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Early Expression of Pro- and Anti-Inflammatory Cytokines in Left Ventricular Assist Device Recipients With Multiple Organ Failure Syndrome

Caruso, Raffaele*; Trunfio, Salvatore†; Milazzo, Filippo†; Campolo, Jonica*; De Maria, Renata*; Colombo, Tiziano†; Parolini, Marina*; Cannata, Aldo†; Russo, Claudio†; Paino, Roberto†; Frigerio, Maria†; Martinelli, Luigi†; Parodi, Oberdan*

doi: 10.1097/MAT.0b013e3181de3049
Clinical Cardiovascular

To assess whether the combined evaluation of total Sequential Organ Failure Assessment (t-SOFA) score and pro- and anti-inflammatory cytokine profiles early after left ventricular assist device (LVAD) implant discriminates patients at high risk for multiple organ failure syndrome (MOFS) in the first month post-LVAD, we analyzed plasma interleukin (IL)-6, IL-8, IL-10, IL-1ra, IL-1β, tumor necrosis factor-α (TNF-α), and urine neopterin levels before (day 0) and at 4 hours, 1, 3, 7, 14, and 30 days after LVAD implant in 23 recipients. Eight patients died of MOFS between days 7 and 30 (nonsurvivors). At preimplant, only blood urea nitrogen and age were higher in nonsurvivors than survivors. At 4 hours, IL-8, IL-10, and IL1-ra levels were higher in nonsurvivors than in survivors; t-SOFA was also higher and peaked on day 3 in nonsurvivors. Only IL-8 levels on day 1 were significantly associated with a t-SOFA ≥10 on day 3 (odds ratio 1.10, 95% confidence interval 1.01–1.21, p = 0.04). Neopterin, marker of monocyte activation, increased significantly only in nonsurvivors (p < 0.001). These findings suggest that an activated inflammatory system soon after LVAD implant is implicated in MOFS development. Early monitoring of inflammatory mediators and t-SOFA score may be a valuable tool for outcome prediction in LVAD recipients.

From the *CNR Clinical Physiology Institute-Milan, and †Cardiovascular Department, Niguarda Ca' Granda Hospital, Milan, Italy.

Submitted for consideration January 2010; accepted for publication in revised form March 2010.

Reprint Requests: Oberdan Parodi, MD, CNR Clinical Physiology Institute-Milan, Niguarda Cà Granda Hospital, Piazza Ospedale Maggiore 3, 20162 Milan, Italy. Email: ifcnig@tin.it.

Left ventricular assist device (LVAD) implant in end-stage heart failure (ESHF) has proven effective as bridge to heart transplantation (HT) by allowing recovery of adequate cardiovascular hemodynamic function. However, current 1-year survival rates of LVAD patients range approximately from 50% to 80%.1–3 The death hazard is highest during the first month after LVAD implant, multiple organ failure syndrome (MOFS) being the major cause of death.

The development of MOFS and its impact on the risk of mortality in the intensive care unit (ICU) after cardiac surgery have been described using the Sequential Organ Failure Assessment (SOFA) scoring system.4,5 Multiple organ failure syndrome is influenced by the degree of the immunoinflammatory response, independent of the presence of infection. In LVAD patients, liver dysfunction was shown to be associated to the progressive release of inflammatory mediators,6 such as interleukin (IL)-6, IL-8, and C-reactive protein (CRP). In the setting of trauma or severe acute pancreatitis, early release of anti-inflammatory cytokines has been detected in patients at high risk for MOFS development.7,8 The impact of an early inflammatory response on MOFS development after LVAD implant still needs to be elucidated.

To assess whether the early postimplant release of pro- and anti-inflammatory cytokines would discriminate LVAD recipients at high risk for MOFS at 1 month, we assessed the profiles of pro- and anti-inflammatory mediators by serial postoperative monitoring and the changes in total SOFA (t-SOFA) score in LVAD recipients during the first postoperative month.

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Materials and Methods

In the study, we included 23 patients with ESHF, not amenable to recovery by pharmacological or conventional surgical therapy, who underwent LVAD implantation as bridge to HT according to guideline indications for mechanical support.9 In 22 patients, continuous flow pumps were implanted: 8 De Bakey (MicroMed, Houston, TX), 6 Incor (Berlin Heart GmbH, Berlin, Germany), 4 HeartMateII (Thoratec, Pleasanton, CA), and 4 Levitronix (Levitronix LLC, Waltham, MA) LVADs. A pulsatile flow pump Novacor LVAD (World Heart Inc., Oakland, CA) was implanted in one patient.

The hemodynamic parameters, cardiac index, pulmonary capillary wedge pressure, right atrial pressure, and mixed venous oxygen saturation, were measured by pulmonary artery Swan-Ganz catheter. Left ventricular ejection fraction was quantified by transesophageal echocardiography. Hemodynamic and echocardiography were assessed preoperatively, before anesthesia induction, at 4 hours and 1, 3, and 7 days after weaning from cardiopulmonary bypass (CPB). We calculated the t-SOFA score according to Pätilä et al.4 from preimplant to 1 week after surgery. In sedated patients, the neurological score was computed retrospectively, when sedatives were stopped or alternatively, after their temporary discontinuation.

We measured plasma IL-6, IL-8, IL-10, IL-1β, tumor necrosis factor-α (TNF-α), IL-1 receptor antagonist (IL-1ra), serum CRP (sCRP), and urine neopterin concentrations serially from day 0 to day 7 and subsequently at 14 and 30 days since LVAD implant. Plasma cytokine levels were measured according to the methods of the manufacturer of the enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, for IL-6, IL-8, IL-10, and IL-1ra; Cayman, Ann Arbor, MI, for TNF-α and IL-1β), whereas sCRP concentrations were measured using a Roche/Hitachi 917 Analyzer by high-sensitive immunonephelometric method (Roche Diagnostic GmbH, Mannheim, Germany).

Urinary neopterin levels were measured by an isocratic high performance liquid chromatography (HPLC) method and were normalized by urine creatinine concentrations (Neo/Cr). Briefly, urine samples, stored at −20°C, were thawed and centrifuged; the supernatant was then adequately diluted with chromatographic mobile phase (15 mM of K2HPO4, pH 3.0). Neopterin and creatinine levels were measured using a Kontron instrument (pump 422-S, autosampler 465) coupled to a fluorimetric detector (JASCO FP-1520, λex = 355 nm and at λem = 450 nm) for neopterin detection and to a UV-VIS detector (BIO-RAD 1706, λ = 240 nm) for creatinine determination. Neopterin and creatinine separations were performed at 50°C on a 5-μm Discovery C18 analytical column (250 × 4.6 mm ID, Supelco, Sigma-Aldrich, Bellofante, PA) at flow rate of 0.9 ml/min. The calibration curves were linear over the range of 0.125–1 μmol/L and 1.25–10 mmol/L for neopterin and creatinine levels, respectively. Inter- and intra-assay coefficients of variation were <5%.

The study protocol was approved by the Local Ethics Committee. All subjects gave written informed consent to participate in the study.

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Statistical Analysis

Results are expressed as median and interquartile range (I–III). Log-transformed values were used for graphic display of inflammatory mediators. Between-group differences were assessed by nonparametric Mann Whitney U test for continuous variables and by χ2 or Fisher exact test for categorical variables. Differences of neopterin time course between patient groups were assessed by nonparametric Friedman test. The association between a critical t-SOFA score value ≥10 and inflammatory parameters was tested by univariable logistic regression. A two-tailed p value <0.05 was considered statistically significant.

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Results

Patient age ranged from 29 to 72 years [median 51 (47–60)]. Sixteen patients had idiopathic dilated cardiomyopathy, 6 ischemic cardiomyopathy (ICM), and 1 acute myocarditis. In none of the ICM patients was acute decompensation leading to LVAD implantation triggered by acute myocardial ischemia, infarction, or postcardiotomy HF. At preimplant, median left ventricular ejection fraction and t-SOFA values were 20% (15%–23%) and 6 (5–8), respectively. Of the 23 patients, 8 died because of MOFS at 12 (11–15) days (nonsurvivors), whereas 15 were alive at 1 month (survivors).

Clinical, hemodynamic, and metabolic parameters at preimplant did not differ between survivors and nonsurvivors, with the exception of age and blood urea nitrogen (BUN) levels (Table 1). The greater prevalence of ICM among nonsurvivors is in agreement with their older age, but this imbalance in ESHF etiology did not achieve statistical significance. Preimplant two nonsurvivors were supported by extracorporeal membrane oxygenation; no other between-group difference was observed (Table 1).

Preimplant median t-SOFA scores were similar between survivors [6 (4–7)] and nonsurvivors [6 (5–11)]. Among survivors, 14 continuous axial flow pumps (93%) and 1 continuous centrifugal LVAD (7%) were implanted; whereas among nonsurvivors, 4 continuous axial flow pumps (50%), 3 continuous centrifugal LVAD (37%), and 1 pulsatile flow pump (13%) were implanted (p = 0.05).

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Postoperative Hemodynamic and SOFA Score Profile

Postoperative recovery and hemodynamic improvement were similar in both groups and were maintained during LVAD support. Cardiac index improved already at 4 hours after LVAD implant in both groups [2.75 (2.23–3.25) in survivors vs. 2.80 (2.50–3.20) L · min−1 · m−2 in nonsurvivors; p = 0.45]. Likewise, pulmonary capillary wedge pressure at 4 hours post-LVAD similarly decreased in both groups [7 (7–10) in survivors vs. 8 (7–12) mm Hg in nonsurvivors, p = 0.33). Postoperative right atrial pressures and inotropic equivalents did not change compared with preimplant values.

During the first postoperative week, t-SOFA score increased progressively in both group (p for time <0.01), but at 24 hours, t-SOFA score was persistently higher in nonsurvivors than in survivors (Figure 1A). In nonsurvivors, t-SOFA peaked at 72 hours, and at this time point, all nonsurvivors and seven survivors showed t-SOFA score ≥10, with an overall 65% mortality rate in subjects who reached this score value.

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Postoperative Inflammatory Profile

The levels of sCRP, an established marker of inflammation, paralleled t-SOFA dynamics and peaked at 72 hours (Figure 1B), without any between-group difference.

Pro-inflammatory cytokine levels at preimplant were not different between survivors and nonsurvivors (Figure 2). Interleukin-6 (Figure 2A) peaked at 4 hours, but between-group differences did not achieve statistical significance (p = 0.065). In addition, TNF-α levels were sustained higher in nonsurvivors than survivors, with a peak on day 1 (Figure 2B). Interleukin-1β concentrations were higher in nonsurvivors than survivors only on day 7 [13.4 (12.2–22.6) vs. 10.3 (5.5–14.8) pg/ml, respectively, p = 0.045]. InterleukinL-8 concentrations peaked at 4 hours with significantly sustained higher levels through day 3 in nonsurvivors than in survivors (Figure 2C).

Concentrations of the anti-inflammatory mediators IL-1ra (Figure 3A) and IL-10 (Figure 3B) levels in nonsurvivors also peaked at 4 hours, were significantly higher than in survivors, and decreased to values comparable with those of survivors by day 1.

Neo/Cr, an established marker of monocyte activation, increased progressively only in nonsurvivors (p for time <0.001) (Figure 4).

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Relations Between t-SOFA Score and Early Inflammatory Response

To assess whether any of the cytokines measured within the first postimplant 24 hours was predictive of a t-SOFA suggestive of MOFS, we tested by univariable logistic regression pro- and anti-inflammatory cytokine concentrations obtained at 4 and 24 hours versus the peak t-SOFA score on day 3 hours. Only IL-8 levels at 24 hours were significantly associated with a t-SOFA score ≥10 at 72 hours (odds ratio 1.10, 95% confidence interval 1.01–1.21, p = 0.04) (Figure 5).

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Discussion

This study investigated the early pro- and anti-inflammatory cytokine profiles in LVAD patients and studied their relationship with both t-SOFA score dynamics and MOFS development during the first postoperative month. The main finding of the study is that an overactive inflammatory response in the first 2 days and a progressive monocyte activation in the first week post-LVAD are associated with MOFS development during the first postoperative month. Patients who developed MOFS showed a worsening t-SOFA score already in the first days after the peak acute inflammatory response, whereas IL-8 levels on day 1 were associated with a critical t-SOFA value on day 3.

Left ventricular assist device implant has become an effective therapeutic option for treatment of deteriorating HT candidates. Although scoring systems have been developed to ameliorate risk stratification of ESHF patients candidate to LVAD implant,9 prediction of MOFS during the first postoperative month is still a challenge. The impact of MOFS on the risk of ICU mortality after cardiac surgery4,5 has been described by the use of models purposely developed to measure its dynamics, such as the SOFA system, a six-organ dysfunction/failure score.4 In our study population, preimplant t-SOFA scores were similar between nonsurvivors and survivors, whereas SOFA dynamics in nonsurvivors worsened early after 24–72 hours of ICU stay. These data suggest that inflammatory mechanisms leading to MOFS were already activated at that time.

In critically ill patients, differences in mortality were better predicted by the maximal t-SOFA in the first days of ICU stay.10 total Sequential Organ Failure Assessment higher than 10 or 11 have been associated with elevated mortality rates (10). Accordingly, in all our nonsurvivors, the peak t-SOFA score on day 3 was >10.

Previous studies reported that LVAD implant induces a release of pro-inflammatory cytokines.11,12 Left ventricular assist device patients, deceased of MOFS during the first 3 postoperative months, had higher postoperative CRP, IL-6, and IL-8 levels than survivors.6 In our series, simultaneous release of peculiar inflammatory mediators was observed already at 4 hours after pump activation. Corry et al.11 and Rothenburger et al.12 also found in event-free LVAD recipients an increase in IL-6 and IL-8 levels during the first postoperative hours similar to that observed in ours. Conversely, the exacerbated release of IL-8 and anti-inflammatory cytokines found in our nonsurvivors compared with survivors, suggestive of an overactive inflammatory status, had not yet been reported. In fact, previous studies6,13 performed blood sampling for inflammatory mediators in a more advanced postoperative phase and furthermore described patients who developed MOFS after the first month postimplant.

Moreover, IL-8 and anti-inflammatory cytokines in our nonsurvivors peaked before t-SOFA increment and were more strictly related to outcome than sCRP, routinely employed for inflammatory monitoring of LVAD recipients. These data underscore the critical role of early IL-8, IL-10, and IL1-ra release on the development of MOFS in LVAD recipients during the first month.

Previous studies reported that high IL-6, IL-8, and TNF levels are associated with severe MOFS and often precede its development in cardiogenic shock or trauma patients.7,14,15 Interleukin-6 and mediators of the immune response were shown to increase early after cardiovascular surgery in patients who experienced postoperative MOFS.16 An increase of anti-inflammatory cytokine IL-1ra and IL-10, in addition to uncontrolled release of pro-inflammatory mediators, was proposed as a peculiar pattern of inflammatory response related to the magnitude of MOFS development in the setting of trauma and severe acute pancreatitis.7,8 The simultaneous elevated release of pro- and anti-inflammatory cytokines might be an index of immunoparalysis, deregulated inflammatory activation leading to cellular and humoral dysfunction and reduced immune competence,8,17,18 conditions that favor the development of organ failure. From our preliminary observations, IL-8, IL-10, and IL-1ra assessment 24 hours postimplant holds promise as a potential biochemical predictors of MOFS later on during ICU stay.

In our experience, the early inflammatory response observed in nonsurvivors was not correlated to preimplant cytokine levels or to HF etiology. Older age and higher BUN values, which reflect long-standing heart failure, renal impairment, and catabolic disorder, emerged as the only preimplant factors related to adverse outcome. A deregulated cytokine response was previously reported in chronic HF patients with co-occurring renal dysfunction,19 as evidenced by elevated BUN levels. Aging has been associated with deterioration of the immunoinflammatory system and chronic inflammatory conditions.20 Sandner et al.21 observed that ESHF patients who developed post-LVAD renal failure were older and had greatly increased overall mortality compared with subjects who did not have renal failure. Because LVAD implant has been described to determine an aberrant activation of T cells and monocytes,22 which are a major source of pro-inflammatory cytokines IL-6 and IL-8, we suppose that our elderly LVAD patients with elevated preimplant BUN were probably more susceptible to develop MOFS by an early overactive and deregulated inflammatory response probably linked to LVAD-induced monocyte or T cells hyperactivity present at preimplant.

The higher prevalence of continuous axial flow pumps than continuous centrifugal and pulsatile flow pumps in survivors versus nonsurvivors hints to a lesser impact of the former LVAD type on the development of an early exacerbated inflammatory response and worsening of t-SOFA status. However, the limited number of patients implanted with continuous centrifugal and pulsatile flow pumps and the nonrandomized nature of our observations mandate caution in interpreting this finding.

The early rise of IL-8, IL-10, and IL-1ra, limited to the first hours after LVAD implantation, could play a critical role in the activation of a downstream inflammatory pathway. This hypothesis is reinforced by the observed IL-1β and TNF-α release and monocyte activation, temporally correspondent to t-SOFA increment later on in our nonsurvivors, which point to a crucial role of these parameters in MOFS development. Indeed, TNF-α is mainly produced from the monocyte/macrophage lineage and is recognized as the pivotal cytokine responsible for the clinical manifestations of shock leading to systemic inflammatory response syndrome after sepsis.7 Similarly, IL-1β is a major product of activated human monocytes, tissue macrophages, and neutrophils and shares many of the TNF-α inflammatory properties.

As observed in severe sepsis and septic shock, the predominant mechanisms leading to the development of MOFS are microcirculatory dysfunction and cytopathic tissue hypoxia associated to an unbalanced inflammatory response.23 In LVAD patients developing MOFS, hepatic microcirculation dysfunction and presence of intravascular coagulation, probably due to monocyte and endothelial activation, were observed.6,24 Therefore, a deregulated inflammatory response in the early post-LVAD phase, as observed in our nonsurvivors, might reflect a key signal activating the biological mechanisms leading to MOFS.

Whether therapeutic interventions to block this hyperinflammatory response may decrease the incidence of MOFS in LVAD recipients remains to be established with an appropriate interventional study. Putative treatments targeted at correcting these mechanisms in the preimplant phase are the intravenous administration of free radical scavenging agent, such as N-acetylcysteine, in view of its anti-inflammatory and antioxidant effects observed in a wide range of clinical applications,25,26 as well as intermittent hemofiltration, which has been shown to reduce the elevated circulating levels of IL-8 and monocyte chemoattractant protein-1 in congestive heart failure patients.27

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Conclusion

Our study indicates that an early release of pro- and anti-inflammatory cytokines after LVAD implant parallels an increased t-SOFA in the first days of LVAD support is related to MOFS development during the first postoperative month. Although our findings require validation in a larger patient population, the combined evaluation of early inflammatory profile, in particular IL-8 level at 24 hours and IL-10 and IL-1ra levels at 4 hours, and t-SOFA score seems a potential tool to identify those patients prone to develop MOFS and to guide clinical decision making early after LVAD implant.

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Acknowledgment

Supported, in part, by grants from both FP7-ICT-2007 project grant agreement 224635 (VPH2—Virtual Pathological Heart of the Virtual Physiological Human) and FP7-ICT-2009 project grant agreement 24863 (SensorART—A Remote Controlled Sensorized ARTificial Heart Enabling Patients Empowerment and New Therapy Approaches).

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