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Published online before print October 17, 2007, 10.1097/PSY.0b013e3181576118
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Psychosomatic Medicine 69:709-716 (2007)
© 2007 American Psychosomatic Society

ORIGINAL ARTICLES

Stimulated Production of Proinflammatory Cytokines Covaries Inversely With Heart Rate Variability

Anna L. Marsland, PhD, RN, Peter J. Gianaros, PhD, Aric A. Prather, MS, J. Richard Jennings, PhD, Serina A. Neumann, PhD and Stephen B. Manuck, PhD

From the Behavioral Immunology Laboratory (A.L.M., A.A.P.), Department of Psychology, University of Pittsburgh; Department of Psychiatry (P.J.G., J.R.J.), University of Pittsburgh School of Medicine, Departments of Psychiatry and Behavioral Sciences and Psychology (S.A.N.), Eastern Virginia Medical School, Behavioral Physiology Laboratory (S.B.M.), Department of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania.

Address correspondence and reprint requests to Anna L. Marsland, Behavioral Immunology Laboratory, Department of Psychology, 3943 O'Hara Street, Pittsburgh, PA 15260. E-mail: marsland{at}pitt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Objective: To examine whether high-frequency heart rate variability, an indirect measure of parasympathetic (vagal) control over variations in heart rate, is associated with immune reactivity to an in vitro inflammatory challenge. Convergent evidence from the animal literature shows that the autonomic nervous system plays a key role in regulating the magnitude of immune responses to inflammatory stimuli. Signaling by the parasympathetic system inhibits the production of proinflammatory cytokines by activated monocytes/macrophages and thus decreases local and systemic inflammation. As yet, no direct human evidence links parasympathetic activity to inflammatory competence.

Methods: We examined the relationship of variations in heart rate, recorded during paced respiration, to lipopolysaccharide-induced production of the inflammatory cytokines interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-{alpha}, and IL-10 among a community sample of 183 healthy adults (mean age = 45 years; 59% male; 92% White, 7% African-American).

Results: Consistent with animal findings, higher derived estimates of vagal activity measured during paced respiration were associated with lower production of the proinflammatory cytokines TNF-{alpha} and IL-6 (r = –.18 to –.30), but were not related to production of the anti-inflammatory cytokine IL-10. These associations persisted after controlling for demographic and health characteristics, including age, gender, race, years of education, smoking, hypertension, and white blood cell count.

Conclusions: These data provide initial human evidence that vagal activity is inversely related to inflammatory competence, raising the possibility that vagal regulation of immune reactivity may represent a pathway linking psychosocial factors to risk for inflammatory disease.

Key Words: proinflammatory cytokines • heart rate variability • inflammation • autonomic nervous system • parasympathetic system

Abbreviations: ANS = autonomic nervous system; HRV = heart rate variability; HF = high frequency; LF = low frequency; rMSSD = root mean square of successive differences; IL = interleukin; TNF = tumor necrosis factor; LPS = lipopolysaccharide; WBC = white blood count; BMI = body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
The autonomic nervous system (ANS) plays a key role in regulating the immune system, specifically by modulating the magnitude of immune responses to inflammatory stimuli (1–3). Emerging clinical and epidemiological evidence indicates that dysregulated activity in the sympathetic and parasympathetic divisions of the ANS is associated with increased risk for inflammatory diseases, such as coronary atherosclerosis, diabetes, rheumatoid arthritis, and other autoimmune disorders (4–7). Animal models suggest that one form of dysregulated autonomic activity—low levels of parasympathetic activity—can increase the magnitude of the inflammatory response, providing evidence for a mechanism by which dysregulated autonomic-immune homeostasis can increase risk for inflammatory diseases. As yet, however, human evidence has not directly linked low levels of parasympathetic activity to immune reactivity to inflammatory stimuli. Accordingly, in the present study of healthy community volunteers, we tested whether greater immune reactivity to an in vitro inflammatory challenge would be associated with lower levels of high-frequency heart rate variability (HF-HRV), an indirect measure of parasympathetic (vagal) control over time-related variations in heart rate.

An inflammatory response begins when monocytes/macrophages are activated by pathogens or tissue damage to release proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-{alpha}. Of these proinflammatory mediators, TNF-{alpha} is a necessary mediator of local and systemic inflammation that is thought to play a role in the early pathogenesis of inflammatory disease. The magnitude of cytokine response to immune activation is critical; insufficient response may leave the organism more vulnerable to infection, and excessive response can increase risk for inflammatory diseases, such as cardiovascular and autoimmune diseases and diabetes. A number of mechanisms suppress macrophage activation and prevent the excessive production of proinflammatory cytokines, including humoral mechanisms, such as the local release of anti-inflammatory mediators by activated immune cells and the systemic release of glucocorticoids after activation of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, recent evidence suggests that the parasympathetic division of the ANS can regulate inflammation in real time via the vagus nerve (8). It is hypothesized that afferent vagal neurons relay sensory information regarding early inflammatory activity to the brain (9), which results in reflexive activation of the efferent vagal fibers leading to the suppression of proinflammatory cytokine release (10). This efferent pathway, termed the "cholinergic anti-inflammatory pathway" (3), is proposed to play an important role in preventing excessive inflammatory responses and maintaining health (10).

The activated efferent vagal nerve exerts anti-inflammatory action through the release of acetylcholine (ACh) in organs of the reticuloendothelial system and by activation of nicotinic ACh receptors expressed on macrophages and other immune cells involved in the inflammatory response (11). Converging evidence from in vivo animal studies provides evidence that activation of the efferent vagal pathway results in the decreased synthesis and release of proinflammatory cytokines. For example, direct electrical stimulation of the efferent vagus nerve inhibits the synthesis of TNF-{alpha} in multiple organs, decreases circulating levels of TNF-{alpha}, and attenuates serum TNF-{alpha} levels during endotoxemia and other diseases associated with excessive cytokine release (1,12). Similarly, cholinergic agonists inhibit the release of TNF and other proinflammatory, but not anti-inflammatory cytokines, from lipopolysaccharide (LPS)-stimulated human macrophages (2,11). Conversely, rats subjected to vagotomy show an increased systemic TNF-{alpha} response to inflammatory stimuli and develop endotoxemic shock more quickly than animals who receive sham surgery (1,2). Further evidence for the cholinergic anti-inflammatory path comes from the study of transgenic mice with deficient ACh receptors. When compared with wild types, these mice show higher serum elevations of TNF, IL-1, and IL-6 in response to LPS and no suppression of serum TNF levels after electrical stimulation of the vagus nerve (11). Taken together, these findings suggest that activation of the efferent vagal pathway plays a role in the tonic inhibitory control of the release of proinflammatory mediators, particularly TNF-{alpha}, and thus the inflammation response.

A growing human literature also links low levels of cardiac vagal activity, as measured by noninvasive indicators of heart rate variability (HRV), both to psychosocial factors known to predict susceptibility to inflammatory diseases, such as trait negative affect and depression (4,13), and to increased risk of infectious disease mortality (14). If, as the animal literature suggests, tonic vagal activity modulates innate immune competence, then individuals who express lower levels of vagal control of heart rate may be more vulnerable to inflammatory disease. To begin to explore this possibility, we extend prior animal literature to an examination of relationships between vagal activity and inflammatory competence in humans.

For this purpose, we examined relationships between indicators of cardiac vagal activity, derived from spectral analyses of time-related variations in resting heart rate measured during paced respiration, and the ability of immune cells to produce pro- and anti-inflammatory cytokines after in vitro stimulation with the bacterial product LPS in a community sample of relatively healthy middle-aged volunteers. Stimulated cytokine production varies substantially across individuals and seems to be a stable trait (15) that may be associated with dispositional differences in parasympathetic (vagal) activity. HF-HRV provides a reasonably stable measure of vagal control over time-related variations in heart rate (16). Based on existing animal evidence, it was hypothesized that lower HF-HRV would be associated with greater stimulated production of TNF-{alpha}, and possibly IL-6 and IL-1ß, but would not be related to the production of the anti-inflammatory cytokine IL-10.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 
Participants
Participants were 183 community volunteers (59% male, 92% White; 7% African American, 1% other), ranging in age between 30 and 54 years, enrolled in the Adult Health and Behavior project—a registry of diverse behavioral and biological measurements among adults recruited by mass-mail solicitation from Western Pennsylvania (principally Allegheny County). Data were collected between 2001 and 2005. Eligibility criteria included good general health, with no history of myocardial infarction or cancer treatment within the past year, chronic kidney or liver disease, major neurological disorders, schizophrenia, or other psychotic illness. Women who were pregnant were also ineligible. Current use of cardiovascular (except antihypertensive and lipid-lowering medications), psychotropic, glucocorticoid, or weight-loss drugs was also exclusionary for the present analyses. Two subjects were dropped from data analysis due to signs and symptoms of current infection, including stimulated cytokine levels and white blood counts that were >2 standard deviations above the sample mean. Four subjects with diabetes and two subjects taking ß blockers were also dropped, resulting in a final sample of 175 subjects. Informed consent was acquired in compliance with guidelines of the University of Pittsburgh Institutional Review Board.

HRV Protocol and Assessment
To measure resting cardiac vagal activity, heart rate was recorded continuously using a 2-lead electrocardiogram (ECG) attached bilaterally to the wrists throughout a 5-minute period of paced respiration. Respiration was paced at a rate of 11 breaths/minute based on pilot observations that affirmed this is a comfortable rate for most people. During pacing, participants were asked to breathe naturally in response to two auditory tones signaling them to inhale and exhale. Respiration was monitored throughout using a thoracic strain-gauge. During the paced respiration, participants were seated and asked to remain stationary in a temperature-controlled recording chamber, thus controlling for the possible contributions of individual differences in respiratory frequency and movements to HRV estimates (17). Before testing, which took place in the morning, participants were asked to avoid caffeine for 4 hours, alcohol and exercise for 12 hours, and over-the-counter medications for 24 hours. ECG signals were digitized at a sampling rate of 1000 Hz (LabView acquisition software, National Instruments Corporation, Austin, Texas). Before calculating estimates of HRV, the digitalized ECG signals were examined and artifactual detections of R-wave occurrences were corrected. ECG data from 11 participants were lost due to cardiac ectopy, equipment malfunction, or noisy ECG signal, resulting in a final sample of 166 participants. All procedures and analyses followed Task Force guidelines (18).

Derivation of power in different frequency bands was accomplished using the point-process analytic approach as realized in the PSPAT program (for spectral analysis of point events) (19). Estimates of integrated spectral power were derived for the high (pacing)-frequency (HF: 0.165–0.195 Hz) and low-frequency (LF: 0.06–0.15 Hz) bandwidths. The HF bandwidth was narrowed from Task Force guidelines to the paced respiration frequency to more accurately target the source of variability. We also ran all analyses using a broader HF bandwidth (0.15–0.39 Hz) and this did not appreciably change any results. As a putative measure of sympathovagal balance, the LF/HF ratio (LF (ms2)/HF (ms2)) was also calculated. In addition to spectral (frequency domain) measures, the square root of the mean of successive differences in IBIs (rMSSD) was calculated to provide a supplementary time-domain measure of cardiac parasympathetic activity, as both HF power and rMSSD have been shown to provide reliable estimates of cardiac vagal activity (18,20,21).

Stimulated Inflammatory Cytokine Levels
Blood samples for the measurement of stimulated production of inflammatory cytokines were collected on a different day 2 to 4 weeks before HRV testing. On this occasion, participants were asked to fast for 8 hours and avoid exercise for 12 hours and alcohol for 24 hours before attending a morning session. At this visit, the project nurse completed a medical history interview, recorded any current symptoms of infection and medication use, obtained measurements of height and weight for the determination of body mass index (BMI) (kg/m2), and took three manual blood pressure measurements, before drawing a 40-ml blood sample in citrate-treated vacutainer tubes.

Whole Blood Stimulation Assay. The stimulation assay was set up within 2 hours of the blood draw. Whole blood was stimulated with LPS (serotype 026:B6, Sigma) at a final concentration of 2.5 µg/ml without antibiotics in polypropylene tubes under sterile conditions (stimulated sample). Control cultures, containing whole blood without LPS, were set up in parallel to measure spontaneous production of cytokines (unstimulated sample). The tubes were incubated at 37°C with 5% CO2 humidified atmosphere for 24 hours. The tubes were then removed and centrifuged at 1000 g for 10 minutes. Supernatants were collected and frozen at –80°C until the study was complete.

Multiplex Assay
At the end of the study, LPS-stimulated and unstimulated plasma samples were thawed and analyzed in batches using the multiplex analysis system, permitting the simultaneous quantification of different cytokines in a small sample volume. Multiplex bead kits (Biosource, Camarillo, California) based on the principle of solid phase sandwich immunoassays were employed. All reagents, working standards, and samples were prepared as per the manufacturer's specifications and were run in duplicates (22). The plates were read within 24 hours using the Bio-plex Reader (Luminex 100, Luminex Corporation, Austin, Texas). Stimulated levels of IL-1ß, IL-6, TNF-{alpha}, and IL-10 were determined using Bio-Plex Manager Software (Bio-rad Corporation, Hercules, California), interpolating from the standard curve (Logistic-5PL curve fit, Brendan Technologies, Carlsbad, California). In addition, pooled plasma controls were included on all plates to further assure assay reliability. The inter- and intra-assay coefficients of variability for IL-1ß, IL-6, and TNF-{alpha} were <10%. Luminex multiplex technology has been demonstrated to be a valid alternative method to ELISA for the quantification of these cytokines (23). As a result of sample dilution, levels of IL-10 were too low to be reliably quantified by these methods. Thus, when possible, we reassessed stimulated levels of IL-10 using a quantitative sandwich enzyme immunoassay kit run per the manufacturer's directions (R & D Systems, Minneapolis, Minnesota). The average CV between samples was <5%. In all cases, stimulated cytokine production was quantified by subtracting cytokine levels in unstimulated samples from the stimulated levels. A complete blood count was also performed to evaluate the absolute numbers of white blood cells (WBCs) in peripheral circulation.

Standard Covariates
A number of variables were assessed that might provide alternative explanations for associations between HRV estimates and cytokine production, including age, gender, race, years of education, smoking status (current smoker versus ex smoker/nonsmoker), BMI, systolic blood pressure (SBP) and diastolic blood pressure (DBP) (average of first two manual pressures), and self-reported medical history and medication use.

Statistical Analyses
All analyses were performed using SPSS for Windows (version 14.0). Measures of stimulated cytokine levels, HRV, SBP, circulating WBC numbers and BMI, were log normal (base e) transformed before analysis to better approximate normal distributions. To test the primary hypothesis that lower cardiac vagal activity would be associated with increased LPS-stimulated production of pro-, but not anti-inflammatory cytokines, Pearson product moment correlations were conducted. Secondary analyses were then conducted to examine whether any associations between HRV measures and cytokine production were independent of the covariates using a series of Pearson product moment correlations for continuous measures and point bi-serial correlations for associations between dichotomous variables. Next, hierarchical linear regression analyses were conducted examining whether HRV measures predicted stimulated cytokine levels after adjustment for any significant covariates. For these analyses, identified covariates were entered in the first step, followed by the HRV measure in the second step, and covariates-HRV interactions in the third step of equations predicting stimulated levels of cytokines. Finally, a series of analyses was conducted to determine whether HRV measures were associated with stimulated cytokine levels independently of circulating numbers of WBCs, a related inflammatory parameter. In these analyses, identified covariates were entered in step 1, WBC in step 2, and the HRV measures in step 3 of a regression equation predicting cytokine production. This hierarchical regression procedure permitted us to systematically examine the independent contribution of variables entered in each step, after taking into account the effects of variables already in the model.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 NOTES
 REFERENCES
 
Cytokine Response to Stimulation
As expected, whole blood stimulation with LPS induced a significant increase in the levels of IL-1ß, IL-6, TNF-{alpha}, and IL-10 (Table 1). Stimulated levels of IL-1ß were highly correlated with concomitant levels of IL-6 (r = .69, p < .001) and TNF-{alpha} (r = .76, p < .001), and IL-6 with TNF-{alpha} (r = .69, p < .001). Production of IL-10 was associated with IL-6 (r = .31, p < .001), but not with TNF-{alpha} or IL-1ß.


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  		TABLE 1. Mean Untransformed Values of Stimulated and Unstimulated Pro- and Anti-inflammatory Cytokines (n = 175)

 

Associations Between HRV Measures and Cytokine Production
Initial correlational analyses between HRV measures and production of the inflammatory cytokines are presented in Table 2. As expected, these analyses revealed the negative associations of time and frequency domain indices of HF-HRV with stimulated production of the proinflammatory cytokines, IL-6, TNF-{alpha}, and to a lesser degree IL-1ß. LF-HRV was also inversely associated with IL-6 and marginally with TNF-{alpha}. The LF/HF ratio was positively associated with stimulated production of IL-6. As hypothesized, there was no significant relationship between measures of HRV and production of the anti-inflammatory cytokine, IL-10. Finally, there were no significant associations between HRV parameters and unstimulated cytokine levels, a measure of the spontaneous production of cytokines during incubation. Figures 1 and 2 show the relationship between indices of HF-HRV and production of IL-6 and TNF-{alpha}.


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  		TABLE 2. Univariate Correlations Between HRV Measures and Stimulated Levels of IL-1ß, IL-6, TNF-{alpha}, and IL-10 (n = 164)

 

Figure 11
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  		Figure 1. Scatterplot of the relationship between HF-HRV and stimulated production of IL-6 showing the mean linear regression line with 95% mean prediction interval (both variables log normalized). HF-HRV = high frequency-heart rate variability; IL = interleukin.

 


Figure 21
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  		Figure 2. Scatterplot of the relationship between rMSSD and stimulated production of TNF-{alpha} showing the mean linear regression line with 95% mean prediction interval (both variables log normalized). rMSSD = root mean square of successive differences; TNF = tumor necrosis factor.

 
Correlations Between Stimulated Cytokine Production and HRV Measures and the Control Parameters
Next, we assessed several control variables that might account for the observed relationships between HRV measures and cytokine production (Table 3). Relative to men, women showed lower stimulated production of IL-6, TNF-{alpha}, and IL-10, a lower LF/HF ratio, and higher HF power and rMSSD. Age was inversely associated with LF and HF power and rMSSD, but was not related to cytokine production. African-American participants showed higher stimulated production of IL-6 than European-American participants. Higher BMI was not associated with cytokine level, but was associated with lower LF and HF power and rMSSD. Higher blood pressure was associated with higher stimulated levels of IL-6 and TNF-{alpha}, a higher LF/HF ratio, and lower HF power and rMSSD. There were no significant associations between years of education or smoking and either cytokine production or HRV.


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  		TABLE 3. Demographic and Health Characteristics of the Sample and Their Correlations With Natural Logarithm Transformed Stimulated Levels of IL-1ß, IL-6, TNF-{alpha}, and IL-10, and HRV Measures (n = 164)

 

In regard to past medical history, there were no associations between stimulated cytokine production or HRV measures and self-reported history of cancer (n = 6), asthma (n = 15), thyroid disease (n = 8), or arthritis (n = 17). There were no associations between stimulated cytokine production and current use of the following medications: calcium channel blockers (n = 1), angiotensin-converting enzyme inhibitors (n = 2), other antihypertensive agents (n = 1), hormone replacement treatment (n = 5), and anti-lipenics (n = 6). No participants reported taking nitrates, diuretics, antiarrhythmics, weight loss medications, proteases, or anti-human immunodeficiency virus medications. On the basis of these relationships, gender, race, age, SBP, and DBP were included as covariates in subsequent statistical models.

Associations Between HRV Parameters and Cytokine Production After Controlling for Demographic Factors and Hypertension
Results of regression analyses are presented in Table 4. After entering gender, race, age, SBP and DBP, regression analyses revealed an inverse relationship between HF power measured during paced respiration and stimulated production of the proinflammatory cytokine IL-6 and a similar trend on analysis of TNF-{alpha} and IL-1ß. Similarly, higher rMSSD was associated with lower production of TNF-{alpha} and a trend for IL-6. LF-HRV also covaried inversely with stimulated production of IL-6 with a trend for TNF-{alpha}. Regression analyses examining stimulated levels of IL-10 revealed no significant relationships with any HRV measures. A further series of regression analyses demonstrated that there were no significant interactions between demographic or health factors and HRV measures in predicting stimulated levels of cytokines.


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  		TABLE 4. Results of Regression Models Examining Natural Logarithm Transformed Measures of HRV as Predictors of Stimulated Cytokine Levels With Standard Covariates Entered in Step 1 and Measures of HRV Entered Into Step 2 of Separate Models

 

Role of WBC
WBC is a measure of systemic inflammation that could account for the observed associations between HRV measures and stimulated production of IL-6 and TNF-{alpha}. Although WBC was not related to HF power, it was associated with lower RMSSD (r = –.19, p = .02), and higher stimulated production of IL-6 (r = .20, p = .01) and TNF-{alpha} (r = .26, p = .001). Thus, we ran a series of regression analyses, entering WBC after the identified covariates in a second step and the HRV measures in a third step. Entering WBC into the model did not significantly reduce the magnitude of the association between HRV indicators of cardiac vagal activity and production of IL-6 (ß changed from –0.25 to –0.21 for HF power and from –0.14 to –0.11 for rMSSD) or TNF-{alpha} (ß changed from –0.17 to –0.15 for HF power and from –0.17 to –0.14 for rMSSD), suggesting that the inverse relationship between vagal control over heart rate and production of IL-6 and TNF-{alpha} is largely independent of WBC.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
NOTES
 REFERENCES
 
This study provides novel evidence for an association between individual differences in cardiac parasympathetic (vagal) activity, as measured by indicators of heart rate variability assessed during paced respiration, and LPS-stimulated production of proinflammatory cytokines by peripheral immune cells among relatively healthy midlife adults. Consistent with a growing body of animal evidence demonstrating that activation of vagal pathways plays a role in the suppression of pro-, but not anti-inflammatory cytokine release (10), we found that higher vagal control of heart rate during paced breathing was associated with lower production of TNF-{alpha}, IL-6, and to a marginal degree IL-1ß, but was not related to the production of the anti-inflammatory cytokine IL-10. These associations were independent of demographic and health characteristics, including age, gender, race, years of education, smoking, hypertension, and WBC count. Thus, the current findings extend the animal literature to humans, providing further support for a "cholinergic anti-inflammatory pathway (2)," with activation of the parasympathetic nervous system (PNS) down-regulating inflammatory responses to endotoxin (1–3). This pathway may partially account for prospective associations between lower HRV, a relatively stable individual difference attribute (16,24), and increased risk of inflammatory disease (4–7).

To our knowledge, this is the first report that interindividual variability in parasympathetic-cholinergic activity is associated with inflammatory potential in humans. To date, research has focused on the role of the SNS, the HPA axis, and health behaviors in explaining how psychosocial factors are linked to immune function and disease. The results of the current study support recent animal literature in suggesting that the PNS suppresses the release of pro-inflammatory cytokines from activated immune cells and thus inhibits inflammation. If corroborated in subsequent investigation, this finding may have important implications for further understanding mind-body relationships and for developing novel therapeutic interventions. For example, vagal nerve stimulation, which is currently being used in the treatment of epilepsy and depression, or the administration of nicotinic ACh receptor agonists may aid in the treatment of inflammatory conditions (8). Alternatively, psychosocial interventions may be effective at increasing parasympathetic activity. A number of interventions are purported to increase parasympathetic function, including biofeedback, acupuncture, relaxation, meditation, and hypnosis. In this regard, initial findings show that hypnosis and meditation increase vagal nerve output and inhibit immediate-type and delayed-type hypersensitivity responses (25,26). The possibility that these and other interventions might influence immunity through the activation of the PNS warrants further investigation and may have valuable clinical implications for the treatment of inflammatory responses.

The present findings may also have implications for understanding the autonomic-immune pathways linking psychosocial risk factors to risk for inflammatory disease. More precisely, a number of studies have demonstrated that acute emotional behavioral states and trait negative affect are typically associated with a reciprocal increase in sympathetic and decrease in parasympathetic activation; however, there are marked individual differences in the patterning and expression of this form of autonomic reactivity (13,27–30). Importantly, such individual differences in autonomic reactivity may relate to risk for a range of health outcomes associated with inflammatory processes. For example, individuals who express a greater stress-induced reduction in cardiac vagal activity (indicated by HF-HRV) show increased subclinical atherosclerosis in the coronary arteries and aorta (31). Individual differences in negative affect have also been associated with increased production of proinflammatory cytokines, including IL-6 and TNF-{alpha} (32–34). To date, research in this area has conceptually focused on the role of the SNS in explaining these relationships; however, the current findings raise the possibility that parasympathetic withdrawal may contribute to emotion-related activation of proinflammatory pathways.

In addition to examining the impact of negative emotional states and traits, recent research has begun to examine the potential health benefit of positive affect (PA), e.g., calm and happy. Here, positive affect has been associated with a pattern of autonomic activation opposite that of negative or aroused emotions, with increases in parasympathetic and decreases in sympathetic activation (27,35). Our findings suggest that increased vagal control of heart rate is associated with decreased peripheral inflammatory responses, which may reduce the risk of inflammatory disease. In support of this possibility, smaller hypersensitivity inflammatory skin reactions to allergens were observed when pleasantness and relaxation were induced by hypnosis among individuals with allergies (36) and when humor was induced by a movie (37). Similarly, a positive emotional style has been associated in a dose-response manner with lower risk of developing a cold post experimental exposure to rhinovirus (38). In this case, further examination revealed that the association of PA and upper respiratory disease is partially mediated by decreased production of IL-6 in nasal secretions in response to rhinovirus (39), which was not explained by markers of SNS activation (e.g., epinephrine or norepinephrine) or cortisol levels. The findings of the current study suggest an alternate pathway, with increased vagal activity mediating relationships between PA and decreased production of IL-6 and thus reduced susceptibility to infection. The possibility that emotions influence inflammatory disease susceptibility via the modulation of parasympathetic activation warrants further investigation.

Consistent with the findings of others (40,41), we observed gender differences in the magnitude of proinflammatory response to LPS, with lower LPS-induced production of IL-6, TNF-{alpha}, and IL-10 among women than men. It is possible that these gender differences are related to sex steroids, with several studies showing that testosterone sensitizes rats to LPS exposure (42), whereas estrogen appears protective (43). In addition to gender differences, we found higher LPS-induced IL-6 production among African-Americans than European-Americans, perhaps as a consequence of ethnic differences in allele frequencies of functional IL-6 gene polymorphisms (44). These findings may help to explain increased infectious disease morbidity and mortality among African-Americans and males, as compared with European-Americans and females (45,46).

Interestingly, the current findings revealed a positive association between LPS-induced levels of IL-6 and IL-10. At first glance, the direction of this association seems surprising; however, recent evidence indicates that IL-6 stimulates IL-10 production in vitro (47), providing a local feedback loop to limit the pro-inflammatory response. Thus, IL-6 can serve as both a pro- and anti-inflammatory cytokine.

There are a number of limitations of the current study. Although it seems likely that vagal activity primes immune cells to respond, causation cannot be attributed confidently on the basis of cross-sectional data. It is hard to imagine how in vitro inflammatory responses would result in increased vagal activity; however, it is plausible that they are independently related to a third factor, such as genetic predisposition. A further limitation of the current study is the single assessment of stimulated cytokine levels and vagal activity, with blood samples for the determination of cytokine production collected 2 to 4 weeks before HRV testing. Although evidence suggests that individuals vary markedly in the magnitude of both stimulated cytokine production by peripheral leukocytes and vagal control of heart rate and that this variability is stable across time (15,16,48), multiple simultaneous assessments over time would provide a more reliable measure of individual differences.

Another limitation of the current study is the assessment of vagal activity at one target organ, the heart. Although HRV is widely employed as a measure of general vagal activity, it is possible that vagal outflow differs to organs involved in immune homeostasis. Thus, the observed and relatively modest relationship between HRV and immune response may actually underestimate potential in vivo effects. It is also possible that stronger associations may accompany situations associated with down- or up-regulation of parasympathetic activation, e.g., acute stress or relaxation.

Another shortcoming of our study is an inherent limitation of the multiplex assay. The amplitude of LPS-induced responses was much higher for the proinflammatory cytokines than for IL-10. Thus, it was impossible to dilute samples so that all cytokines fell within the range of detection and IL-10 levels had to be reassessed by ELISA. Finally, the clinical significance of observed differences in stimulated production of proinflammatory cytokines remains to be determined. Proinflammatory cytokine production is assumed to provide a measure of the functional ability of the body's circulating WBCs to mount an acute inflammatory response in the face of injury or an invading pathogen, a response that is typically local in vivo and difficult to access.

Despite these challenges, our novel findings provide initial evidence that vagal activity, as measured at the level of the heart and under paced respiration conditions, is inversely related to inflammatory competence. This is an important potential pathway in understanding relationships between psychosocial factors and risk of inflammatory disease. Thus, further testing is indicated using large, normative samples and longitudinal designs to examine whether vagal activity predicts inflammatory competence and susceptibility to the development of inflammatory conditions in humans.

The expert technical assistance of Ramasri Saathanoori, MS, and Adele Marrangoni at the University of Pittsburgh Cancer Institute Luminex Core Facility is gratefully acknowledged.


   NOTES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
REFERENCES
 
Because co-author J. Richard Jennings is an associate editor of this journal, the review of this paper was overseen by a guest editor. Dr. Jennings was not involved in the decision-making process and, like all authors, was blinded to the identity of the peer reviewers.

Received for publication January 22, 2007; revision received July 2, 2007.

This study was supported by a Central Research Development Grant provided by the Vice Provost for Research in the Office of the Provost at the University of Pittsburgh (A.L.M.) and by Grants P01HL40962 and HL007560 from the National Heart Lung and Blood Institute (S.B.M. and S.A.N.), and Grant NR008237 from the National Institute of Nursing Research (A.L.M.).

DOI:10.1097/PSY.0b013e3181576118


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 NOTES
 REFERENCES
 

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