Subpopulation of Hyperresponsive Polymorphonuclear Neutrophils in Patients with Adult Respiratory Distress Syndrome Role of Cytokine Production1-3
SYLVIE CHOLLET-MARTIN, PHILIPPE MONTRAVERS, CLAUDE GIBERT, CAROLE ELBIM, JEAN MARIE DESMONTS, JEAN YVES FAGON, and MARIE ANNE GOUGEROT-POCIDALO
Introduction
Adult respiratory distress syndrome (ARDS) is a term used to describe the consequences of diffuse, acute parenchymal lung injury that results in increased permeability pulmonary edema, severe hypoxemia, and mechanical abnormalities. Although the pathogenesis of ARDS is undoubtedly complex, and poorly understood, several observations point to an important role of polymorphonuclear neutrophils (PMN) (1, 2). First, the vast majority of the cells recovered by bronchoalveolar lavage (BAL) in most patients with ARDS are PMN (3). Second, PMN secretoryproducts such as elastase, collagenase, and myeloperoxidase can be detected in BAL fluid (3, 4). Finally, it has been reported that circulating PMN from patients with ARDS are activated, as assessed by their chemiluminescence response and superoxide anion production (5, 6). By contrast, impaired alveolar neutrophil function has recently been described by Martin and coworkers (7), and the syndrome can occur in patients with neutropenia (1, 8). The results of several recent investigations have suggested that certain cytokines - interleukin 1/3 (IL-l/3), tumor necrosis factor-a (TNFa), and interleukin 6 (Il-6)-play a critical role in the pathogenesis of ARDS because these humoral substances have powerful effects on PMN function and endothelial integrity (9). For example, BAL fluid from patients with ARDS contains high levels of IL-l/3 (10), which has been attributed to increased release by alveolar macrophages (11). In addition, TNFa has been implicated in the pathogenesis of experimental acute lung injury (12, 13),and high levels have been found in both bronchopulmonary secretions (14) and BAL 990
SUMMARY Togain further Insight Into the pathogenesis of the edult respiratory distress syndrome (ARDS), we studied possible relationships among the activation sIBtus of clrculBtlng polymor. phonuclear neutrophlls (PMN), cytoklne levals, and the sevarlty of lung Injury In 31 petlenIB: 15 with ARDS, nine with sevare pneumonia uncomplicated by ARDS, and sevan mechanically vantllated with neither ARDS nor pneumonia. Nine healthy sUbjectB served as controls. Using flow cytometry, we Identified e subpopulBtlon of PMNwith an Increased capacity to generate hydrogen peroxide after stimulation ex "/"0 In all three patient groups; significantly higher values were found In those with ARDS. The PMN stimulation Index, a reflection of the degree of hyperresponslveness, correlat· ed with elevated levals of tumor necrosis factor-a (TNFa) In plasma, and both spontaneoua and llpopolyseccharlde-Induced TNFa production by cultured monocytes. These biologic expresslona of PMNactivation and cytoklne generation both correlated with Indices of the sevarlty of lung Injury, but not with the ovarall cllnlcelsevarlty. In contrast, IL-6 and 1l.-113 showed little or no relationship with either the degree of lung Injury or PMN hyperresponslvaness. Weconclude that TNFa-primed PMN msy play a major role In the pathogenesis of ARDS-assoclated lung Injury. AM REV RESPIR DIS 1992; 148:990-lIt8
fluid (15, 16) from patients with ARDS. More recently,Marks and coworkers (17) and Roten and colleagues (18) found elevated TNFa plasma levels in both patients with ARDS and those at risk for ARDS because of septic shock. To our knowledge, IL-6levels have not been investigated in ARDS, but high concentrations have been found in the blood of patients with sepsis (19). To gain further insight into the pathogenesis of ARDS, we measured (1) hydrogen peroxide (H 202 ) production by resting and phorbol myristate acetate (PMA)-stimulated PMN, using a flow cytometric assay that permits the study of single cells and, therefore, the identification of subpopulations; (2) plasma levels of TNFa, IL-l/3, and IL-6, together with the production of these cytokines by cultured monocytes, both unstimulated and stimulated with lipopolysaccharide (LPS). We studied three well-characterized groups of mechanically ventilated patients: patients with ARDS with and without pneumonia, patients with-
out ARDS but with pneumonia, and patients with neither ARDS nor pneumonia. Healthy subjects served as controls. Methods Patients and Control Subjects The 31 mechanically ventilated patients enrolled in this study were recruited from the medical and surgical intensivecare units (leU) of the Hopital Bichat. Informed consent was
(Receivedin originalform February 24, 1992 and in revisedform May 19, 1992) 1 From the Laboratoire d'Immunologie et INSERM U294, the Departement d'Anesthesie Reanimation Chirurgicale, and the Service de Reanimation Medicale, Faculte Xavier Bichat, Paris Cedex, France. 2 Presented in part at the Annual Meeting of the American Thoracic Society, Anaheim, CA, May 1991. 3 Correspondence and requests for reprints should be addressed to Pro M. A. GougerotPocidalo, Laboratoire d'Immunologie et d'Hematologie, CHU Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France.
991
CHOLLET-MARTIN, MONTRAVERS, GIBERT, ELBIM, DESMONTS, FAGON, AND GOUGEROT·POCIDALO
obtained from each of the patients or their closest relative. All procedures were in strict accordance with the ethical standards of the committee for the protection of human subjects at our institution. Fifteen patients had ARDS, eight without pneumonia (Group la) and seven with pneumonia (Group lb), nine patients had pneumonia without ARDS (Group 2), and seven patients had neither ARDS nor pneumonia (Group 3). Diagnosis was made according to criteria described below. Nine healthy volunteers served as control subjects. All the patients were studied within 3 days of the onset of ARDS or pneumonia, and they were assigned to the various groups before the results of the study were known. ARDS was defined using the lung injury score described by Murray and associates (20), which includes the extent of roentgenographic densities, the severity of hypoxemia, and the level of positive end-expiratory pressure. The 15patients with a score of 2.5 or more during the 2 days before or after blood sampling for ex vivo testing were defined as having ARDS and were included in Group 1; all had SwanGanz catheters inserted to assist in their clinical management; pulmonary capillary wedge pressure was always < 18 mm Hg. All patients included in Groups 1 and 2 underwent fiberoptic bronchoscopy during the 2 days before or the same day as blood sampling. The diagnosis of lung infection was based on clinical findings and the results of protected brush specimen cultures, as follows: fever> 38.3 0 C, presence of a new and persistent (more than 24-h) infiltrate on chest roentgenograms, presence of macroscopically purulent tracheal aspirates, and growth of ~ 103 cfu/ml in protected brush specimens (21). Patients who did not meet the criteria for pneumonia and/or ARDS were excluded. Group 3 patients had been admitted to the ICU for a variety of reasons, had been ventilated for more than 2 days, had a lung injury score
SYLVIE CHOLLET-MARTIN, PHILIPPE MONTRAVERS, CLAUDE GIBERT, CAROLE ELBIM, JEAN MARIE DESMONTS, JEAN YVES FAGON, and MARIE ANNE GOUGEROT-POCIDALO
Introduction
Adult respiratory distress syndrome (ARDS) is a term used to describe the consequences of diffuse, acute parenchymal lung injury that results in increased permeability pulmonary edema, severe hypoxemia, and mechanical abnormalities. Although the pathogenesis of ARDS is undoubtedly complex, and poorly understood, several observations point to an important role of polymorphonuclear neutrophils (PMN) (1, 2). First, the vast majority of the cells recovered by bronchoalveolar lavage (BAL) in most patients with ARDS are PMN (3). Second, PMN secretoryproducts such as elastase, collagenase, and myeloperoxidase can be detected in BAL fluid (3, 4). Finally, it has been reported that circulating PMN from patients with ARDS are activated, as assessed by their chemiluminescence response and superoxide anion production (5, 6). By contrast, impaired alveolar neutrophil function has recently been described by Martin and coworkers (7), and the syndrome can occur in patients with neutropenia (1, 8). The results of several recent investigations have suggested that certain cytokines - interleukin 1/3 (IL-l/3), tumor necrosis factor-a (TNFa), and interleukin 6 (Il-6)-play a critical role in the pathogenesis of ARDS because these humoral substances have powerful effects on PMN function and endothelial integrity (9). For example, BAL fluid from patients with ARDS contains high levels of IL-l/3 (10), which has been attributed to increased release by alveolar macrophages (11). In addition, TNFa has been implicated in the pathogenesis of experimental acute lung injury (12, 13),and high levels have been found in both bronchopulmonary secretions (14) and BAL 990
SUMMARY Togain further Insight Into the pathogenesis of the edult respiratory distress syndrome (ARDS), we studied possible relationships among the activation sIBtus of clrculBtlng polymor. phonuclear neutrophlls (PMN), cytoklne levals, and the sevarlty of lung Injury In 31 petlenIB: 15 with ARDS, nine with sevare pneumonia uncomplicated by ARDS, and sevan mechanically vantllated with neither ARDS nor pneumonia. Nine healthy sUbjectB served as controls. Using flow cytometry, we Identified e subpopulBtlon of PMNwith an Increased capacity to generate hydrogen peroxide after stimulation ex "/"0 In all three patient groups; significantly higher values were found In those with ARDS. The PMN stimulation Index, a reflection of the degree of hyperresponslveness, correlat· ed with elevated levals of tumor necrosis factor-a (TNFa) In plasma, and both spontaneoua and llpopolyseccharlde-Induced TNFa production by cultured monocytes. These biologic expresslona of PMNactivation and cytoklne generation both correlated with Indices of the sevarlty of lung Injury, but not with the ovarall cllnlcelsevarlty. In contrast, IL-6 and 1l.-113 showed little or no relationship with either the degree of lung Injury or PMN hyperresponslvaness. Weconclude that TNFa-primed PMN msy play a major role In the pathogenesis of ARDS-assoclated lung Injury. AM REV RESPIR DIS 1992; 148:990-lIt8
fluid (15, 16) from patients with ARDS. More recently,Marks and coworkers (17) and Roten and colleagues (18) found elevated TNFa plasma levels in both patients with ARDS and those at risk for ARDS because of septic shock. To our knowledge, IL-6levels have not been investigated in ARDS, but high concentrations have been found in the blood of patients with sepsis (19). To gain further insight into the pathogenesis of ARDS, we measured (1) hydrogen peroxide (H 202 ) production by resting and phorbol myristate acetate (PMA)-stimulated PMN, using a flow cytometric assay that permits the study of single cells and, therefore, the identification of subpopulations; (2) plasma levels of TNFa, IL-l/3, and IL-6, together with the production of these cytokines by cultured monocytes, both unstimulated and stimulated with lipopolysaccharide (LPS). We studied three well-characterized groups of mechanically ventilated patients: patients with ARDS with and without pneumonia, patients with-
out ARDS but with pneumonia, and patients with neither ARDS nor pneumonia. Healthy subjects served as controls. Methods Patients and Control Subjects The 31 mechanically ventilated patients enrolled in this study were recruited from the medical and surgical intensivecare units (leU) of the Hopital Bichat. Informed consent was
(Receivedin originalform February 24, 1992 and in revisedform May 19, 1992) 1 From the Laboratoire d'Immunologie et INSERM U294, the Departement d'Anesthesie Reanimation Chirurgicale, and the Service de Reanimation Medicale, Faculte Xavier Bichat, Paris Cedex, France. 2 Presented in part at the Annual Meeting of the American Thoracic Society, Anaheim, CA, May 1991. 3 Correspondence and requests for reprints should be addressed to Pro M. A. GougerotPocidalo, Laboratoire d'Immunologie et d'Hematologie, CHU Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France.
991
CHOLLET-MARTIN, MONTRAVERS, GIBERT, ELBIM, DESMONTS, FAGON, AND GOUGEROT·POCIDALO
obtained from each of the patients or their closest relative. All procedures were in strict accordance with the ethical standards of the committee for the protection of human subjects at our institution. Fifteen patients had ARDS, eight without pneumonia (Group la) and seven with pneumonia (Group lb), nine patients had pneumonia without ARDS (Group 2), and seven patients had neither ARDS nor pneumonia (Group 3). Diagnosis was made according to criteria described below. Nine healthy volunteers served as control subjects. All the patients were studied within 3 days of the onset of ARDS or pneumonia, and they were assigned to the various groups before the results of the study were known. ARDS was defined using the lung injury score described by Murray and associates (20), which includes the extent of roentgenographic densities, the severity of hypoxemia, and the level of positive end-expiratory pressure. The 15patients with a score of 2.5 or more during the 2 days before or after blood sampling for ex vivo testing were defined as having ARDS and were included in Group 1; all had SwanGanz catheters inserted to assist in their clinical management; pulmonary capillary wedge pressure was always < 18 mm Hg. All patients included in Groups 1 and 2 underwent fiberoptic bronchoscopy during the 2 days before or the same day as blood sampling. The diagnosis of lung infection was based on clinical findings and the results of protected brush specimen cultures, as follows: fever> 38.3 0 C, presence of a new and persistent (more than 24-h) infiltrate on chest roentgenograms, presence of macroscopically purulent tracheal aspirates, and growth of ~ 103 cfu/ml in protected brush specimens (21). Patients who did not meet the criteria for pneumonia and/or ARDS were excluded. Group 3 patients had been admitted to the ICU for a variety of reasons, had been ventilated for more than 2 days, had a lung injury score
