Dopamine induces neutrophil apoptosis through a ... - Surgery

Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor– independent mechanism S. Sookhai, FRCSI, J. H. Wang, PhD, M. McCourt, FRCSI, D. O’Connell, PhD, and H. P. Redmond, FRCSI, Cork, Ireland

Background. For the normal resolution of an acute inflammatory response, neutrophil (PMN) apoptosis is essential to maintain immune homeostasis and to limit inappropriate host tissue damage. A delay in PMN apoptosis has been implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS). Dopamine, a biogenic amine with known cardiovascular and neurotransmitter properties, is used in patients with SIRS to maintain hemodynamic stability. We sought to determine whether dopamine may also have immunoregulatory properties capable of influencing PMN apoptosis, function, and activation state in patients with SIRS. Methods. PMNs were isolated from healthy volunteers and patients with SIRS and treated with varying doses of dopamine and a dopamine D-1 receptor agonist, fenoldopam. PMN apoptosis was assessed every 6 hours with use of propidium iodide DNA staining and PMN function was assessed with use of respiratory burst activity, phagocytosis ability, and CD11a, CD11b, and CD18 receptor expression as functional markers. Results. There was a significant delay in PMN apotosis in patients with SIRS compared with controls. Treatment of isolated PMNs from both healthy controls and patients with SIRS with 10 and 100 µmol/L dopamine induced apoptosis. PMN ingestive and cytocidal capacity were both decreased in patients with SIRS compared with controls. Treatment with dopamine significantly increased phagocytic function. Fenoldopam did not induce PMN apoptosis. Conclusion. Our data demonstrate for the first time that dopamine induces PMN apoptosis and modulates PMN function both in healthy controls and in patients with SIRS. These results indicate that dopamine may be beneficial during SIRS through a nonhemodynamic PMN-dependent proapoptotic mechanism. (Surgery 1999;126:314-22.) From the Departments of Surgery and Pharmacology, Cork University Hospital and National University of Ireland, Cork, Ireland

THE SYSTEMIC INFLAMMATORY RESPONSE SYNDROME (SIRS) is characterized by a massive systemic proinflammatory reaction that often leads to the development of the multiple organ dysfunction syndrome (MODS). Although there have been many attempts made at trying to avert the development of MODS, it still remains the leading cause of death in the intensive care unit, affecting as many as 40% to 50% of critically ill patients.1 Neutrophils (PMNs) are the most abundant circulating proinflammatory leukocytes and they constitute the “first line of defense” against infectious agents or “non-self” substances that penetrate the body’s physical barriers.2 Although PMNs play a benPresented at the 60th Annual Meeting of the Society of University Surgeons, New Orleans, La, Feb 11-13, 1999. Reprint requests: H. P. Redmond, FRCSI, Department of Surgery, Cork University Hospital, Wilton, Cork, Ireland. Copyright © 1999 by Mosby, Inc. 0039-6060/99/$8.00 + 0

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eficial role in host immune defense, paradoxically they also have potential deleterious effects on normal host tissue. The human PMN is known to have a relatively short half-life in circulation, estimated to be between 8 and 16 hours. This life span is short because circulating PMNs constitutively undergo apoptosis. For the normal resolution of an acute inflammatory reaction to occur, PMN apoptosis with subsequent ingestion by tissue macrophages is required; this process plays a critical role in minimizing the autotoxic potential of this cell.3 A delay in the apoptotic program of activated PMN results in the failure to terminate the acute inflammatory response, which has been suggested as a precipitant of SIRS.4 In addition, activated PMN-mediated endothelial cell damage has been implicated in the development of increased vascular permeability and the capillary leak syndrome associated with SIRS.5 For more than two decades, dopamine, administered by continuous intravenous infusion in the intensive care unit, has been frequently used in

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Fig 1. Spontaneous PMN apoptosis from healthy adult volunteers and patients with SIRS. Human PMNs isolated from healthy adult volunteers (n = 6) and patients with SIRS (n = 6) were cultured in complete RPMI 1640 medium at 37°C in 5% carbon dioxide for 24 hours. PMN apoptosis was assessed at 0, 6, 12, 18, and 24 hours according to percent of cells with hypodiploid DNA by flow cytometry as described in the “Material and Methods.” Data are expressed as mean ± SD and are representative of 6 separate experiments. Statistical significance was compared with healthy controls (asterisk, P < .02; dagger, P < .05; double dagger, P < .005; section, P < .004; parallel, P < .002 respectively).

septic patients to maintain adequate urinary output, cardiac output, and blood pressure because dopamine appeared to improve short-term survival.6 This endogenous catecholamine influences different catecholamine receptors in a dose-dependent manner.7 Data suggest that stimulation and inhibition of these receptors can modulate SIRS as evidenced by in vitro and in vivo studies and that the levels of catecholamines increase during sepsis. In this study we investigated whether dopamine may also have a role to play in SIRS by modulating apoptosis, microbicidal capacity, and activation state in PMN isolated from patients with SIRS. Regulation of PMN clearance mechanism and apoptosis may provide novel therapeutic strategies capable of limiting host tissue damage and ultimately inflammatory disease states such as SIRS. MATERIAL AND METHODS Reagents. The following reagents were used for the isolation of human PMN and assessment of PMN apoptosis and function: RPMI 1640, phosphatebuffered saline solution (PBS) without calcium and magnesium, fetal calf serum (FCS), penicillin, streptomycin sulfate, amphotericin (Fungizone), and glutamine were purchased from Gibco-BRL (Paisely, UK). Sodium citrate, propidium iodide (PI), Percoll, dextran, Triton X-100, EDTA (ethylenediaminetetraacetic acid), TRIS (tromethamine), and dopamine were purchased from Sigma (St Louis, Mo). FicollPaque and fenoldopam were purchased from Pharmacia (Uppsala, Sweden) and Neurex Corporation (Menlon Park, Calif), respectively. Mouse antihuman

CD11a, CD11b, and CD18 monoclonal antibodies (mAbs) were purchased from Becton Dickinson (Mountain View, Calif). Rabbit antihuman dopamine D-1 receptor subtype antibody was kindly supplied by the Department of Pharmacology, Cork University Hospital, Cork, Ireland. Patients. The study population consisted of critically ill patients admitted to the Medical/Surgical Intensive Care Unit of Cork University Hospital, Cork. Heparinized venous blood was obtained from 6 patients who met the clinical criteria for SIRS8 and compared with that of healthy controls. Patients receiving dopamine treatment were excluded from the study. This study was approved by the Ethics committee of Cork University Hospital. Isolation of human PMN. Whole venous blood was collected from healthy adult volunteers and patients with SIRS (matched for age and weight) in the intensive care unit, with lithium heparin used as an anticoagulant. The PMN were isolated with use of dextran sedimentation (6% dextran in 0.9% sodium chloride) followed by Ficoll-Paque gradient centrifugation. Contaminating erythrocytes were removed by centrifugation through an 81% isotonic Percoll gradient. The granulocyte layer at the interface was collected, washed in RPMI 1640, and resuspended in complete RPMI 1640 medium containing 10% FCS, penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin (Fungizone) (0.25 µg/mL), and 2 mmol/L glutamine. PMNs were counted, and cell viability as determined by trypan blue exclusion was >98%. PMN purity was >97% as determined by Rapi-Diff

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Fig 2. Effect of dopamine on spontaneous PMN apoptosis from healthy controls. Isolated human PMNs from healthy controls (n = 6) were treated with varying concentrations of dopamine (0.001 to 100 µmol/L) and incubated at 37°C in 5% carbon dioxide for 24 hours. PMN apoptosis was assessed at 6, 12, 18, and 24 hours according to percent of cells with hypodiploid DNA by flow cytometry as described in “Material and Methods.” Data are expressed as mean ± SD and are representative of 6 separate experiments. Statistical significance was compared with PMN treated with medium as control. Asterisk, P < .02.

II (DiaCheM, Lancashire, UK) staining on cytocentrifuged samples. Assessment of PMN apoptosis. Isolated human PMN from healthy adult volunteers and patients with SIRS were treated with or without dopamine (0.001 to 100 µmol/L) or fenoldopam (the agonist of dopamine D-1 receptor, 0.001 to 100 µmol/L). Determination of PMN apoptosis by flow cytometry was performed after 6, 12, 18, and 24 hours of incubation at 37°C in humidified 5% carbon dioxide conditions. PMN apoptosis was further confirmed by morphologic assessment with Wright’s Giemsa staining as described by Martin et al.9 PMN apoptosis was quantified according to the percentage of cells with hypodiploid DNA by use of

the PI staining technique as previously described.10 Briefly, after centrifugation PMN (0.5 × 106 cells) in 17 × 100 mm polypropylene tubes (Falcon, Lincoln Park, NJ) were gently resuspended in 0.5 mL of hypotonic fluorochrome solution (50 µg/mL PI, 3.4 mmol/L sodium citrate, 1 mmol/L TRIS, 0.1 mmol/L EDTA, 0.1% Triton X-100), incubated in the dark at 4°C for 2 hours before they were analyzed by a FACScan flow cytometer (Becton Dickinson). The forward and side scatter of PMN particles were simultaneously measured. The PI fluorescence of individual nuclei with an acquisition of fluorescence channel (FL) 2 was plotted against forward scatter, and the data were registered on a logarithmic scale. The minimum number of 5000

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Fig 3. Induction of human PMN apoptosis from patients with SIRS by dopamine treatment. Isolated human PMNs from patients with SIRS (n = 6) were treated with 10 and 100 µmol/L dopamine (DA) and incubated at 37°C in 5% carbon dioxide for 24 hours. PMN apoptosis was assessed at 6, 12, 18, and 24 hours according to percent of cells with hypodiploid DNA by flow cytometry as described in “Material and Methods.” Data are expressed as mean ± SD and are representative of 6 separate experiments. Statistical significance was compared with PMN treated with medium. Asterisk, P < .001.

Table I. Patient demographics from patients diagnosed with SIRS Patient No. 1 2 3 4 5 6 Statistics (mean ± SD)

Clinical diagnosis

Age (y)

No. of SIRS criteria

Survival status

Esophageal varices Pneumonia Renal failure Multiple fractures Pneumonia Pancreatitis

53 73 61 28 70 69 59 ± 17

3 3 4 3 2 3 3.1 ± 0.4

Survived Died Died Survived Survived Survived 66.7%

events was collected and analyzed on the software Lysis II (Becton Dickinson, Mountain View, Calif). Apoptotic PMN nuclei were distinguished by their hypodiploid DNA content from the diploid DNA content of normal PMN nuclei. Cell debris were excluded from analysis by raising the forward threshold. All measurements were performed under the same instrument settings. Measurement of PMN respiratory burst and phagocytosis. Human PMNs from healthy adult volunteers and patients with SIRS were treated with or without dopamine (10 and 100 µmol/L) for 1 hour at 37°C in humidified 5% carbon dioxide conditions. PMN respiratory burst was assessed with use of a Bursttest (Orpegen, Heidelberg, Germany). Briefly, after incubation with 20 µL of stabilized and opsonized dead Escherichia coli suspension at 37°C for 10 minutes, 20 µL of the fluorogenic substrate dihydrorhodamin 123 was added to 100 µL of PMN suspension (1 × 106 cells/mL) and incubated at 37°C for a further 10 minutes. After centrifugation the cell pellets were resuspended in 100 µL of DNA staining solution and incubated at 4°C for 15 minutes. A Phagotest (Orpegen) was used to determine PMN

phagocytosis. Briefly, 20 µL of stabilized and opsonized fluorescence isothiocyanate (FITC)–labeled E coli suspension was added to 100 µL of PMN suspension (1 × 106 cells/mL) and incubated at 37°C for 10 minutes. After the addition of 100 µL of quenching solution, the sample was washed twice with 3 mL of washing solution in an ice-water bath. After centrifugation 100 µL of DNA staining solution was added to cell pellets and incubated for 15 minutes in an icewater bath. The analysis of PMN respiratory burst and phagocytosis was performed on a FACScan flow cytometer. The mean channel fluorescence was detected with FL 1 with use of logarithmic amplification on the basis of a minimum number of 10,000 cells collected and analyzed with the software Lysis II. Fluorescence-activated cell sorter analysis of immunofluorescence. After incubation of human PMN from healthy volunteers and SIRS patients with or without dopamine (10 and 100 µmol/L) for 1 hour at 37°C in humidified 5% carbon dioxide conditions, the expression of CD11a, CD11b, and CD18 on human PMN was assessed by addition of 20 µL of FITC-conjugated anti-LFA-1α (anti-CD11a), phycoerythrin-conjugated anti-Leu-15 (anti-CD11b), and

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avidin-biotin-peroxidase method (Vectastain, Burlingame, Calif). The signal was detected with diaminobenzidine and counterstained with Mayer’s hematoxylin. Statistical analysis. All data are presented as the mean ± SD. Statistical analysis was performed with use of Student’s t test. Differences were judged statistically significant when the P value was less than .05.

Fig 4. Effect of dopamine (DA) on human PMN respiratory burst and phagocytosis. Human PMN from healthy controls (n = 6) and patients with SIRS (n = 6) were treated with 10 and 100 µmol/L dopamine at 37°C in 5% carbon dioxide for 1 hour. PMN respiratory burst (A) and phagocytosis (B) were assessed with use of flow cytometry as described in “Material and Methods” and were expressed as mean channel fluorescence (MCF) per cell. Results are presented as mean ± SD, representative of 6 separate experiments. Statistical significance was compared with PMN from healthy controls. Asterisk, P < .02, and compared with PMN from SIRS patients treated with medium, P < .02.

FITC-conjugated anti-LFA-1β (anti-CD18) mAbs to 100 µL of PMN suspension (1 × 106 cells/mL). FITCand phycoerythrin-conjugated isotype immunoglobulin (Ig) G1 and IgG2a mAbs were used as a negative control. After incubation at 4°C for 30 minutes, CD11a, CD11b, and CD18 expression on PMN were analyzed on a FACScan flow cytometer for detecting the log of the mean channel fluorescence intensity with an acquisition of FL 1 and FL 2, respectively. The minimum number of 10,000 events was collected and analyzed on the software Lysis II. Immunohistochemistry detection of dopamine D-1 receptor expression on human PMN. PMNs isolated from healthy donors and patients with SIRS were spun onto slides, acetone fixed, washed in PBS, and air dried overnight. The cells were then incubated overnight with a rabbit antihuman dopamine D-1 receptor subtype antibody with use of the

RESULTS Demographic data for patients with SIRS. Six patients who met the clinical criteria for SIRS were enrolled in the study. The demographic characteristics of these patients are demonstrated in Table I. Delayed spontaneous PMN apoptosis in patients with SIRS. Time-course studies of spontaneous PMN apoptosis were conducted to compare those isolated from healthy volunteers and those isolated from patients with SIRS. Flow cytometric analysis demonstrated that PMN apoptosis was significantly delayed in patients with SIRS compared with normal controls (P < .05). This phenomenon occurred at all the various time points studied (0, 6, 12, 18, and 24 hours) (Fig 1). These findings correlated with the morphologic changes associated with apoptosis as assessed under a light microscope with Wright’s Giemsa staining (data not shown). The effect of dopamine on PMN apoptosis in controls and in patients with SIRS. Treatment of human PMN isolated from healthy volunteers with varying concentrations of dopamine resulted in a significant induction of apoptosis (P < .02). This occurred both in a time- (12, 18, and 24 hours) and dose- (10 and 100 µmol/L) dependent manner (Fig 2, A to D). To clarify whether this induction of PMN apoptosis in healthy controls may also occur in PMN isolated from patients with SIRS, PMNs isolated from these patients were incubated with 10 and 100 µmol/L dopamine for the various time points described above. There was a significant increase in PMN apoptosis at 6, 12, 18, and 24 hours (P < .001), as shown in Fig 3. Unlike PMN isolated from controls, there was also a significant induction in apoptosis at 6 hours in patients with SIRS (P < .001). The effect of dopamine on human PMN cytocidal function. To evaluate the effect of dopamine on human PMN cytocidal function, PMN respiratory burst and phagocytosis were assessed after a 1-hour incubation of PMN from patients with SIRS with 10 and 100 µmol/L dopamine. As shown in Fig 4, A, PMN respiratory burst activity was significantly downregulated in patients with SIRS compared with healthy controls (P < .02). Treatment with 10

Surgery Volume 126, Number 2 and 100 µmol/L dopamine did not alter PMN respiratory burst activity in the SIRS group. PMN phagocytic ability in patients with SIRS was again significantly decreased compared with healthy control values (P < .02). Dopamine at 100 µmol/L resulted in a significant attenuation of this response (P < .02 vs SIRS patients) returning PMN ingestive capacity toward control values (Fig 4, B). However, there was no alteration in cytocidal function after the treatment of PMN from healthy controls with dopamine (data not shown). The effect of dopamine on human PMN adhesion receptor expression. There was no significant difference in CD11a receptor expression in isolated PMN from patients with SIRS compared with healthy controls. Addition of dopamine did not alter this response (Fig 5, A). However, CD11b receptor expression was significantly up-regulated in PMNs from patients with SIRS compared with control values (P < .001) (Fig 5, B). Dopamine at 10 and 100 µmol/L significantly reduced PMN CD11b receptor expression in patients with SIRS (P < .003) (Fig 5, B). In addition, CD18 adhesion receptor expression showed no significant difference in PMNs from patients with SIRS compared with control values; however, after incubation of PMNs from SIRS patients with 100 µmol/L of dopamine, there was a significant down-regulation in CD18 receptor expression (P < .003) (Fig 5, C). In control healthy subjects, after the incubation of PMN with dopamine for 1 hour, there was no difference in CD11a receptor expression; however, there was a significant down-regulation in both CD11b and CD18 receptor expression compared with PMNs not treated with dopamine (P < .05) (data not shown). Effect of a dopamine D-1 receptor agonist on PMN apoptosis. Fig 6 shows the positive expression of dopamine D-1 receptor on human PMNs. To ascertain whether the induction of PMN apoptosis by dopamine is mediated through a dopamine D-1 receptor pathway, isolated normal PMNs from healthy volunteers were incubated with varying concentrations of the dopamine D-1 receptor agonist fenoldopam for 6, 12, 18, and 24 hours. There was no significant difference in percent of PMN apoptosis isolated from healthy controls compared with those treated with fenoldopam at any of the time points or at any of the doses of fenoldopam (Table II). DISCUSSION PMNs are the primary effector cells mediating host defense against a variety of infectious agents. It is through oxygen-dependent as well as oxygen-independent mechanisms such as the production of significant amounts of reactive oxygen metabolites and

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Fig 5. Effect of dopamine (DA) on human PMN adhesion receptor expression. Human PMNs from healthy controls (n = 6) and patients with SIRS (n = 6) were treated with 10 and 100 µmol/L dopamine at 37°C in 5% carbon dioxide for 1 hour. Expression of PMN CD11a (A), CD11b (B), and CD18 (C) was assessed with use of flow cytometry as described in “Material and Methods” and was expressed as mean channel fluorescence (MCF) per cell. Results are mean ± SD, representative of 6 separate experiments. Statistical significance was compared with PMNs from healthy controls (asterisk, P < .001) and compared with PMNs from patients with SIRS treated with medium (dagger, P < .003).

proteolytic enzymes, respectively, that these ubiquitous cells are capable of functioning efficiently. However, the inappropriate release of these same proinflammatory mediators into the surrounding tissue spaces may be responsible for the deleterious effects on normal host tissue that is all too commonly seen.11 Programmed cell death or apoptosis followed

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Fig 6. Expression of dopamine D-1 receptor on human PMNs. Isolated PMNs from healthy controls were treated with rabbit antihuman dopamine D-1 receptor subtype antibody with use of avidin-biotin-peroxidase method. Signal was detected with diaminobenzidine and counterstained with Mayer’s hematoxylin. Brown staining represents positive expression of dopamine D-1 receptor expression on human PMNs.

Table II. The effect of fenoldopam on human PMN apoptosis Fenoldopam (µmol/L) Time (h)

0

0.001

0.01

0.1

1.0

10

100

6 12 18 24

7.4 ± 4 11 ± 3 22 ± 7 33 ± 10

6.6 ± 3 9±4 23 ± 5 42 ± 4

5.1 ± 2 10 ± 6 27 ± 8 39 ± 3

4.9 ± 2 11 ± 6 23 ± 7 34 ± 8

5.8 ± 3 9±3 21 ± 7 38 ± 8

5.2 ± 3 10 ± 7 22 ± 4 40 ± 8

5.0 ± 2 10 ± 6 18 ± 6 37 ± 7

Human PMN isolated from healthy control subjects were treated with a dopamine D-1 receptor agonist, fenoldopam (0.001-100 µmol/L) at 37°C in 5% CO2 conditions for different time points. PMN apoptosis was assessed according to the percent of the cells with hypodiploid DNA by flow cytometry as described in the “Materials and Methods.” Data are expressed as mean ± SD and are representative of 6 separate experiments. The statistical significance was compared with PMN treated with medium as control.

subsequently by phagocytosis by tissue macrophages is now generally accepted as the mechanism by which effete PMNs at the site of an inflammatory focus are removed.12 When this process is allowed to occur undisturbed, minimal host tissue injury ensues. SIRS is a massive inflammatory reaction resulting from an imbalance between the systemic release of proinflammatory and anti-inflammatory mediators that often leads to the development of MODS or multiple organ failure (MOF).13 As already alluded to, a delay in PMN apoptosis has been implicated in the pathogenesis of SIRS. In this study we confirmed that PMNs isolated from patients with SIRS had a significant delay in their apoptotic program compared with those of healthy controls. The exposure of various cells including chick sympathetic neurons, rat cerebellar granular cells, and

mouse thymocytes and splenocytes to dopamine causes the characteristic biochemical, histochemical, and morphologic electron microscopic changes of apoptosis.14 The effect of dopamine on PMN apoptosis has never been previously studied. The effects of dopamine on PMN apoptosis were assessed flow cytometrically with use of propidium iodide DNA staining and were confirmed by PMN morphologic characteristics. It was demonstrated that there was a significant induction of apoptosis in PMNs isolated from healthy volunteers after incubation with 10 and 100 µmol/L concentrations of dopamine for 12, 18, and 24 hours. The dose of dopamine used in this experiment correlated with that used in previous studies where dopamine was shown to induce apoptosis in cultured chick sympathetic neurons as a possible pathogenetic mechanism in Parkinson’s disease.15 It

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has been previously shown that dopamine triggers the apoptotic death program through an oxidative stress-involved JNK activation signaling pathway.16 To investigate whether dopamine had any effect on PMNs isolated from patients diagnosed with SIRS in the intensive care unit, cells were treated with the same concentrations of dopamine and incubated for the same time periods as previously described. We found that dopamine significantly up-regulated apoptosis at all the time points assessed. PMN microbicidal mechanisms consist of a combination of oxidative and enzymatic processes that appear to be activated simultaneously on initiation of phagocytosis. Not surprisingly, the phagocytic ability of isolated PMNs was significantly reduced in patients with SIRS compared with healthy controls. Furthermore, incubation of SIRS PMNs with dopamine for 1 hour resulted in an increased PMN ingestive capacity. One of the primary mechanisms by which PMNs kill intracellular organisms is through the respiratory burst pathway.17 The oxidative or respiratory burst in isolated PMNs from healthy volunteers and patients with SIRS was triggered by activating the intracellular killing cascade after ingestion of opsonized E coli by the isolated PMNs in vitro. The respiratory burst results in the sequential production of a variety of microstatic and microbicidal reactive oxygen species. PMNs isolated from patients with SIRS demonstrated a significant down-regulation in respiratory burst activity compared with healthy controls. Incubation of isolated PMNs with dopamine for 1 hour did not attenuate this response. These effects indicate that the phagocytic ability of isolated PMN from patients with SIRS impaired and the subsequent intracellular microbicidal capacity are impaired, as is evident by a significant decrease in both phagocytosis and intracellular reactive oxygen intermediate generation. PMN-endothelial interactions play a critical role in sepsis, SIRS, and MODS.18 PMN adherence to activated endothelium comprises a critical step for both transendothelial migration and initiation of potentially deleterious release of PMN products.19 The endothelial molecule intercellular adhesion molecule-1 interacts with the PMN integrin CD11b/CD18 (also known as Mac-1) to strengthen adherence and subsequent cell migration.20 In this study we clearly demonstrated a significant up-regulation of CD11b adhesion receptor expression in patients with SIRS compared with controls. This has profound implications in SIRS in that there is now an enhanced potential for PMN to transmigrate across normal vascular endothelium, potentially provoking host tissue damage. After incubation of PMNs from SIRS patients with dopamine (10 and 100 µmol/L concentrations) for 1 hour, there was a reversal of this enhancement

of CD11b receptor expression toward control values. Although there was no significant difference in CD18 adhesion receptor expression between PMNs from healthy volunteers and those from SIRS patients, after incubation of PMNs from SIRS patients with 100 µmol/L dopamine there was a significant downregulation in CD18 receptor expression. Functionally, dopamine acts by a receptor-mediated mechanism (α- and β-adrenergic or dopaminergic), direct uptake into the cell by an amine uptake transport system, or directly at a gene level. To ascertain whether the effects exhibited on PMNs by dopamine was by a receptor-mediated mechanism, the specific dopamine D-1 receptor agonist fenoldopam was used to mimic the functional effect of dopamine. Although PMNs expressed the dopamine D-1 receptor on their cell surfaces, fenoldopam did not induce PMN apoptosis. This suggests, at least in part, that some of the functional changes seen in PMNs treated with dopamine may not involve specific dopamine receptor activation. This experimental study comparing normal PMNs with those isolated from critically ill patients with SIRS demonstrates a significant diminution in SIRS PMN apoptosis with an associated impairment in both phagocytic potential and bactericidal function. These patients may be further compromised because their circulating PMN population expresses significantly higher levels of CD11b, which is associated with increased PMN adhesion to endothelial cells. These PMNs may damage normal vascular endothelium or transmigrate across a variety of vascular beds, resulting in systemic organ damage. Clinically relevant concentrations of inotropes such as dopamine that increase cyclic adenosine monophosphate may also inhibit cytokine-stimulated up-regulation of endothelial adhesion proteins.21 In conclusion, this study confirms altered PMN function and apoptotic rates in patients meeting the criteria for SIRS. Dopamine, in pharmacologic doses, induces apoptosis in PMN isolated from healthy volunteers and reverses delayed apoptotic rates in PMN isolated from patients with SIRS. Further studies are now required to evaluate the effects of dopamine on PMN apoptosis in vivo and to elucidate the precise proapoptotic mechanism of action of this biogenic amine. REFERENCES 1. Bone RC. The sepsis syndrome: definition and general approach to management. Clin Chest Med 1996;17:175-81. 2. Smith JA. Neutrophils, host defense, and inflammation: a double-edged sword. J Leukoc Biol 1994;56:672-86. 3. Cox G, Crossley J, Xing Z. Macrophage engulfment of apop-

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Surgery August 1999 13. Bone RC. Immunologic dissonance: a continuing evolution in our understanding of the systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Ann Intern Med 1996;125:680-7. 14. Offen D, Ziv I, Gorodin S, Malik Z, Barzilai A, Malamed E. Dopamine-induced programmed cell death in mouse thymocytes. Biochem Biophys Acta 1995;1268:171-7. 15. Ziv I, Melamed E, Nardi N, Luria D, Offen D, Barzilai A. Dupamine induces apoptosis-like cell death in cultured chick sympathetic neurons—A possible novel pathogenetic mechanism in Parkinson’s disease. Neuroscie Lett 1994;170:136-40. 16. Luo Y, Umegaki H, Wang X, Abe R, Roth GS. Dopamine induces apoptosis through an oxidation-involved SAPK/JNK activation pathway. J Biol Chem 1998;273:3756-64. 17. Root RK, Cohen MS. The microbicidal mechanisms of human neutrophils and eosinophils. Rev Infect Dis 1981;3:565-9. 18. Malik AB. Endothelial cell interactions and integrins. New Horizons 1993;1:37-51. 19. Wortel CH, Doerschuk CM. Neutrophils and neutrophilendothelial cell interactions in adult respiratory distress syndrome. New Horizons 1993;1:631-7. 20. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994;84:2068-101. 21. Fortenberry JD, Huber AR, Owens ML. Inotropes inhibit endothelial cellsurface surface adhesion molecules induced by interleukin-1-beta. Crit Care Med 1997;25:303-8.