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• W2031519092 abstract "Sepsis syndrome frequently results in endothelial injury in many organ systems. To evaluate neutrophil-pulmonary endothelial cell interaction in the sepsis syndrome, we studied 39 critically ill patients prospectively and 20 normal volunteers. Thirteen patients with sepsis (mean age, 71.4 years), 14 patients in an intensive care unit control group (mean age 65.4 years), and 12 patients admitted with acute myocardial infarction (mean age, 66.8 years) were evaluated. Blood samples were drawn from septic patients within 24 hours and from ICU and MI patients within 72 hours of admission. All sepsis patients were culture positive, 6 of 13 from the blood. Both renal failure and ARDS developed in 54 percent of septic patients. 51Cr-labelled neutrophils were prepared and added to bovine pulmonary endothelial cell monolayers with and without added phorbol myristate acetate. Endothelial cells with adherent PMA and nonadherent PMN's, were harvested and radioactivity in each fraction measured with a gamma scintillation counter. Baseline and maximally stimulated (PMA, 3.0 ng/ml) neutrophil adherence to endothelial cells were similar in all patient groups. However, in septic patients, PMA-stimulated PMN adherence was reduced at lower doses, most significantly in those who developed ARDS within 24 to 48 hours of admission (p<0.05). Seventy-one percent of patients who developed ARDS had reduced stimulated adherence (PMA 1.0 ng/ml) compared to 22 percent of critically ill patients who did not. We conclude that diminished adherence of neutrophils to endothelium in response to low-level PMA stimulation is significantly more common in patients with sepsis who develop ARDS. Our findings suggest that PMN-endothelial cell interaction is altered by the time sepsis is clinically recognized but before the development of ARDS. We speculate that the observed reduction in adherence of the PMN to endothelial cells may be a consequence of down-regulation by mediators generated in the inflammatory response to sepsis and/or the need for active participation of septic endothelium in this interaction. Sepsis syndrome frequently results in endothelial injury in many organ systems. To evaluate neutrophil-pulmonary endothelial cell interaction in the sepsis syndrome, we studied 39 critically ill patients prospectively and 20 normal volunteers. Thirteen patients with sepsis (mean age, 71.4 years), 14 patients in an intensive care unit control group (mean age 65.4 years), and 12 patients admitted with acute myocardial infarction (mean age, 66.8 years) were evaluated. Blood samples were drawn from septic patients within 24 hours and from ICU and MI patients within 72 hours of admission. All sepsis patients were culture positive, 6 of 13 from the blood. Both renal failure and ARDS developed in 54 percent of septic patients. 51Cr-labelled neutrophils were prepared and added to bovine pulmonary endothelial cell monolayers with and without added phorbol myristate acetate. Endothelial cells with adherent PMA and nonadherent PMN's, were harvested and radioactivity in each fraction measured with a gamma scintillation counter. Baseline and maximally stimulated (PMA, 3.0 ng/ml) neutrophil adherence to endothelial cells were similar in all patient groups. However, in septic patients, PMA-stimulated PMN adherence was reduced at lower doses, most significantly in those who developed ARDS within 24 to 48 hours of admission (p<0.05). Seventy-one percent of patients who developed ARDS had reduced stimulated adherence (PMA 1.0 ng/ml) compared to 22 percent of critically ill patients who did not. We conclude that diminished adherence of neutrophils to endothelium in response to low-level PMA stimulation is significantly more common in patients with sepsis who develop ARDS. Our findings suggest that PMN-endothelial cell interaction is altered by the time sepsis is clinically recognized but before the development of ARDS. We speculate that the observed reduction in adherence of the PMN to endothelial cells may be a consequence of down-regulation by mediators generated in the inflammatory response to sepsis and/or the need for active participation of septic endothelium in this interaction. phorbol myristate acetate sepsis and ARDS sepsis without ARDS Sepsis syndrome1Bone RC Fisher CJ Clemmer TP Slotman GJ Metz CA Balk RA The methylprednisolone severe sepsis study group: sepsis syndrome—a valid clinical entity.Crit Care Med. 1989; 17: 389-393Crossref PubMed Scopus (604) Google Scholar is a profound systemic physiologic and inflammatory response to infection. This response characteristically includes fever or hypothermia, tachycardia, and tachypnea, as well as dysfunction of multiple organ systems. The adult respiratory distress syndrome (ARDS) is often the most severe, and sometimes the only, organ failure resulting from sepsis. Recent hypotheses have suggested that ARDS results from diffuse endothelial cell injury mediated in part by neutrophils. Leukocyte chemotaxis and adherence to endothelial cells lining small blood vessels is essential for the initiation of an inflammatory response, a response usually thought to be beneficial to host defense. However, studies in animals2Shasby DM Vanbenthuysen KM Tate RM Shasby SS Mc-Murtry I Repine JE Granulocytes mediate acute endomatous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals.Am Rev Respir Dis. 1982; 125: 443-447PubMed Google Scholar and in vitro3Shasby DM Shasby SS Peach MJ Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs: role of oxygen radicals and granulocyte adherence.Am Rev Respir Dis. 1983; 127: 72-76Crossref PubMed Scopus (99) Google Scholar,4Smedly LA Tonnesen MG Sandhaus RA Haslett C Guthrie LA Johnston Jr, RB et al.Neutrophil-mediated injury to endothelial cells: enhancement by endotoxin and essential role of neutrophil elastase.J Clin Invest. 1986; 77: 1233-1243Crossref PubMed Scopus (480) Google Scholar have indicated that this polymorphonuclear (PMN) leukocyte-endothelial cell interaction, under some conditions, can result in endothelial injury from the combined effects of reactive oxygen species and proteolytic enzymes released at the site of adherence. Haslett, Worthen, and colleagues5Haslett C Worthen GS Giclas PC Morrison DC Henson JE Henson PM The pulmonary vascular sequestration of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit.Am Rev Respir Dis. 1987; 136: 9-18Crossref PubMed Scopus (130) Google Scholar,6Worthen GS Haslett C Rees AJ Gumbay RS Henson JE Henson PM Neutrophil-mediated pulmonary vascular injury: synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung.Am Rev Respir Dis. 1987; 136: 19-28Crossref PubMed Scopus (198) Google Scholar have demonstrated increased pulmonary sequestration of radioactively labeled PM Ns in rabbits treated with endotoxin, a response that could be enhanced by chemotactic factors. Moreover, Warshawski et al7Warshawski FJ Sibbald WJ Driedger AA Cheung H Abnormal neutrophil-pulmonary interaction in the adult respiratory distress syndrome: qualitative and quantitative assessment of pulmonary neutrophil kinetics in humans with “in vivo” indium neutrophil scintigraphy.Am Rev Respir Dis. 1986; 133: 797-804PubMed Google Scholar have reported increased localization of 111In-labeled neutrophils in the lungs of septic patients with ARDS compared to those with less severe lung injury. MacGregor8MacGregor RR Granulocyte adherence changes induced by hemodialysis, endotoxin, epinephrine, and glucocorticoids.Ann Intern Med. 1977; 86: 35-39Crossref PubMed Scopus (100) Google Scholar observed that rabbit leukocytes from whole blood became more adherent to nylon fibers for up to four hours in response to endotoxin. However, after 24 hours, the leukocytes were significantly less adherent than those from unexposed animals. In patients with bacteremia, Venezio et al9Venezio FR Westenfelder GO Phair JP The adherence of polymorphonuclear leukocytes in patients with sepsis.J Infect Dis. 1982; 145: 351-357Crossref PubMed Scopus (21) Google Scholar used the same whole blood assay 24 to 36 hours after antibiotic therapy was begun and found PMN adherence to nylon fiber columns to be increased only when shock developed. Zimmerman et al10Zimmerman GA Renzetti AD Hill HR Granulocyte adherence in pulmonary and systemic arterial blood samples from patients with adult respiratory distress syndrome.Am Rev Respir Dis. 1984; 129: 798-804Crossref PubMed Scopus (34) Google Scholar also reported no increase in PMN adherence in a study of patients with ARDS secondary to a variety of underlying diseases. While these studies examined neutrophil adherence to artificial surfaces, no studies to our knowledge have examined PMN-endothelial cell interaction directly with human neutrophils recovered from patients with the sepsis syndrome. The purpose of our study was to examine neutrophil-endothelial cell interaction in the acute phase of human sepsis. We report that while baseline adherence of neutrophils to endothelium was not increased, neutrophils isolated from septic patients demonstrated a marked inability to increase their adherence in response to the nonspecific activator phorbol myristate acetate (PMA), an observation that was most pronounced in those who developed progressive lung injury. We prospectively studied 39 patients admitted to the critical care services of Winthrop-University Hospital during 1988 and 1989 (Table 1). Thirteen patients with the sepsis syndrome were studied, (six men and seven women; mean age, 71.4±3.9 years). Underlying chronic illness was common among these patients and included ischemic heart disease in five, chronic obstructive pulmonary disease (COPD) in two, diabetes in two, and cirrhosis, hypothyroidism, and lymphoma (in remission) in one each. Chronic disease of two organ systems was present in five patients. While none of the patients with the sepsis syndrome had clinically recognizable ARDS upon entry into the study, ARDS developed in three within 24 hours, and in another four within 48 hours. We, therefore, subsequently analyzed data for the septic patients in two categories: those who developed ARDS (SA group) and those who did not (SO group).Septic patients were compared to two other groups of critically ill patients and to a group of 20 normal volunteers (mean age, 30.9±1.6 years). The intensive care unit (ICU) control subjects consisted of a group of 14 patients (eight men and six women, mean age, 65.4±3.7 years). Of these, eight patients had ischemic cardiovascular disease and were admitted for monitoring because of suspected acute myocardial infarction (MI), but this diagnosis was excluded. Four other patients had worsening of preexisting heart failure, and two were admitted for a noninfectious exacerbation of COPD (no infiltrates on chest roentgenogram with negative blood and sputum cultures). A second control group of patients with acute MI was also evaluated. In this group, there were four men and eight women with a mean age 66.8±3.6 years. Nine had inferior MI, one anterior MI, and two non-Q wave infarctions, with a peak creatine phosphokinase level of 1,372±318 units. With one exception, their courses were uncomplicated by heart failure, life-threatening arrhythmias, or cardiac arrest. None of the patients in either the ICU or MI groups experienced other organ failure (liver or kidney). There was one death among the MI patients and two deaths in the ICU group.There were no significant differences among the four patient groups in age and sex distributions. Steroids were used in only three of the 39 patients, and dopamine or dobutamine was used for hemodynamic support in eight of the septic patients. Antibiotic treatment for the septic patients had been given for less than 24 hours. Outcomes evaluated in the patients included development of ARDS (Table 1), renal failure (creatinine >2.0 mg/dl), liver failure (total bilirubin >5.0 mg/dl), and death. Blood samples were drawn within 24 hours from the septic patients and within 72 hours for the other patient groups. The study was approved by the Institutional Research Review Committee of Winthrop-University Hospital, and informed consent was obtained from all patients or from next of kin. Twenty milliliters of citrate-anticoagulated peripheral venous blood was obtained from each study patient. Red blood cells were removed by dextran sedimentation, and neutrophils were then isolated by continuous colloidal silica density gradient centrifugation using a mononuclear separation medium. After isolation, PMNs were washed with Dulbecco's phosphate-buffered saline without calcium and magnesium, containing 1 percent fetal bovine serum. The PMNs were radioactively labeled with 51Cr, 1 pCi/106Worthen GS Haslett C Rees AJ Gumbay RS Henson JE Henson PM Neutrophil-mediated pulmonary vascular injury: synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung.Am Rev Respir Dis. 1987; 136: 19-28Crossref PubMed Scopus (198) Google Scholar cells, for one hour at 37°C as described by Gallin et al.11Gallin JI Clark RA Kimball HR Granulocyte chemotaxis: an improved in vitro assay employing 51Cr-labeled granulocytes.J Immunol. 1973; 110: 233-240PubMed Google Scholar After subsequent washing with PBS/FBS, any remaining red blood cells in the radioactively labeled cell suspension were removed by hypotonic lysis, and the PMNs were then diluted to 2 × 106Worthen GS Haslett C Rees AJ Gumbay RS Henson JE Henson PM Neutrophil-mediated pulmonary vascular injury: synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung.Am Rev Respir Dis. 1987; 136: 19-28Crossref PubMed Scopus (198) Google Scholar cells/ml in minimal essential medium containing 10 percent FBS. Neutrophil preparations exhibiting cell aggregation at any stage of the preparation were not used. The endotoxin level in the FBS used in these experiments was 76±12 pg/ml. This trace level of endotoxin was lower than could be achieved with “low-endotoxin” serum substitutes, does not induce cytokine synthesis by human monocytes, and is one tenth the level of endotoxin which is capable of “priming” PMN response.12Guthrie LA McPhail LC Henson PM Johnston Jr, RB Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide.J Exp Med. 1984; 160: 1656-1671Crossref PubMed Scopus (466) Google Scholar All other reagents were endotoxin free. Bovine pulmonary artery endothelial cells were grown in MEM, alpha modification with 10 percent FBS; penicillin, 200 U/ml; and streptomycin, 200 μg/ml, in 75 cm2 tissue culture flasks. Sixteenthto nineteenth-passage cells were passed with trypsin-EDTA and plated on 4.5 cm2, 12-well plates and grown to confluence for use in the adherence experiments described below. Frozen stock (1 mg/ml in DM SO) of phorbol 12-myristate 13-acetate was used to prepare a working stock of 100 ng/ml in MEM. Aliquots were then added to wells of endothelial cells in doses ranging from 0 to 3.0 ng/ml in 0.5-ng increments. After addition of PMA to the cell monolayers, 0.5-ml aliquots of 51Cr-labeled PMN cell suspensions were then added. The 12-well plates were incubated without agitation for 30 minutes at 37°C, in an atmosphere of 5 percent CO2, balance air. After incubation, the assay wells were assessed by phase-contrast microscopy to confirm the integrity of the monolayers and the adherence of the activated neutrophils. The super nates containing nonadherent 51Cr-labeled PMNs were aspirated and the assay wells washed gently three times with MEM plus 10 percent FBS. The supernates and washes were pooled (supernate cpm). Endothelial monolayers and the adherent PMNs were then harvested with cotton-tipped swabs (adherent cpm), and radioactivity in each fraction was measured with a gamma scintillation counter. All assays were done in triplicate and the data expressed as: %adherence=[adherent cpm/(adherent cpm+supernatant cpm)]×100. All data are expressed as mean±standard error of the mean. Quantitative measurements were evaluated by analysis of variance, and discrete data were analyzed using chi-square analysis. Individual comparisons between group means were made by the Fisher PSLD test using Statview 512. Differences were considered significant at p<0.05. Clinical data for the patients in the study groups are given in Table 2. There were two deaths out of 14 (14 percent) in the ICU group due to the development of cardiogenic shock, and one patient in the MI group also died. Seven septic patients died of multiorgan failure, four in the SA and three in the SO group. Blood cultures were positive in six of the 13 septic patients. Organisms cultured included Hemophilus influenzae, enterococcus, Escherichia coli, Klebsiella pneumoniae, Salmonella, and Corynebacterium (JK species). In the seven patients with negative blood cultures, sputum cultures were positive in four, peritoneal fluid in two, and urine in one. The source of the sepsis was presumed to be the lung (infiltrate on chest roentgenogram plus positive sputum culture) in eight, the abdomen in four, and line sepsis in one. Among the 13 septic patients, renal failure (creatinine >2.0) developed in seven (54 percent), while liver failure (bilirubin >5.0) occurred in only one patient.Table 1—Clinical Diagnostic CriteriaSepsis syndrome 1. Temperature >38.3°C or <35.5°C 2. Tachycardia (>90 beats/min) 3. WBC >12/mm3 or >20 percent band forms 4. Positive blood cultures or known source of infection and hypotension or acidosisMl 1. Persistent ECG changes compatible with an ischemic event 2. Chest pain 3. Elevated creatine phosphokinaseARDS 1. Diffuse bilateral infiltrates without heart failure 2. PaO2/FIo2 <200 Open table in a new tab Table 2—Clinical Characteristics: All Patient Group*Data are expressed as mean±SEM.Patient GroupsParameterICUMISOSANumber141267Age, yr65.4±3.766.8±3.672.0±4.870.9±6.3Sex, M/F8/64/81/55/2Temperature36.7±0.336.9±0.336.6±1.137.5±0.8>38.3°C0/140/123/64/7<35.5°C0/140/123/63nSystolic BP, mm Hg121±5112±6.0127±3883±21¶p<0.05 compared to SO.Pulse, beats/min79.0±5.275.8±4.5114.0±17‡p<0.05 compared to ICU and MI.98±7.6BUN, mg/dl17.9±1.815.9±1.746.5±14.3§p<0.01 compared to ICU and MI.31.7±9.4Blood pH7.36±0.097.36±0.097.40±0.047.44±0.02Platelets, × HP/mm3307.0±53.7295.2±20.9187.0±39.8‡p<0.05 compared to ICU and MI.125.7±31.0§p<0.01 compared to ICU and MI.WBC, cells/mm38.1±0.312.5±1.1†p<0.05 compared to ICU.16.4±2.4‖p<0.001 compared to ICU.11.8±1.9†p<0.05 compared to ICU.¶p<0.05 compared to SO.PMN, cells/mm35.3±0.69.8±1.1†p<0.05 compared to ICU.13.8±3.2§p<0.01 compared to ICU and MI.7.7±1.7¶p<0.05 compared to SO.Liver failure (bilirubin >5 mg/ml)0001Renal failure (creatinine >2 mg/ml)0343Death2134* Data are expressed as mean±SEM.† p<0.05 compared to ICU.‡ p<0.05 compared to ICU and MI.§ p<0.01 compared to ICU and MI.‖ p<0.001 compared to ICU.¶ p<0.05 compared to SO. Open table in a new tab Among the septic patients, the mean PaO2/FIO2 was 162±25 mm Hg in the SA group compared to 220±27 mm Hg in the SO patients. Systolic blood pressure was significantly lower in the SA group than in the ICU and the SO groups (p<0.02). The SO patients were more tachycardiac than the SA, ICU, and MI groups (p<0.05). Blood pH was similar among all the groups, although serum bicarbonate was reduced in septic patients (18.3±3.6 mEq/L). Platelet levels were greatly reduced in both septic groups. The SO group had only 62 percent of the platelet numbers found in the ICU and MI groups (p<0.05), while in the SA group, the levels were even lower, 42 percent of the two control groups (p<0.01), although not statistically different from the SO group (p>0.1). Both of the septic groups and the MI group had elevated WBCs compared to the control ICU population. Septic patients without ARDS had the highest level, 39 percent higher than septic patients who went on to develop ARDS (p<0.05). The numbers of circulating neutrophils were elevated in these groups (Fig 1). While mean PMN numbers were increased in MI patients by 1.85-fold and in SO patients by 2.6-fold when compared to ICU patients, the SA patients had only a 1.45-fold increase, significantly lower than the SO group (p<0.05). Unstimulated baseline adherence of PMNs to endothelial cells (PMA = 0), shown in Table 3, was similar in normal volunteers and all four patient groups. When increasing doses of PMA were added to the incubation wells, increasing numbers of 51Cr-labeled PMNs adhered tightly to the endothelial monolayers in all patient groups. Figure 2 illustrates the dose-response curves for PMA-stimulated adherence by PMNs in the ICU, SA, MI, and SO groups. In both of the septic groups, PMA-stimulated PMN adherence was reduced, significantly so for the SA patients compared to the ICU group at PMA doses from 0.5 to 1.0 ng/ml (p<0.05). The PMNs from the ICU group tended to be more adherent than those from normal subjects, although the difference was not significant. Those from MI patients responded identically to those of the normal subjects.Table 3—Unstimulated Neutrophil Adherence to Endothelial Cells*Data expressed as mean±SEM; no significant difference between any groups (p>0.6).Patient GroupNeutrophil Adherence, %Normal14.24±2.92ICU12.47±1.41MI10.68±1.93SA9.42±1.80SO12.34±2.42* Data expressed as mean±SEM; no significant difference between any groups (p>0.6). Open table in a new tab Reduced neutrophil adherence in the low PMA dose range, shown in Figure 3, was common in those who developed ARDS. At 1.0 ng/ml of PMA, only two of seven patients who developed ARDS had PMN adherence above 20 percent, compared to four of six septic patients who did not develop ARDS and 21 of 26 nonseptic patients without ARDS (p = 0.05 by chi square). Thus, 71 percent of ARDS patients had reduced adherence contrasted with only 22 percent of the septic and nonseptic patients who did not develop ARDS. At the higher concentrations of PMA, those doses which induced maximal adherence to endothelium, there was no difference among all study groups (Fig 2), indicating that the neutrophils had retained their ability to adhere to endothelium when fully stimulated. There was no significant relationship between the PMN response to PMA and other organ failure, mortality, or drug therapy. Our study reports the direct interaction of neutrophils obtained from critically ill patients with pulmonary artery endothelial cells. Patients who met the criteria for the sepsis syndrome were compared to a normal group and to two nonseptic control populations with other critical illness. The first was composed primarily of patients with ischemic cardiovascular disease admitted for monitoring, while the other was a similar group in whom an acute uncomplicated MI was diagnosed. These groups were used to control for the effects of stress and accompanying hormonal alterations on neutrophil function that might occur in critically ill patients. Neutrophils isolated from patients with sepsis syndrome who develop ARDS, when stimulated by low levels of PMA, were significantly less adherent to normal endothelium than were those from the nonseptic ICU population. Baseline and maximally stimulated adherence were similar among normal subjects and all patient groups. Acute lung injury is one of many manifestations of organ failure that may accompany the sepsis syndrome. In this lung injury, endothelial damage results in compromise of permeability barrier function of the alveolar blood vessels, severe pulmonary edema, and, often, progressive pulmonary fibrosis and hypertension. Activated neutrophils are believed to be important in initiating the endothelial injury. Neutrophils respond to both local and systemic stimuli first by sequestering in the pulmonary microvasculature, then adhering tightly to the endothelium.5Haslett C Worthen GS Giclas PC Morrison DC Henson JE Henson PM The pulmonary vascular sequestration of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit.Am Rev Respir Dis. 1987; 136: 9-18Crossref PubMed Scopus (130) Google Scholar, 6Worthen GS Haslett C Rees AJ Gumbay RS Henson JE Henson PM Neutrophil-mediated pulmonary vascular injury: synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung.Am Rev Respir Dis. 1987; 136: 19-28Crossref PubMed Scopus (198) Google Scholar,13Gimbrone Jr, MA Brock AF Schafer AI Leukotriene B4 stimulates polymorphonuclear leukocyte adhesion to cultured vascular endothelial cells.J Clin Invest. 1984; 74: 1552-1555Crossref PubMed Scopus (153) Google Scholar Once in close proximity to the endothelial cells, neutrophils can release reactive oxygen species, proteases, and lipid mediators which act together to produce endothelial injury.2Shasby DM Vanbenthuysen KM Tate RM Shasby SS Mc-Murtry I Repine JE Granulocytes mediate acute endomatous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals.Am Rev Respir Dis. 1982; 125: 443-447PubMed Google Scholar, 3Shasby DM Shasby SS Peach MJ Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs: role of oxygen radicals and granulocyte adherence.Am Rev Respir Dis. 1983; 127: 72-76Crossref PubMed Scopus (99) Google Scholar, 4Smedly LA Tonnesen MG Sandhaus RA Haslett C Guthrie LA Johnston Jr, RB et al.Neutrophil-mediated injury to endothelial cells: enhancement by endotoxin and essential role of neutrophil elastase.J Clin Invest. 1986; 77: 1233-1243Crossref PubMed Scopus (480) Google Scholar,14Harlan JM Schwartz BR Reidy MA Schwartz SM Ochs HD Harker LA Activated neutrophils disrupt endothelial monolayer integrity by an oxygen radical-independent mechanism.Lab Invest. 1985; 52: 141-150PubMed Google Scholar A number of in vivo and in vitro studies have demonstrated that enhanced PMN endothelial adherence results from an increase in the expression of interacting adhesion proteins on the surfaces of both the neutrophils (CDw18 complex, Mac-1)15Arnaout MA Hakin RM Todd RF Dana N Colten HR Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis.N Engl J Med. 1985; 312: 457-462Crossref PubMed Scopus (195) Google Scholar,16Harlen JM Killen PD Senecal FM Schwartz BR Yee EK Taylor RF et al.The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro.Blood. 1985; 66: 167-178PubMed Google Scholar and the endothelial cells (ELAM-1).17Pohlman TH Stanness KA Beatty PG Ochs HD Harlan JM An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1, and tumor necrosis factor increases neutrophil adherence by a CDw 18-dependent mechanism.J Immunol. 1986; 136: 4548-4553PubMed Google Scholar,18Pober JS Gimbrone Jr, MA Lapierre LA Mendrick DL Liers W Rothlein R et al.Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon.J Immunol. 1986; 137: 1893-1896Crossref PubMed Google Scholar These alterations in the neutrophil and the endothelial cell which promote adherence are induced by a wide variety of inflammatory mediators including activated complement,15Arnaout MA Hakin RM Todd RF Dana N Colten HR Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis.N Engl J Med. 1985; 312: 457-462Crossref PubMed Scopus (195) Google Scholar LTB-4,13Gimbrone Jr, MA Brock AF Schafer AI Leukotriene B4 stimulates polymorphonuclear leukocyte adhesion to cultured vascular endothelial cells.J Clin Invest. 1984; 74: 1552-1555Crossref PubMed Scopus (153) Google Scholar bacterial cell wall LPS (endotoxin), and the cytokines TNF19Gamble JR Harlen JM Klebanoff SJ Vadas MA Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor.Proc Natl Acad Sci USA. 1985; 82: 8667-8671Crossref PubMed Scopus (786) Google Scholar and IL-117Pohlman TH Stanness KA Beatty PG Ochs HD Harlan JM An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1, and tumor necrosis factor increases neutrophil adherence by a CDw 18-dependent mechanism.J Immunol. 1986; 136: 4548-4553PubMed Google Scholar,18Pober JS Gimbrone Jr, MA Lapierre LA Mendrick DL Liers W Rothlein R et al.Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon.J Immunol. 1986; 137: 1893-1896Crossref PubMed Google Scholar,20Bevilacqua MP Pober JS Wheeler ME Mendrick D Cotran RS Gimbrone Jr, MA Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.J Clin Invest. 1985; 76: 2003-2011Crossref PubMed Scopus (813) Google Scholar all of which are present as sepsis progresses. In addition to enhancing PMN adherence, these mediators also stimulate neutrophil granule secretion and oxidant release21Larrick JW Graham D Toy K Lin LS Senyk G Fendly BM Recombinant tumor necrosis factor causes activation of human granulocytes.Blood. 1987; 69: 640-644PubMed Google Scholar,22Shalaby MR Aggarwal BB Rinderknecht E Svedersky LP Finkle BS Palladino Jr, MA Activation of human polymorphonuclear neutrophil functions by interferon and tumor necrosis factors.J Immunol. 1985; 135: 2069-2073PubMed Google Scholar and enhance endothelial cell killing.23Varani J Bendelow MJ Sealey DE Kunkel SL Gannon DE Ryan US et al.Tumor necrosis factor enhances susceptibility of vascular endothelial cells to neutrophil-mediated killing.Lab Invest. 1988; 59: 292-295PubMed Google Scholar Evidence for enhanced neutrophil-endothelial cell adherence in human sepsis is limited. Warshawski et al,7Warshawski FJ Sibbald WJ Driedger AA Cheung H Abnormal neutrophil-pulmonary interaction in the adult respiratory distress syndrome: qualitative and quantitative assessment of pulmonary neutrophil kinetics in humans with “in vivo” indium neutrophil scintigraphy.Am Rev Respir Dis. 1986; 133: 797-804PubMed Google Scholar using lung scanning with 111In-labeled neutrophils 17 to 20 hours after a septic insult, demonstrated significantly more PMN sequestration in patients with active septic ARDS compared to patients with resolving lung injury or to septic patients who did not develop ARDS. This study provided a macroscopic picture of neutrophil kinetics in septic ARDS but no information regarding the specific neutrophil-endothelial cell interaction. Venezio et al9Venezio FR Westenfelder GO Phair JP The adherence of polymorphonuclear leukocytes in patients with sepsis.J Infect Dis. 1982; 145: 351-357Crossref PubMed Scopus (21) Google Scholar studied unstimulated adherence to nylon fibers of leukocytes from whole blood of patients with sepsis. They observed no difference between bacteremic patients without shock and normal control subjects. However, leukocytes from patients with septic shock had significantly enhanced adherence using this assay when studied 24 to 36 hours after initiation of antibiotic therapy. Data from this study also suggested that the enhanced adherence was mediated, at least in part, by factors circulating in the plasma. When normal neutrophils were mixed with the plasma of patients from the septic shock group, their adherence was enhanced. Interestingly, steroid administration was associated with diminished adherence of the neutrophils, an inhibition of PMN activation related to effects reported in vitro24Strieter RM Remick DG Lynch JP Genord M Raiford C Spengler R et al.Differential regulation of tumor necrosis factor-alpha in human alveolar macrophages and peripheral blood monocytes: a cellular and molecular analysis.Am J Respir Cell Mol Biol. 1989; 1: 57-63Crossref PubMed Scopus (79) Google Scholar and in animals.25Fukushima K Ando M Suga M Araki S Impaired function of polymorphonuclear leukocytes exuded into bronchoalveolar spaces infected with Pseudomonas aeruginosa in steroid-treated rabbits.Exp Lung Res. 1987; 13: 141-155Crossref PubMed Scopus (5) Google Scholar Our results (Table 3) indicate no difference between baseline adherence of leukocytes from septic or nonseptic patients and from our normal group. Results differing from those of Venezio et al9Venezio FR Westenfelder GO Phair JP The adherence of polymorphonuclear leukocytes in patients with sepsis.J Infect Dis. 1982; 145: 351-357Crossref PubMed Scopus (21) Google Scholar might be due to the different surfaces used in the two studies (nylon vs endothelial cells) or to differences in the timing of the blood sampling in the course of the septic process. We also found no difference in maximal adherence among our study groups. PMA, a nonspecific protein kinase C activating agent,26Niedel JE Kuhn LJ Vanderbark GR Phorbol diester copurifies with protein kinase C.Proc Natl Acad Sci USA. 1983; 80: 36-40Crossref PubMed Scopus (1034) Google Scholar stimulates PMN adherence in a dose-dependent manner. When neutrophils were exposed to this agent, the PMN response was attenuated in the septic group as a whole (Fig 2) and particularly in those patients who would develop progressive lung injury within 24 to 48 hours. The PMN adherence at a PMA dose of 1.0 ng/ml was more commonly reduced in septic patients who developed ARDS (Fig 3). Previous attempts to find an objective circulating marker for the development of ARDS have proved disappointing and less useful than evaluation of clinical risk factors such as shock and acidosis. Parsons et al27Parsons PE Worthen GS Moore EE Tate RM Henson PM The association of circulating endotoxin with the development of the adult respiratory distress syndrome.Am Rev Respir Dis. 1989; 140: 294-301Crossref PubMed Scopus (145) Google Scholar recently reported that C5 and C3 fragment levels were similar in ARDS patients and in a group of those at risk who did not develop the syndrome. However, ARDS did develop more frequently when both complement fragments and circulating endotoxin were detectable. These investigators propose that the combination of endotoxin priming and chemoattractant stimulation may be necessary for endothelial injury to progress, a suggestion supported by previous animal studies from the same group.6Worthen GS Haslett C Rees AJ Gumbay RS Henson JE Henson PM Neutrophil-mediated pulmonary vascular injury: synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung.Am Rev Respir Dis. 1987; 136: 19-28Crossref PubMed Scopus (198) Google Scholar While no single circulating inflammatory mediator has been found to predict progressive lung injury, the use of neutrophil activation response as a marker for ARDS allows for integration of the effects of multiple mediators. The neutrophil-endothelial cell interaction in sepsis and ARDS occurs in a complex and changing environment. The more pronounced reduction in stimulated PMN adherence in septic patients prior to developing florid ARDS suggests that the neutrophil is altered functionally before clinical recognition of the acute lung injury. Our results are in agreement with preliminary data of Parsons et al,28Parsons PE Worthen GS Moore FA Moore FE Henson PM Activity levels of circulating neutrophils in acute lung injury.Am Rev Respir Dis. 1990; 141: 511Crossref PubMed Scopus (24) Google Scholar who have recently reported decreased stimulated superoxide anion production in patients who developed ARDS. The reason for the attenuated response to PMA of neutrophils from our septic patients is unclear. First, it is possible that we have sampled PMNs which could not sequester in the lung because they were inherently less capable of responding to stimuli and thus remained in the circulation. That the septic patients who developed ARDS had significantly lower numbers of circulating neutrophils (Fig 1) is consistent with this possibility and with studies which demonstrate an early neutropenia in animals in response to bolus endotoxin administration8MacGregor RR Granulocyte adherence changes induced by hemodialysis, endotoxin, epinephrine, and glucocorticoids.Ann Intern Med. 1977; 86: 35-39Crossref PubMed Scopus (100) Google Scholar and in patients developing ARDS.29Thommason HW Boyko WJ Russell JA Hogg JC Transient leukopenia associated with adult respiratory distress syndrome.Lancet. 1984; 1: 809-812Abstract PubMed Scopus (57) Google Scholar However, because these septic neutrophils did not have reduced baseline adherence and were able to respond normally to high levels of PMA stimulation (2.5 to 3.0 ng/ml), this does not appear to be the case. Another possible explanation is down-regulation of expression of adhesion receptors in response to prior exposure to inflammatory mediators. Because PMA bypasses specific mediator receptors and stimulates protein kinase C directly,26Niedel JE Kuhn LJ Vanderbark GR Phorbol diester copurifies with protein kinase C.Proc Natl Acad Sci USA. 1983; 80: 36-40Crossref PubMed Scopus (1034) Google Scholar the differences we observed must relate to desensitization of this signal transduction mechanism or to a deficit in specific granule stores of adhesion proteins available to be transferred to the PMN surface. The ability of TNF and IL-1 to desensitize immune effector cells has been demonstrated in other systems.30Wallah D Holtmann H Engelmann H Nophar Y Sensitization and desensitization to lethal effects of tumor necrosis factor and IL-1.J Immunol. 1988; 140: 2994-2999PubMed Google Scholar An unstimulated bovine endothelial cell line was used as the substrate for adhesion in our study. This more closely approximates the in vivo situation than does adherence to artificial surfaces previously used in such studies. However, the endothelial cells had not been exposed to significant levels of endotoxin or to other factors clearly present in sepsis. The endothelium in sepsis is changed both by expressing its own adhesion receptors in response to inflammatory mediators17Pohlman TH Stanness KA Beatty PG Ochs HD Harlan JM An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1, and tumor necrosis factor increases neutrophil adherence by a CDw 18-dependent mechanism.J Immunol. 1986; 136: 4548-4553PubMed Google Scholar and by actively secreting factors which can act on other cells.31Rinaldo JE Basford RE Neutrophil-endothelial interactions: modulations of neutrophil activation responses by endothelial cells.Tissue Cell. 1987; 19: 599-606Crossref PubMed Scopus (10) Google Scholar,32Wheeler ME Luscinskas FW Bevilacqua MP Gimbrone Jr, MA Cultured human endothelial cells stimulated with cytokines or endotoxin produce an inhibitor of leukocyte adhesion.J Clin Invest. 1988; 82: 1211-1218Crossref PubMed Scopus (39) Google Scholar Thus, while the septic neutrophil binding to unstimulated endothelium may be attenuated, this might not be true for an endothelium altered by the septic environment. Our data suggest that, if neutrophil-endothelial cell adherence is necessary to initiate injury, the endothelium must be an important active participant in this process. Our data from the MI patient group are also of interest. We have recently reported the development of acute lung injury following acute MI.33Niederman MS Fein AM Sklarek HM Mantovani R Rosen H Schettini B et al.Pulmonary edema with low pulmonary capillary wedge pressure after acute myocardial infarction: clinical features and prognostic implications.J Crit Care. 1989; 4: 194-201Abstract Full Text PDF Scopus (4) Google Scholar Previous investigators have demonstrated complement activation following infarction,34Earis JE Marcuson EC Bernstein A Complement activation after myocardial infarction.Chest. 1985; 87: 186-189Crossref PubMed Scopus (21) Google Scholar and we reasoned that neutrophil adherence to pulmonary endothelium might be increased in these patients. The ability of neutrophils from patients with acute uncomplicated MI to bind to endothelial cells was similar to that in normal subjects. This suggests that, while systemic inflammation may occur in acute MI, inflammatory mediators are not at levels sufficient to promote PMN adherence to endothelium in the absence of endotoxin or endotoxin-induced mediators. In summary, we have studied the interaction of neutrophils isolated from septic patients with a pulmonary artery endothelial cell surface. These cells demonstrated diminished adherence in response to low levels of PMA stimulation, a deficit that was associated with the development of progressive lung injury 24 to 48 hours later in septic patients. These data suggest that neutrophils are functionally altered at a relatively early stage of sepsis, and that progressive multiorgan failure may already have begun at a cellular level by the time sepsis is clinically recognized. We thank John Mordente and James DiMeo for technical assistance." @default.
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