein may help downregulate PMN migration, it was of interest to determine if changes in PMN function might precede the clinical recognition of ARDS. The results of our longitudinal studies suggest that increased CDllb/CD18 expression and MTT-Formazan production may provide markers for the incipient development of ARDS. Whether this will correlate with specific causes of ARDS or improve clinical outcome remains unclear. In conclusion, post-traumatic ARDS is associated with an upregulation of circulating pulmonary artery PMN and changes in these PMN may provide markers for the subsequent development of ARDS.
VOL.
2.
Anonymous.
and Mason, Adult
respiratory
G. R. An end to “ARDS.” distress
syndrome.
Chest Lancet
89:
3.
Bernard, G. R., and Brigham, K. L. Pulmonary edema. physiologic mechanisms and new approaches to therapy. 89: 594,1986.
J. J., Pohl, D. F., Randall, C. B., et al. Incidence, site, and outcome of infections in patients with the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 134: 12, 1986.
6. Vercollotti,
G. M., Yin, H. Q., Gustafson, K. S., Nelson, R. D., and Jacob, H. S. Platelet-activating factor primes neutrophil responses to agonists: Role in promoting neutrophil-mediated endothelial damage. Blood 71: 1100, 1988.
7. McDonald, derived monary perfused
R. J., Berger, E. M., and Repine, J. E. Neutrophiloxygen metabolites stimulate thromboxane release, pulartery pressure increases, and weight gains in isolated rat lungs. Am. Reu. Respir. Dis. 135: 957, 1987.
8. Anderson,
9.
B. O., Brown, J. M., Bensard, D. D., Grosso, M. A., Banerjee, A., Patt, A., Whitman, G. J. R., and Harken, A. H. Reversible lung neutrophil accumulation can cause lung injury by elastase-mediated mechanisms. Surgery lOS(2): 262, 1990. Harlan, J. M., Killen, P. D., Harker, L. A., et al. Neutrophil-mediated endothelial injury in vitro. J. Clin. Znuest. 68: 1394,198l.
10.
Malech, H. L., and Gallin, J. I. Neutrophils Engl. J. Med. 317: 687, 1987.
11.
Kohl, S., Springer, T. A., Schmalstieg, F. C., et al. Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-l/ OKM-1 deficiency. J. Zmmunol. 133: 2972,1984.
12.
Boyum, human
13. 14.
A. Isolation of mononuclear blood. J. Clin. Lab. Znuest.
in human
disease.
cells and granulocytes
2l(Suppl.):
N.
from
77, 1968.
Markwell, M. A. K. A new method of protein iodination. Anal. Biochem. 125: 427-432, 1968. Rex, J. H., Bennett, J. E., Gallin, J. I., Malech, H. L., and Melnick, D. A. Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J. Infect. Dis. 162: 523, 1990.
15.
Baehner, R. C., and Nathan, D. G. Quantitative nitroblue lium test in CGD. NET Med. 278: 971, 1968.
16.
Auniri, P. W., Heck, K. L., Browne, C., et al. Stimulated nitroblue tetrazolium test to assess neutrophil antibacterial function prediction of wound sepsis in burned patients. Surgery 74: 6, 1973. Bass, D. A., Parce, J. W., Dechatelet, L. R., Szejda, P., Seeds, M. C., and Thomas, M. Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J. Zmmurwl. 130(4): 1910, 1983. Solomkin, J. S., Cotter, L. A., Brodt, J. K., and Hurst, J. M. Regulation of neutrophil superoxide production in sepsis. Arch. Surg. 120: 93, 1985. Maunder, R. J., Hackmann, R. C., Riff, E., Albert, R. K., and Springmeyer, S. C. Occurrence of the adult respiratory distress syndrome in neutropenic patients. Am. Rev. Respir. Dis. 133: 313, 1986. Heffner, J. E., Sahn, S. A., and Repine, J. E. The role of platelets in the adult respiratory distress syndrome. Culprits or bystanders? Am. Rev. Respir. Dis. 135: 482, 1987. Modig, J., and Hallgren, R. Pathophysiologic significance of lung granulocytes in human adult respiratory distress syndrome induced by septic or traumatic shock. Acta Chir. Stand. 153: 267, 1987. Thommasen, H. V. The role of the polymorphonuclear leukocyte in the pathogenesis of the adult respiratory distress syndrome. Clin. Znuest. Med. 8(2): 185, 1985. Weiland, J. E., Davis, W. B., Holtar, J. F., Mohammed, J. R., Dorinsky, P. M., and Gadek, J. E. Lung neutrophils in the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 133: 218, 1986.
17.
18.
19.
20.
22. 23.
PathoChest
K. N., and Prognosis
5. Siedenfeld,
1: 301,
1986.
1991
A. A., Hamman, R. F., Zerbe, G. O., Benson, Hyers, T. M. Adult respiratory distress syndrome. after onset. Am. Rev. Respir. Dis. 132: 472, 1985.
REFERENCES Effros, R. M., 162,1986.
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4. Fowler,
21.
1.
50, NO.
tetrazo-
SIMMS 24.
25.
26.
27.
28.
AND
D’AMICO:
INCREASED
PMN
FOLLOWING
POST-TRAUMATIC
ARDS
367
McDonald, R. J., Bruckner, L. V., and Repine, J. E. Neutrophil elastase augments acute edematous injury in isolated rat lungs perfused with neutrophil cytoplasts. Am. Reu. Respir. Dis. 140: 1825,1989. Davis, M. D., Meyers, J. D., Barie, P. S., Yurt, R. W., Duhaney, R., Dineen, P., and Shires, T. Elevated production of neutrophil leukotriene B, precedes pulmonary failure in critically ill surgical patients. Surg. Gynecol. O&et. 170: 495, 1990.
29.
Tennenberg, S. D., Jacobs, M. P., and Solomkin, J. S. Complement-mediated neutrophil activation in sepsis- and trauma-related adult respiratory distress syndrome. Arch. Surg. 122: 26, 1987.
30.
Kreuzfelder, E., Joka, T., Keinecke, H. O., Obertacke, U., Schmit-Neuerburg, K. P., Nakhosteen, J. A., Paar, D., and Scheiermann, N. Adult respiratory distress syndrome a specific manifestation of a general permeability defect in trauma patients. Am. Rev. Respir. Dis. 137: 95, 1988.
31.
Stevens, J. H., O’Hanley, P., Shapiro, J. M., Mihm, F. P. S., Collins, J. A., and Raffin, T. A. Effects of anti-C5a ies on the adult respiratory distress syndrome in septic J. Clin. Znuest. 77: 1812, 1986. Rothe, G., Kellermann, W., andvalet, G. Flow cytometric eters of neutrophil function as early indicators of trauma-related pulmonary or cardiovascular organ
Duchateau, M., Haas, M., Schreyen, H., Radoux, L., Sprangers, I., Noel, F. X., Braun, M., and Lamy, M. Complement activation in patients at risk of developing the adult respiratory distress syndrome. Am. Reu. Respir. Dis. 130: 1058, 1984. Hosea, S., Brown, E., Hammer, C., and Frank, M. Role of complement activation in a model of adult respiratory distress syndrome. J. Clin. Inuest. 66: 375, 1980.
Lab. Clin. Med. 115(l): 32.
33.
G., Satoh, antibodprimates. paramsepsis- or failure. J.
52, 1990.
Schleimer, R. P., and Rutledge, B. K. Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin and tumor-promoting phorbol diesters. J. Immunol. 136: 649, 1986. Kew, R. R., Hyers, T. M., and Webster, R. 0. Human C-reactive protein inhibits neutrophil chemotaxis in vitro: Possible implications for the adult respiratory distress syndrome. J. Lab. Clin. Med. 115(3): 339, 1990.
OF SURGICAL
RESEARCH
(1991)
60,362-367
Increased PMN CD1 1 b/CD1 8 Expression following Post-traumatic ARDS H. HANK Division
of Surgical
Presented
Research,
at the Annual
Rhode Meeting
Island
SIMMS, Hospital,
of the Association
M.D., AND R. D’AMICO, Brown
University
School
for Academic
Surgery,
B.S.
of Medicine, Houston,
Providence, Texas,
Rhode
November
Island
14-17,
02903 1990
part by the surface expression of the receptor CDllb/ CD18 (CR3) [g-lo]. In this study, the components of this hypothesis were dissecting by assessing the state of activation of circulating pulmonary artery PMN before and after the clinical recognition of ARDS. Second, we performed longitudinal studies on patients at risk for the development of ARDS to determine if changes in PMN function might precede the development of ARDS. To investigate the ability of PMN to adhere to pulmonary artery endothelial cells and subsequently release reactive oxygen species, we measured the number of CDllb/CDlE) receptors per PMN, the production of MTT-Formazan, intracellular H,O, production, and superoxide anion production by circulating pulmonary artery PMN. Our results suggest that post-traumatic ARDS is associated both with an increased number of CDllb/CD18 receptors on the PMN cell surface and an upregulation of the oxidative burst. Second, both the numbers of CDllb/CD18 receptors and MTT-Formazan production increased in six of seven patients prior to the clinical recognition of ARDS. These results suggest that alterations in circulating artery PMN may provide markers for the incipient development of ARDS in the critically ill traumatized patient.
We investigated the role of polymorphonuclear leukocytes (PMN) in the pathogenesis of post-traumatic adult respiratory distress syndrome (ARDS). Two groups of patients were studied: Group I (n = 29) represents trauma patients studied within 24 hr of admission to the SICU, and Group II (n = 10) represents a subset of Group I patients who subsequently developed ARDS. Circulating pulmonary artery PMN were then assayed for CDllb/CD18 expression, MTT-Formazan production, intracellular H,Oz production, and superoxide anion release. PMN from Group II patients were upregulated with regard to all of the PMN functions assayed within 24 hr of the diagnosis of ARDS being made. Subsequently, longitudinal assays were performed on 17 patients at risk for the development of ARDS. In 6 of 7 patients prior to the clinical recognition of ARDS, CDllb/CD18 expression and MTT-Formazan production increased significantly over baseline. These results suggest that: (i) ARDS coincides with increased CDllb/CDlS expression on PMN cell surfaces, (ii) PMN oxidative metabolism increases at the onset of ARDS, and (iii) changes in circulating pulmonary artery PMN may provide markers for the development of ARDS in the traumatized patient. o 1991 Academic PWS, I~C.
INTRODUCTION
The adult respiratory distress syndrome (ARDS) remains a common and potentially lethal problem in modern ICU patient care. While a number of disease processeshave been associated with ARDS, the exact etiology of the syndrome remains unknown [l-5]. Among several potential theories, there is increasing evidence that activated polymorphonuclear neutrophils (PMN) may play a role in the pathogenesis of ARDS [6-71. Specifically, adherence of PMN to pulmonary artery endothelial cells followed by release of reactive oxygen species and subsequent endothelial cell basement membrane damage may be the trigger that induces a leaky capillary state in the pulmonary circulation [8]. PMN adherence to endothelial cells is mediated in large 0022-4804/91$1.50 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
362 Inc. reserved.
MATERIALS
AND
METHODS
Reagents and Buffers Hanks’ balanced salt solution with and without Ca2+ and Mg2+ ( HBSS2+, HBSS2-) and RPM1 1640 containing L-glutamine were obtained from GIBCO Laboratories (Grand Island, NY). Reagents were obtained from the following sources: Lymphocyte separation medium, Litton Bionetics, Kensington, Maryland; C5a, MTTFormazan, ferricytochrome c, FMLP, superoxide dismutase, Sigma Chemical Co., St. Louis, Missouri; Dextran T500, Pharmacia Fine Chemicals, Uppsala, Sweden; and 2-ethylhexylphthalate and dibutyl phthalate
SIMMS
were purchased New York.
AND
D’AMICO:
from Eastman-Kodak
INCREASED
PMN
Co., Rochester,
Study Population Trauma patients admitted to the Surgical Intensive Care Unit at the Rhode Island Hospital were considered for study. Group I (n = 29) was composed of trauma patients studied on admission to SICU. The mean ISS score of these patients was 27.2 + 2.3. Group II (n = 10) represented a subset of Group I patients who subsequently developed ARDS. The mean ISS score of these patients was 30.6 f 2.9. ARDS was defined as the presence of all of the following: hypoexemia (P,O, sat < 90%) requiring F,O, > 0.5 with PEEP 2 5, Q,/Q, 2 15%, decreased static and dynamic compliance, PCWP < 15, and bilateral interstitial infiltrates seen in chest X-ray. The mean length of stay until the development of ARDS was 5.2 + 0.8 days and all Group II patients were studied within 24 hr of the diagnosis of ARDS. At the time the diagnosis of ARDS was made, invasive bacterial infection had not yet been documented in any of these patients. A second series of trauma patients (n = 17) was studied prospectively to determine if alterations in circulating pulmonary artery PMN function would precede the clinical development of ARDS. Of these 17 patients, 7 developed ARDS and the mean ISS score of these patients was 29.8 * 3.0. The mean length of stay until the development of ARDS in these patients was 4.9 + 0.8 days. Patients were studied every other day until discharge from the ICU, removal of the Swan-Ganz catheter, or the development of ARDS. Phagocyte
Preparation
Correct placement of the Swan-Ganz catheter was confirmed by chest X-ray and appropriate pulmonary artery pressure wave forms. Whole blood (15 cc) was then withdrawn from the PA port of the Swan-Ganz catheter. PMN were separated from venous blood samples by Ficoll-Hypaque density centrifugation and dextran sedimentation [12]. The erythrocytes were removed by two hydroponic saline lysis steps. The remaining PMN were washed two additional times and resuspended in RPM1 or HBSS2- depending upon the particular assay (vide infra). The Ficoll-Hypaque media was found to be free of endotoxin using a chromogenic substrate in the Limulus amebocyte lysate assay. The limit of endotoxin detection in this assay was 10 pg/ml (Kit No. QCL-1000, MA-Bioproducts, Walkerville, MD). Monoclonal
Antibodies
Anti-Macl, which recognizes the CDllb/CD18 surface receptor, was provided by Dr. Michael Frank (LCI, NIAID, NIH). Anti-Mac1 was radiolabeled using the Io-
FOLLOWING
POST-TRAUMATIC
dobead iodination procedure of the Mab was 1.25 &i/pg. Binding
ARDS
[13]. The specific
363 activity
of Iz51Anti-Mac1
For “‘1 anti-Mac1 binding assays, PMN at 20 X 106/ ml were prepared in HBSS2-. One hundred microliters of cells was added to 12 X 75-mm polypropylene tubes, followed by the addition of 1251anti-Macl. In parallel, 1251anti-Mac1 was added in the presence of 50- to lOOfold excess of unlabeled anti-Macl. The cells were incubated for 60 min at 0°C and then pelleted by centrifuging through an oil mixture consisting of one part bis-2-(ethylhexyl)phthalate to three parts dibutyl phthalate. Bound anti-Mac1 was determined from the level of radioactivity in the cell pellets and specific binding was calculated by subtracting 1251anti-Mac1 bound in the presence of 50- to loo-fold excess unlabeled anti-Macl. PMN MTT-Formazan
Production
The utilization of oxygen by PMN was measured using the production of MTT-Formazan [ 14-161. PMN at concentrations ranging from 1 to 8 X 106/ml were adhered for 60 min at 37°C in flat-bottom 96-well microtiter plates. MTT in DMSO was then added to the PMN and the OD at 550 mm was recorded after 60 min at room temperature. The MTT-Formazan produced was calculated based upon known standards and the results were recorded as nanomoles MTT-Formazan produced/ PMN/GO min. PMN Oxidative Response The production of intracellular H,O, by both baseline and stimulated PMN was assessedby the formation of intracellular 2’,7’-dichlorofluorescein [ 171. PMN (1 X lo7 ml) in calcium- and magnesium-free GHBSS were loaded by incubation with 2.5 PM 2’,7’-dichlorofluorescein diacetate (DCF-DA; Eastman-Kodak) for 15 min at 37°C then washed, and resuspended in 1 ml GHBSS. Aliquots of 100 ~1 of PMN were then incubated with buffer or FMLP (final concentration 5 X 10e7M) for 30 min at 37°C. The reaction was stopped by addition of 200 ~1 ice-cold PBS, and intracellular fluorescence was determined by flow cytometry. Results are expressed as the fold increase in fluorescence over Group I PMN in buffer alone. Superoxide Anion Production The amount of superoxide anion generated was assayed by measurement of the reduction of ferricytochrome c [18]. Assays were performed in 96-well flatbottom plates (Costar, Cambridge, MA). One hundred microliters of cytochrome c, with and without C5a at 5 X lo-‘M, was added to each chamber and 5 ~1 of superoxide dismutase (60,000 U/ml) was added to control chambers. After the addition of 100 ~1 of PMN at 5
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CDllb/CD18 expression increased from 3.32 f 0.58 X lo3 to 8.62 f 1.08 X lo3 in Groups I and II, respectively (P < 0.001 comparing Group I vs Group II.) The nanomoles MTT-Formazan/PMN increased from 1.42 f 0.31 to 3.04 + 0.26 in Groups I and II, respectively (data shown for PMN at 8 X 106; P < 0.001 comparing Group I vs Group II). Finally, both intracellular H,O, and superoxide anion production increased by both baseline and stimulated PMN. Fluorescence obtained from Group I PMN in buffer (baseline H,O, production-mean channel fluorescence 68.98) was arbitrarily assigned a value of 1.0. PMN from Group II patients in buffer demonstrated a 2.5-fold increase in fluorescence over Group I PMN in buffer (P < 0.001 comparing Group I vs Group II in buffer). While FMLP induced an increase in fluorescence for both groups of patients, a 2.45-fold and a 4.92fold increase in fluorescence over baseline was seen in Groups I and II, respectively (P = 0.028 comparing Group I vs Group II for H,O, production with FMLP). Superoxide anion production (nM/PMN/hr) from baseline PMN was 0.52 + 0.06 and 0.89 k 0.09 for Groups I and II, respectively. Superoxide anion production from
GROUP
FIG. 1. (Top) PMN at 20 X 106/ml were incubated with “‘1 antiMac1 in the presence of unlabeled anti-Macl. Results represent the number of CDllb/CDlS per PMN in patient Groups I and II. The number of CDllb/CD18 per PMN increased significantly between patient groups. *P < 0.001 comparing Group I vs Group II. (Bottom) PMN at 8 x 106/ml were incubated with MTT in DMSO for 60 min and the nM MTT-Formazan produced/PMN/hr was recorded. The nM MTT Formazan produced/PMN/hr increased significantly between patient groups. *P < 0.001 comparing Group I vs Group II. 2
1
X 105/ml, the plate was incubated for 60 min at 37°C in a 5% CO, incubator. Optical density was assayed at 550 mm on a dual beam microplate reader (Model MR600; Dynatech Laboratories). Production of superoxide anion was determined using the molar extinction coefficient of cytochrome c (6.3 with a light path of 3 mm).
PATIENT
GROUP
PATIENT
GROUP
Statistical Analysis Experiments conducted between Group I and Group II patients were analyzed by the unpaired t test. Differences over time in the group of patients studied longitudinally were analyzed using the one-way ANOVA with the Newman-Keuls test. A P value < 0.05 was considered statistically significant. RESULTS
ARDS Is Associated with PMN
Upregulation
The results of experiments designed to assessthe state of activation of pulmonary artery PMN at the time the diagnosis of ARDS was made are shown in Figs. 1 and 2.
2
1
FIG. 2. (Top) PMN at 1 X lO’/ml were incubated with 2.5 pM 2’,7’-dichlorofluorescein diacetate and the production of intracellular H,O, was measured. Comparing Group I vs Group II, PMN incubated in buffer or with FMLP demonstrated increased intracellular H,O, production. (*P < 0.001 comparing Group I vs Group II for PMN in buffer; +P = 0.028 comparing Group I vs Group II for PMN incubated with FMLP). (Bottom) PMN at 5 x 105/ml were incubated with or without C5a in the presence of ferricytochrome c and superoxide anion production was measured. Comparing Group I vs Group II, PMN incubated in buffer or with C5a demonstrated increased superoxide anion production. (*P < 0.001 comparing Group I vs Group II for bufferor C5a-stimulated PMN).
SIMMS
AND
D’AMICO:
INCREASED
PMN
FOLLOWING
POST-TRAUMATIC
TABLE
365
ARDS
1
Increased CDllb/CDlS Expression and MTT-Formazan Production Precedes the Development of ARDS Control
I
I ::‘I
0
.
’ 1
”
2
”
3
.
” 4
’ 5
I 6
I 7
DAYS
FIG. 3. The seven patients who developed ARDS are each represented by a separate symbol over Days l-7. CDllb/CDlS expression increased on six of seven patients prior to the clinical recognition of ARDS (all patients except that patient represented by the solid triangles). The mean length of time until the onset of ARDS is demonstrated by the vertical bar on Day 4.8. CDllb/DClS expression in patients who did not develop ARDS is shown by the open triangles. *P < 0.01 comparing patients with ARDS-open triangles vs the six patients who did develop ARDS.
C5a (final concentration 5 x lo-’ M) stimulated PMN was 1.08 +- 0.09 and 2.10 +- 0.04 for Groups I and II, respectively (P < 0.01 comparing Group I vs Group II for superoxide anion production). Longitudinal
(x103)
3
ARDS-Day
3.5 + 0.35
5.7 f 0.46*
8.3 + 0.61
1.45 r?r: 0.07
2.5 f 0.18.l
3.8 f 0.24t
6
Note. Control, patients who did not develop ARDS. * P < 0.003 comparing control vs ARDS-Day 3; ARDS-Day 3 vs ARDS-Day 6. t P = 0.02 comparing ARDS-Day 3 vs control; P < 0.001 comparing ARDS-Day 3 vs ARDS-Day 6.
prior to the development of ARDS. Significant differences between patients with or without ARDS with respect to these two PMN functions appeared by Hospital Day 3 and these data are summarized in Table 1. Intracellular H,O, production and superoxide anion production increased significantly in only two of seven patients prior to the clinical recognition of ARDS (data not shown).
Studies
The results of experiments designed to determine if changes in circulating pulmonary artery PMN would precede the clinical diagnosis of ARDS are shown in Figs. 3 and 4. CDllb/CD18 expression increased significantly over baseline in six of seven patients 24-48 hr prior to the clinical recognition of ARDS. Concomitantly, MTT-Formazan production increased significantly over baseline in six of seven patients 24-48 hr
F
CDllb/CD18 MTT-Formazan production (nM/PMN/hr)
ARDS-Day
I 11~1~~~u~u~4~r~~~’ 0 1 2 3 4
I 5
6
7
8
DAYS
FIG. 4. The seven patients who developed ARDS are each represented by a separate symbol over Days l-7. The nM MTT-Formazan/ PMN increased in six of seven patients prior to the clinical recognition of ARDS (all patients except that patient represented by the solid triangles). The mean length of time until the onset of ARDS is demonstrated by the vertical bar on Day 4.8. MTT-Formazan production in patients who did not develop ARDS is shown by the open triangles. *P < 0.01 comparing patients without ARDS-open triangles vs the six patients who did develop ARDS.
DISCUSSION
Polymorphonuclear neutrophils are a unique component of the immune system largely because of their capacity to markedly increase oxidative metabolism and intracellular bacteriacidal activity. There is increasing evidence, however, that active oxidative metabolism may be deleterious. Although the presence of PMN is not absolutely required for the development of ARDS [19-201, recent experimental evidence has implicated neutrophils as an important element in the pathogenesis of ARDS. Increased concentrations of PMN have been found in the bronchoalveolar lavage fluid of patients with ARDS [21-221. Furthermore, increased concentrations of neutrophil collagenase and myeloperoxidase activity have been found to correlate with lung permeability changes in patients with ARDS [23]. Neutrophil elastase has been shown to augment acute edematous lung injury [24] and neutrophil leukotriene B, precedes the development of pulmonary failure and has been implicated as a potential marker in the development of ARDS [25]. Although the exact etiology of ARDS remains unclear, it is apparent that a pulmonary permeability defect is present. This permeability defect is not limited to the lungs and in fact ARDS may represent a particular manifestation of a generalized permeability defect [26]. While complement activation has been shown to play a role in the subsequent upregulation of PMN function,
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complement activation per se does not appear to be a sufficient stimulus to cause ARDS [27-301. Upregulation of PMN function has been seen during early periods of sepsis and is manifested by increased phagocytosis and elevated intracellular pH [31]. Various authors have demonstrated that PMN are capable of binding to pulmonary endothelial cells [32] primarily via ICAM-1, a surface receptor expressed by pulmonary endothelial cells. Subsequent release of reactive oxygen species in close proximity to endothelial cells may then damage these cells and induce a leaky capillary state. We have attempted to analyze the components of this hypothesis by measuring the PMN surface receptor expression of CDllb/CD18 (CR3) which is the primary adhesion molecule on the PMN cell surface and various aspects of the PMN oxidative burst. Our results suggest that post-traumatic ARDS is associated with an increased number of CDllb/CD18 on the PMN cell surface. While this result does not prove that enhanced PMN adhesiveness to endothelial cells is occurring during ARDS, it does provide experimental evidence that upregulation of pulmonary artery PMN with regard to potential adhesiveness has occurred. Furthermore, there is a marked upregulation of various components of the PMN oxidative burst following posttraumatic ARDS. These changes were seen with both intracellular (H202) and extracellular (superoxide anion) oxidative products. Kew et al. have proposed that elevated C-reactive protein found in patients with ARDS might help downregulate PMN migration and be a marker for patients with ARDS [33]. Their results, however, looked at C-reactive protein levels after the diagnosis of ARDS had been made. While C-reactive protein may help downregulate PMN migration, it was of interest to determine if changes in PMN function might precede the clinical recognition of ARDS. The results of our longitudinal studies suggest that increased CDllb/CD18 expression and MTT-Formazan production may provide markers for the incipient development of ARDS. Whether this will correlate with specific causes of ARDS or improve clinical outcome remains unclear. In conclusion, post-traumatic ARDS is associated with an upregulation of circulating pulmonary artery PMN and changes in these PMN may provide markers for the subsequent development of ARDS.
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2.
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