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(Chest. 2005;128:1706-1712.)
© 2005 American College of Chest Physicians

Neutrophils, Nitric Oxide, and Microvascular Permeability in Severe Sepsis*

Samjot S. Dhillon, MD; Karthik Mahadevan, MD; Venkata Bandi, MD; Zhilan Zheng, MS; C. Wayne Smith, MD and Rolando E. Rumbaut, MD, PhD

* From the Departments of Medicine (Drs. Dhillon, Mahadevan, and Bandi) and Pediatrics (Ms. Zheng and Dr. Smith), Baylor College of Medicine, Houston; and Medical Care Line (Dr. Rumbaut), VA Medical Center, Houston, TX.

Correspondence to: Rolando E. Rumbaut, MD, PhD, Baylor College of Medicine, CNRC Building, Room 6014, 1100 Bates St, Houston, TX 77030; e-mail: rrumbaut{at}bcm.tmc.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Alterations in microvascular permeability are prevalent in patients with sepsis; a recent study reported that patients with septic shock had increased capillary filtration coefficient (Kf), a noninvasive index of microvascular permeability. We aimed to determine whether patients with severe sepsis had increased Kf, and whether the magnitude of Kf correlated with indexes of nitric oxide activity and neutrophil activation.

Design: Single-center, prospective study.

Setting: Twenty-five–bed ICU of a medical college-affiliated teaching hospital.

Patients: Fifteen ICU patients with severe sepsis based on the American College of Chest Physicians/Society of Critical Care Medicine consensus criteria of 1992, and 10 nonseptic ICU patients as control subjects.

Interventions: Kf was measured by venous congestion plethysmography, plasma nitrate/nitrite (NOx) by chemiluminescence, and neutrophil expression of {alpha}4-integrin (an index of neutrophil activation) by flow cytometry.

Measurements and results: Septic patients had higher Kf than nonseptic control subjects. Kf of septic patients was 5.6 ± 0.6 x 10-3 mL·min-1·100 mL tissue-1·mm Hg-1 (mean ± SEM, mL·min-1·100 mL tissue-1·mm Hg-1 = Kf units [KfU]) as compared to 3.9 ± 0.5 x 10-3 KfU in nonseptic ICU patients (p < 0.05). There was no correlation between plasma NOx and Kf, or between neutrophil {alpha}4-integrin expression and Kf in patients with sepsis. Septic patients with clinical evidence of edema had significantly higher Kf (p < 0.05) than nonedematous septic patients.

Conclusions: ICU patients with severe sepsis have increased Kf, a noninvasive index of microvascular water permeability. The magnitude of hyperpermeability did not correlate with NOx levels or one index of neutrophil activation ({alpha}4-integrin expression). Presence of peripheral edema in these patients was associated with increased Kf, and may represent a simple, clinical indicator of altered microvascular permeability in sepsis.

Key Words: {alpha}4-integrin • capillary permeability • neutrophils • nitric oxide • plethysmography • sepsis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Microvascular permeability refers to the ease of passage or transport of volume and solutes across exchange vessels (eg, capillaries and postcapillary venules) and is a major determinant of nutrient delivery to tissues. Pathologic alterations of this process, or hyperpermeability, contribute to the pathogenesis of sepsis,123 a leading cause of death in adult ICUs.4 Several lines of evidence support a role for nitric oxide (NO) in hemodynamic alterations in sepsis.5 Experimental sepsis is characterized by enhanced expression and activity of inducible NO synthase,67 and both experimental and clinical sepsis are associated with enhanced plasma markers of NO (nitrate/nitrite [NOx]).7891011 Further, plasma NOx levels correlate inversely with arterial BP,11 and NO synthase inhibitors improve some hemodynamic abnormalities in several sepsis models.812 Although work from our laboratory and others131415 demonstrate that NO can enhance single-vessel permeability coefficients in animal models, it is unclear whether NO contributes to hyperpermeability in human sepsis. Similarly, although an association between neutrophil activation and altered permeability has been described in experimental sepsis,1617 the role of activated neutrophils on these abnormalities in septic humans is unknown. Neutrophil {alpha}4-integrin expression has been described as a marker of neutrophil activation in sepsis, elevated in septic patients, but infrequently detected in healthy subjects or those with focal infections.18 Whether neutrophil {alpha}4-integrin expression correlates with severity of disease or hyperpermeability in sepsis remains to be determined.

The aim of this study was to understand the relationship between neutrophil activation, NO, and microvascular hyperpermeability in septic patients. The global hypothesis was that NO and activated neutrophils contribute causally to the systemic permeability alterations in patients with sepsis. Thus, we anticipated positive correlations between capillary filtration coefficient (Kf) [an index of microvascular permeability], NOx, and neutrophil {alpha}4-integrin expression.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The study protocol was approved by the Institutional Review Board of Baylor College of Medicine and performed in accordance with the Declaration of Helsinki19 and Good Clinical Practice Code. Informed consent was obtained from all subjects. Both septic and nonseptic control subjects were recruited prospectively from the 25-bed medical ICU/coronary care unit of Ben Taub General Hospital, a 647-bed, medical college-affiliated teaching hospital, from August 2002 through June 2003. The recruitment was done within the first 48 h of admission to the ICU or within 48 h of clinical onset of sepsis if the subjects were ICU inpatients.

Inclusion Criteria
Septic subjects were recruited based on the American College of Chest Physicians/Society of Critical Care Medicine consensus criteria of 1992 for severe sepsis.20 Nonseptic control patients were adults (> 18 years of age) recruited from the same ICU who did not fulfill the above definition of sepsis and who did not meet any of the exclusion criteria shown below.

Exclusion Criteria
Patients who met one or more of the following criteria were excluded: (1) confused, agitated, or unable to cooperate for Kf measurements; (2) pregnant or breastfeeding; (3) diastolic BP < 50 mm Hg; (4) acute hemodynamic instability or rapidly escalating doses of vasopressors; (5) receiving oral or IV nitrates; (6) body mass index < 17 kg/m2 or > 35 kg/m2; (7) deep venous thrombosis; (8) immediate postoperative period; (9) acute clinical pancreatitis without a proven source of infection; (10) receiving chemotherapeutic agents within the prior 21 days; and (11) having undergone cardiopulmonary resuscitation during the current hospital admission.

Assessment of Microvascular Permeability
We measured Kf, a noninvasive index of microvascular water permeability, using venous congestion plethysmography (VCP), a well-validated method to quantify Kf.21222324 The assumptions and limitations of this method have been described in detail.21

Briefly, the method involves sequential cumulative step increases in limb venous pressure with an occlusive cuff placed around the thigh. It is connected to a built-in air pump, which allows the application a stepwise increase in pressure based on a preset protocol. This results in a change in limb volume, measured with an electromechanical sensor (Filtrass 2001, Software version 2.03 d; DOMED Medizintechnik GmbH; Munich, Germany) placed around the calf. Cuff pressure (Pcuff) and sensor signals are passed via an analog/digital converter card to a personal computer and recorded continuously. The VCP recordings are analyzed off-line.

The application of external pressure results in an increase in limb volume due to changes in both vascular volume and fluid filtration. Pressure steps of ≤ 10 mm Hg are applied to prevent venoarteriolar reflex that can decrease blood flow to the extremity on application of external pressure.2122 When Pcuff is elevated above the existing venous pressure, venous outflow ceases while blood continues to flow into the limb. A rapid increase in limb circumference attributable to this altered vascular volume can be seen. This is followed by a slow increase in limb volume, which reflects fluid filtration across the microvasculature (Fig 1 ). As long as venous pressure is maintained significantly lower than arterial diastolic pressure, limb circumference (ie, limb volume) changes as a linear function of the change in venous pressure.21 The value of limb fluid filtration components (rate of fluid filtration [Jv]) are obtained from a series of pressure steps, as shown in Figure 1. The slope of the relationship (Jv/change in pressure) represents Kf (volume flux/tissue volume/pressure changes), as depicted in Figure 2 .21 The intercept on the x-axis is the Pcuff that must be exceeded to generate net fluid filtration (Pvi). The time constant of the initial rapid vascular volume change is < 15 s, and the total completion time of the change in vascular compliance is assumed at five time constants (75 s) for small pressure steps.21 As a result, if the slope of the limb circumference record is estimated after this time (we used 2 min as the initial time for slope determination), it will almost exclusively reflect microvascular fluid filtration.



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  		Figure 1. Tracing from VCP at four Pcuffs. The dashed lines reflect the slope used to determine Jv at each pressure.

 


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  		Figure 2. Calculation of Kf as the slope of the relationship between rate of Jv and Pcuff.

 
To avoid bias, interpretation of all VCP data and determination of Kf and Pvi was done off-line by an independent investigator, blinded to the patient’s group or diagnosis. During this analysis, tracings exhibiting motion or other artifacts, which precluded accurate assessment of Kf were excluded.

Plasma NOx
Blood was collected in a heparinized blood collection tube and centrifuged at 3,000g for 15 min at room temperature. Plasma was stored at – 80°C until analyzed for NOx. NOx levels in plasma were determined using a NO chemiluminescence analyzer (Model 280; Sievers; Boulder, CO), calibrated with serial dilutions of 1 mmol/L NaNO3. This device has high sensitivity for measuring NO based on a gas-phase chemiluminescent reaction between NO and ozone.25 Vanadium III-HCl (Aldrich Chemical Co.; Milwaukee, WI) was used to reduce nitrite and nitrate to NO, and chemiluminescence was analyzed per the specifications of the manufacturer.

{alpha}4-Integrin Expression
Neutrophil expression of {alpha}4-integrin was determined by flow cytometry.18 Mouse monoclonal anti-human {alpha}4-integrin antibody conjugated with phycoerythrin (BD Pharmingen; San Diego, CA) was added to 100 µL of blood (20 µL antibody/106 leukocytes) anticoagulated with citrate-phosphate-dextrose (Baxter; Deerfield, IL). After incubation in the dark at 4°C for 20 min, cells were washed in phosphate-buffered saline solution, and then the erythrocytes were lysed (FACS lysing solution; Becton Dickinson; Palo Alto, CA). Leukocytes were again washed three times in phosphate-buffered saline solution and fixed with 1% paraformaldehyde. Neutrophil fluorescence was analyzed on a flow cytometer (FACScan; Becton Dickinson); neutrophils were identified and gated by forward- and side-scatter patterns. Phycoerythrin-labeled mouse IgG1 isotype controls were used for each set of experiments. As an additional control, {alpha}4-integrin expression of neutrophils derived from either of eight healthy volunteers (routine blood donors of the Leukocyte Biology Laboratory of Baylor College of Medicine, with appropriate informed consent) was determined on each day that a patient’s sample was processed.

Protocol
All eligible septic subjects had Kf measurements planned for days 0, 3 and 7, and blood samples for days 0 and 7, for markers of increased NO production and neutrophil activation. The days for Kf measurements were selected to provide serial measures during 1 week, while minimizing disruptions to care in a busy teaching ICU (each measure required approximately 1 h of uninterrupted access to the patient’s bedside). The study was performed only for the duration of subjects’ stay in the ICU; the protocol was terminated if the subjects were transferred out of the ICU. Kf measurements were done only once in control subjects. Presence of peripheral edema (pedal or sacral) was noted on the days of Kf measurements.

Statistical Analysis
Data are shown as mean ± SEM. A Pearson correlation coefficient was used to analyze relationship between markers of NO activity, neutrophil activation, and Kf for values determined simultaneously. For the comparison of Kf between the septic and control groups, we obtained an average Kf for each patient who had multiple determinations of Kf (ie, three values were measured in some patients; others had only one or two values due to shorter ICU stays or tracings excluded due to motion artifacts). One-way analysis of variance with a Scheffé post hoc test was performed with statistical software (StatView 5.0; SAS Institute; Cary, NC) and a personal computer. A p value < 0.05 was considered statistically significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A total of 23 septic and 20 control patients were recruited. Four patients in each group were excluded prior to obtaining Kf data, as they were unable to remain still for the duration of procedure. In addition, Kf data obtained from four septic patients and from six control patients were rejected because the independent observer determined that motion artifacts precluded accurate determination of Kf. Therefore, data from 15 septic and 10 control patients were included in the final analysis. The mean time interval from the onset of severe sepsis to data collection was 30.1 ± 3.4 h. The characteristics of patients with severe sepsis are given in Table 1 . The control patients were admitted for upper GI bleeding (n = 2), diabetic ketoacidosis (n = 3), pneumonia (n = 1), COPD (n = 1), acute renal failure/abdominal wall abscess (n = 1), diffuse alveolar hemorrhage (n = 1), and angioedema (n = 1). There was no difference between age and sex distribution of the patients with severe sepsis or nonseptic ICU control subjects (Table 2 ). The septic patients had significantly higher mean APACHE (acute physiology and chronic health evaluation) II scores and lower serum albumin level.


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  		Table 1. Clinical Characteristics of Patients With Severe Sepsis

 

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  		Table 2. Comparison between Septic Patients and Nonseptic Control Subjects*

 
The patients with severe sepsis had a Kf of 5.6 ± 0.6 x 10–3 (mean ± SEM, mL·min–1·100 mL tissue-1·mm Hg–1 = Kf units [KfU]), significantly higher than that of nonseptic ICU control subjects (3.9 ± 0.5 x 10-3 KfU, p < 0.05). The mean Pvi of septic patients was higher than that of nonseptic control subjects (16.9 ± 2.4 mm Hg vs 13.2 ± 2.0 mm Hg, respectively), although the difference was not statistically significant (p = 0.3). Ten of the 15 septic patients received vasopressors at the time of Kf measures and 5 septic patients did not; mean Kf did not differ statistically between these groups (5.9 ± 0.7 x 10–3 KfU vs 4.8 ± 1.0 x 10–3 KfU, respectively, p = 0.3)

Mean plasma NOx level in the septic group was 133.6 ± 47.7 µmol/L. However, there was considerable variability in NOx levels, including three patients with values < 20 µmol/L; no correlation was evident between Kf and plasma NOx levels during ICU stay (Fig 3 , top, A).



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  		Figure 3. Top, A: Correlation between Kf expressed in KfU (KfU = mL·min-1·100 mL tissue-1·mm Hg-1) and plasma NOx, shown on a logarithmic scale. Bottom, B: Correlation between Kf and percentage of neutrophil (PMN) {alpha}4-integrin expression. NS = not significant.

 
Septic patients had enhanced neutrophil {alpha}4-integrin expression as compared to healthy volunteers (14.9 ± 5.2% vs 2.7 ± 0.5%, p < 0.05). The peak value for {alpha}4-integrin expression was 54.5%; two thirds of all samples from septic patients (and none from the healthy volunteers) had > 7% neutrophil {alpha}4-integrin expression. However, there was no correlation between Kf and neutrophil {alpha}4-integrin expression (Fig 3, bottom, B).

Peripheral edema was absent in the control patients, while it was evident in 7 of the 15 patients with severe sepsis. Septic patients with edema had significantly higher Kf than those without edema (Fig 4 ). There was no difference in serum albumin concentrations between septic patients with edema and those without edema (2.0 ± 0.2 g/dL and 2.0 ± 0.4 g/dL, respectively). Further, no correlation was present between Kf and serum albumin concentration.



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  		Figure 4. Kf expressed in KfU (mL·min-1·100 mL tissue-1·mm Hg-1) in patients with severe sepsis with and without edema. *p < 0.05.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The phenomenon of increased microvascular permeability in sepsis has been of considerable interest to clinicians and researchers. Increases in microvascular permeability to solutes and water have been described in animal models of sepsis by a variety of techniques.2627 Clinically, noncardiogenic pulmonary edema (ARDS) and peripheral edema are frequent manifestations of sepsis.228 Objective increases in systemic microvascular permeability in sepsis have been described in limited human studies, by demonstrating increased transvascular escape of albumin,3 as well as by increases in edema fluid/serum protein ratios in skin.2

Christ et al29 used venous congestion plethysmography and demonstrated increased microvascular permeability in 10 patients with septic shock, compared to 18 patients with shock of nonseptic origin (cardiogenic, hemorrhagic, multiple trauma). The mean Kf value of patients with septic shock in that study was 6.1 ± 0.4 x 10-3 KfU and of nonseptic shock patients was 3.5 ± 0.3 x 10-3 KfU. In our study, Kf of patients with severe sepsis and septic shock was 5.6 ± 0.6 x 10-3 KfU, and of nonseptic ICU control subjects (not in shock) was 3.9 ± 0.5 x 10-3KfU. Thus, our findings are consistent with those of Christ et al29 and confirm that Kf is increased in severe sepsis as well as septic shock. Although we found no difference in mean Kf between patients with severe sepsis and those with septic shock, a larger study would be needed to determine whether systemic microvascular permeability differs between these two groups. Further, it is worth noting that the control group in our study included two patients with localized infections (pneumonia and abdominal wall abscess) but without systemic manifestations of sepsis. Of interest, their Kf was 5.0 and 7.2 x 10-3 KfU, comparable to the mean Kf of the septic group of 5.6 x 10-3 KfU. Although it is tempting to speculate that systemic alterations in permeability may be evident in patients with localized infections prior to the onset of sepsis, additional studies are warranted to test this hypothesis.

A limitation in the assessment of microvascular permeability in this study was a significant rate of data rejection. The VCP takes approximately 35 min, and subjects have to remain still during the measurements; several subjects were excluded because they were unable to do this. Even minor head or arm movements may shift the baseline, impairing data interpretation; similar rejection rates have been reported previously.21 The duration of the test makes it impractical for hemodynamically unstable patients. In addition, patients with hypotension are not suitable candidates for these measures (we excluded those with diastolic BP < 50 mm Hg), since insufficient pressure steps are possible to obtain adequate Kf values. Despite these limitations, however, this method is perhaps the most practical noninvasive method available to obtain quantitative measures of microvascular permeability in humans.

Although some animal data suggest that NO increases single-vessel permeability coefficients to water and solutes,131415 the role of NO in the permeability alterations of human sepsis is unclear. Due to the short half-life of NO, its end products (NOx) are measured frequently. Increased NOx has been described in several studies on sepsis.7891011 The mean value of NOx determined in the septic patients of our study was 133.6 ± 47.7 µmol/L. The maximum value of NOx observed was 529 µmol/L, while three patients had NOx values < 20 µmol/L. These results are similar to those of Strand et al,10 who found a mean level of 144 ± 39 µmol/L in septic patients as compared to 20 ± 3 µmol/L in control subjects. The peak NOx level in that study was 662 µmol/L, and 3 of 16 patients with hyperdynamic septic shock had normal or subnormal NOx levels.

We found no correlation between Kf and plasma NOx; this finding is not totally unexpected. Several studies910113031 have demonstrated wide variability of changes in NOx of septic patients. Classifying septic patients into high and low NOx responders did not show pathophysiologic differences between the two groups.30 Further, NOx levels are subject to significant fluctuation as a result of dietary changes. In one study, NOx levels of healthy subjects increased fivefold following a meal rich in nitrate and nitrite, reaching levels comparable to those of patients with sepsis.30 NOx levels may also be influenced by plasma protein concentrations, changes in distribution volumes, and renal failure during sepsis.1030 In view of these limitations, despite the lack of correlation between NOx and Kf, we cannot exclude a role for NO in microvascular permeability alterations in human sepsis.

We selected {alpha}4-integrin expression as a marker of neutrophil activation, based on data from Ibbotson et al,18 who studied eight septic patients and found enhanced levels of {alpha}4-integrin on all of them, A functional role for this integrin was demonstrated in mediating neutrophil adhesion onto vascular cell adhesion molecule-1 under flow. On average, septic patients had 30 to 40% neutrophil {alpha}4-integrin expression in their study, as compared to 0 to 5% in control subjects. Our findings were consistent with those of Ibbotson et al18; septic patients had 15% neutrophil {alpha}4-integrin expression compared to < 3% in healthy volunteers. However, we found no correlation between Kf and neutrophil {alpha}4-integrin expression. As in the case of NOx, our data argue against the presence of a large correlation between Kf and neutrophil {alpha}4-integrin expression. However, given the sample size, a small or moderate correlation between these parameters cannot be excluded. Further, {alpha}4-integrin expression is one of many markers of neutrophil activation, and the relationship between Kf and other indicators of neutrophil activation remain to be determined.

Peripheral edema was present in seven of the septic patients in our study, a finding that has been described previously.228 This may occur due to alterations in permeability and reductions in plasma colloid osmotic pressure, with increased fluid leakage into the interstitium that overcomes the resorptive capacity of the lymphatics.32 Reductions in plasma colloid osmotic pressure, in the absence of increased permeability, would result in no change in Kf,21 though may lead to a reduction in the pressure axis intercept, Pvi (Fig 2). Although the septic patients had lower serum albumin concentration (and thus lower colloid osmotic pressure) than nonseptic patients, there was no correlation between serum albumin and Kf. Similarly, there was no difference in serum albumin concentration between septic patients with and without edema. Interstitial edema is present in several organs in sepsis; however, only peripheral (eg, pedal, sacral) edema can be detected on physical examination. Of note, tissue edema due to increased microvascular permeability has been reported to develop prior to the onset of organ dysfunction.33 In the present study, septic patients with peripheral edema had a significantly higher Kf than patients without edema. This suggests that peripheral edema may be a simple, practical index of increased microvascular permeability in sepsis. Whether this clinical finding might help identify patients at higher risk for hyperpermeability-associated organ dysfunction remains to be determined.

In summary, we show that ICU patients with severe sepsis have increased Kf, an index of microvascular water permeability. The magnitude of hyperpermeability did not correlate with NOx levels or one index of neutrophil activation ({alpha}4-integrin expression). The presence of peripheral edema in these patients was associated with increased Kf and may represent a simple and practical clinical indicator of altered microvascular permeability in sepsis.


   Footnotes
 
Abbreviations: Jv = rate of fluid filtration; Kf = capillary filtration coefficient; KfU = capillary filtration coefficient unit; NO = nitric oxide; NOx = nitrate/nitrite; Pcuff = cuff pressure of congestion plethysmography device; Pvi = cuff pressure that must be exceeded to generate net fluid filtration; VCP = venous congestion plethysmography

Support was provided by National Heart, Lung, and Blood Institute HL-070537.

Received for publication December 17, 2004. Accepted for publication March 23, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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