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ANESTHETIC PHARMACOLOGY

The Inhibition of Human Neutrophil Phagocytosis and Oxidative Burst by Tricyclic Antidepressants

Annette Ploppa, MD^*, Donald M. Ayers, MD, Tanja Johannes, MD^*, Klaus E. Unertl, MD^*, and Marcel E. Durieux, MD, PhD

From the *Department of Anesthesiology and Intensive Care Medicine, Eberhard-Karls University, Tuebingen, Germany; and Department of Anesthesiology, University of Virginia, Charlottesville, Virginia.

Address correspondence to Ploppa Annette, MD, Department of Anesthesiology and Intensive Care Medicine, Eberhard-Karls University Tuebingen, Hoppe-Seyler-Str. 3, 72 076 Tuebingen, Germany. Address e-mail to annette.ploppa{at}uni-tuebingen.de.

	Abstract

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BACKGROUND: Tricyclic antidepressants are being investigated as long-acting analgesics for topical application in wounds or IV for postoperative pain relief. However, it remains unclear if tricyclic antidepressants affect the host defense and if reported toxic effects on neutrophils are of relevance in this setting. We therefore investigated the effects of amitriptyline, nortriptyline, and fluoxetine on human neutrophil phagocytosis, oxidative burst, and neutrophil toxicity in a human whole blood model.

METHODS: Heparinized blood samples from healthy volunteers were incubated with amitriptyline, nortriptyline, or fluoxetine (10^–6 to 10^–3 M) for 0, 1, or 3 h. Staphylococcus aureus in a bacteria:neutrophil ratio of 5:1 and dihydroethidium (for the determination of oxidative burst) were added. Phagocytosis was stopped after 5, 10, 20, and 40 min. After lysis of red blood cells, samples were analyzed by flow cytometry.

RESULTS: In concentrations up to 10^–4 M, none of the compounds affected neutrophil phagocytosis and oxidative burst. At 10^–3 M, all three compounds were highly toxic for neutrophils. Amitriptyline preserved morphological integrity, but completely suppressed neutrophil function. Nortriptyline and fluoxetine caused a marked disruption of neutrophils. The effects of the investigated antidepressants were not time-dependent.

CONCLUSIONS: Phagocytosis and intracellular host defense are largely unaffected by antidepressants in concentrations of 10^–4 M and below. Our results confirm that antidepressants are highly toxic to neutrophils in millimolar concentrations. The neurotoxic effects and clinical side effects, but not effects on neutrophil functions, therefore, are likely to be the limiting factors in using antidepressants as analgesics.

	Introduction

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Tricyclic antidepressants (TCAs) are used commonly as analgesic drugs in chronic pain therapy. They exhibit structural similarity with local anesthetics and share several properties with these compounds, in particular the ability to block neuronal sodium channels.1 The half-life of TCAs, however, is much longer than that of local anesthetics, (e.g., >16 h for amitriptyline), and therefore antidepressants are currently being investigated as long-acting local anesthetics for local injection, neuronal blockade, or for perioperative IV infusion.2,3 Potentially clinically beneficial local anesthetic properties of TCAs have been demonstrated.4 However, concerns remain about their toxicity: toxic effects on neurons and neutrophils have been reported.5–8 The latter toxicity is of particular concern after local administration, as it might result in an increase in wound infections.9 Although the concentrations required for inducing neutrophil toxicity in isolated cell models are high,10 it is conceivable that an attenuation of critical neutrophil functions might take place at lower, clinically used concentrations.

One of the most relevant neutrophil functions is the defense against bacteria, and this host defense is particularly important in the postsurgical setting. Local anesthetics inhibit two key host-defense functions, phagocytosis of bacteria and oxidative burst, which is required to destroy ingested pathogens, at concentrations approximately 1/100 of those that are overly toxic to cells.11,12 By analogy, TCAs might induce a significant impairment of neutrophil functions at micromolar concentrations, i.e., orders of magnitude lower than concentrations attained after local injection or nerve blockade.

We therefore investigated the ability of amitriptyline to affect bacterial phagocytosis and respiratory burst of human neutrophils. For comparative purposes, we also studied the TCA nortriptyline, and a nontricyclic compound, the selective serotonin reuptake inhibitor fluoxetine. To mimic the clinical situation as closely as possible, we performed this study in whole blood from volunteers, and used a clinically relevant pathogen, Staphylococcus aureus, as stimulus and target for neutrophil responses. We hypothesized that antidepressants, at concentrations below the toxic range, would dose-dependently inhibit neutrophils functions, and that this inhibition would be time dependent, analogous to that observed with local anesthetics.

	METHODS

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Reagents
All antidepressants, N-ethylmaleimide, phosphate buffered saline, RPMI, and dihydroethidium were purchased from Sigma (St. Louis, MO). Heparin was purchased from Baxter (Deerfield, IL), Caltag Cal-Lyse TM lysing solution from Invitrogen Corporation (Carlsbad, CA), mannitol salt agar from Edge Biological (Memphis, TN), standard methods agar from Hardy Diagnostics (Santa Maria, CA), tryptic soy broth from MP Biomedicals (Solon, OH), and Calcein-AM from Molecular Probes (Eugene, OR).

Blood Samples and Antidepressants
We studied the effects of the TCAs amitriptyline, nortriptyline, and fluoxetine on phagocytosis of S. aureus and oxidative burst as two key functions in the neutrophil host defense.

With IRB approval, written informed consent was obtained from healthy volunteers. Ten milliliters of blood samples were drawn and immediately heparinized (5 IU/mL). Autologous serum was prepared from an additional 5 mL blood sample. Two milliliters of heparinized blood samples were then incubated with amitryptiline, nortryptiline, or fluoxetine in a final working concentration of 10^–3 to 10^–6 M or NaCl 0.9% in controls.

Bacteria
S. aureus ATCC 25923 (American Type Culture Collection, Manassas, VA) were grown on mannitol salt agar for 24 h at 37°C. Thirty colony forming units were transferred in tryptic soy broth and incubated for 3 h at 37°C. After centrifugation (375g, 5 min), the pellet was incubated with calcein-AM (12.5 µg) for 3 h. Stained bacteria were washed twice and adjusted to a final concentration of 1 x 10⁹ colony forming units per milliliter. Bacterial counts were performed by plating appropriate dilutions of bacterial suspension on mannitol salt agar. Stained bacteria were frozen in RPMI at –20°C until use.

For each assay, bacteria were thawed and adjusted to a constant ratio of 5:1 bacteria per leukocyte, according to leukocyte counts of each donor. Bacteria were incubated in RPMI with 10% autologous serum for 30 min.

Phagocytosis and Oxidative Burst
All experiments were performed in quadruplicate, with blood samples of the same donor for all antidepressant concentrations. Blood samples were incubated with antidepressants or NaCl 0.9% for 0, 1, or 3 h. Thereafter, dihydroethidium (2.5 µg/mL blood) and preopsonized bacteria were added. Phagocytosis was stopped at 5, 10, 20, or 40 min by adding N-ethylmaleimide (10 mM) and keeping samples on ice. After lysis of erythrocytes, samples were washed with phosphate buffered saline and fixed with paraformaldehyde 4% before flow cytometry. For each sample, 10,000 neutrophils were analyzed. Neutrophils were identified by their characteristic scatter properties. Compensation of the flow cytometer was performed using unstained blood samples, and samples with ingested stained bacteria or dihydroethidium (stimulated with unstained bacteria) before each experiment.

Statistics
Data were processed in FlowJo V 7.1.3 (Tree Star Inc., Ashland, OR) and are presented as mean and standard deviation. Statistical analysis was performed using SigmaPlot 10/SigmaStat 3.5 (Systat Software, Point Richmond, CA). After confirming normal distribution with Kolmogorov–Smirnov test, analysis of variance for repeated measurements was performed to compare the different concentrations and time points. The level for significance was set at 0.05. For multiple comparisons, Holm–Sidak correction was applied.

	RESULTS

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Effects on Phagocytosis
In control samples, the ingestion of bacteria increased monotonically over time (Fig. 1A). No differences were observed among the incubation times of 0, 1, and 3 h, indicating constant experimental conditions. Neither amitriptyline (Fig. 1A) nor the other antidepressants (Fig. 1B) altered phagocytosis in concentrations of 10^–6 M to 10^–4 M. In contrast, 10^–3 M amitriptyline, nortriptyline, and fluoxetine induced a profound suppression of phagocytosis. Nortriptyline almost completely, and fluoxetine partially, disrupted leukocytes. This was observed immediately after addition of the compound and even in the absence of neutrophil stimulation with bacteria. Prolonged incubation in the antidepressants did not increase their inhibitory action.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effects of amitriptyline, nortriptyline, and fluoxetine on phagocytosis. A, Phagocytosis of Staphylococcus aureus after 0, 1, or 3 h incubation with amitriptyline. Fluorescence intensity of bacteria was determined after 0, 5, 10, 20, and 40 min phagocytosis. Data are presented as mean and standard deviation. B, Dose-dependent effects of the three antidepressants on phagocytosis. Data refer to exposure of samples to antidepressants for 1 h. Data were calculated as % inhibition in comparison to respective controls without antidepressant and are presented as mean and standard deviation.

Effects on Oxidative Burst
In the absence of antidepressants, oxidative burst continuously increased over time, with no differences among the incubation times of 0, 1, and 3 h (Fig. 2A). Similar to the effects on phagocytosis, none of the antidepressants affected oxidative burst in concentrations of 10^–6 to 10^–4 M, but at 10^–3 M complete suppression of oxidative burst was observed (Fig. 2B) immediately after addition of the compound.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of amitriptyline, nortriptyline and fluoxetine on oxidative burst. A, Oxidative burst after stimulation with Staphylococcus aureus after 0, 1, or 3 h incubation with amitriptyline. Fluorescence intensity of oxidative burst was determined after 0, 5, 10, 20, and 40 min. Data are presented as mean and standard deviation. B, Dose-dependent effects of the three antidepressants on oxidative burst. Data refer to exposure of samples to antidepressants for 1 h. Data were calculated as % inhibition in comparison to respective controls without antidepressant and are presented as mean and standard deviation.

Effects on Cell Morphology
Flow cytometry side scatter values were determined to assess changes in cell morphology. Side scatter represents the refractive index of a cell and can be used as a measure of membrane ruffling and degranulation. We determined changes in side scatter after exposure to antidepressants and bacteria. In control samples, side scatter values decreased during phagocytosis, due to ingestion of bacteria and formation of phagolysosomes (Fig. 3A). Again, none of the antidepressants affected this process in concentrations up to 10^–4 M. Amitriptyline at 10^–3 M caused a significant decrease in side scatter values even in the absence of phagocytosis. During exposure to bacteria, no further degranulation was observed (Fig. 3A), which is in agreement with the inhibition of phagocytosis observed at this concentration. Because of disintegration of neutrophils after exposure to nortriptyline and fluoxetine at 10^–3 M, side scatter was not determined for this concentration (see below).

View larger version (40K): [in this window] [in a new window]   Figure 3. A, Effects of amitriptyline on side scatter values (in arbitrary units [AU]) after stimulation with Staphylococcus aureus after 0, 1, or 3 h incubation with antidepressants. Values were determined after 0, 10, and 40 min. Data are presented as mean and standard deviation. B, Dot plots show characteristic patterns of cell necrosis after incubation with amitriptyline (middle row), nortriptyline (lower row) and the respective control (upper row) after 0, 10, and 40 min phagocytosis. In control samples neutrophils can be identified by their characteristic scatter properties. In amitriptyline treated samples, cells are split into two subpopulations: one containing neutrophils, which however present with lower forward-scatter values and side scatter values, and a second lower left population of necrotic cells. In nortriptyline-treated samples, the majority of cells is being disrupted and can no longer be identified by their scatter properties. C, Overlay histogram of the fluorescence intensities of the amitriptyline-treated samples and controls from B. Whereas in control samples fluorescence intensity of dihydroethidium for oxidative burst increases with longer exposure to bacteria (10 and 40 min), in the amitriptyline-treated samples no increase of oxidative burst can be observed. In both populations, the original neutrophils gate and in the lower left (necrotic) population no metabolic action can be observed.

Neutrophil Toxicity
Figure 3B shows the dot plots in forward and side scatter for representative samples of the controls, amitriptyline 10^–3 M, and nortriptyline 10^–3 M. In controls, neutrophils can be clearly detected by their scatter properties. Also, the increase in forward scatter and the decrease in side scatter of the neutrophils population at 10 and 40 min indicates the ingestion of bacteria (Fig. 3B). The scattergram was similar after exposure to each of the antidepressants in concentrations up to 10^–4 M (data not shown).

After exposure to 10^–3 M amitriptyline, two neutrophil subpopulations were observed. The location in the lower left is typical for necrotic (shrunken and degranulated) cells, and in the presented amitriptyline-treated sample, roughly 30% of the neutrophils are located in this area (Fig. 3B). After exposure to nortriptyline and fluoxetine, the majority of neutrophils appeared dead or disrupted.

For the evaluation of phagocytosis and oxidative burst, only the neutrophils in the right upper area had been gated. We also determined the fluorescence intensity for oxidative burst in the lower left population in the presence of antidepressants 10^–3 M to confirm that cells had undergone an immediate, and not a late and slow, necrosis within increasing incubation times where the generation of oxidative burst might be conceivable (Fig. 3C). There were no differences in fluorescence intensity of oxidative burst in the two populations; in other words, oxidative burst was completely and immediately suppressed in both populations after exposure to amitriptyline 10^–3 M according to the immediate decrease of sideward scatter that indicated a toxic effect.

To compare the toxic effects of the antidepressants, we performed a neutrophil count in whole blood by flow cytometry after incubation of blood samples with each of three antidepressants. This method was chosen because it does not require any washing or sample preparation steps and therefore directly reveals the number of necrotic leukocytes. In amitriptyline-treated samples 92% ± 5%, in nortriptyline-treated samples 0.5 ± 0.4, and in fluoxetine-treated samples 49% ± 16%, neutrophils could be detected. This indicates that amitriptyline induces cell necrosis but preserves cell morphology, whereas cells partially disintegrate in the presence of fluoxetine and almost completely in the presence of nortriptyline.

	DISCUSSION

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Our results indicate that amitriptyline, nortriptyline, and fluoxetine, at concentrations up to 10^–4 M, are essentially without effect on neutrophil phagocytosis of S. aureus and respiratory burst. Concentrations of only one order of magnitude above induce severe neutrophil toxicity. Although amitriptyline at 10^–3 M preserves neutrophil integrity, but essentially suppresses cellular functionality, nortriptyline and fluoxetine induce neutrophil disintegration at this concentration. These effects were observed immediately after addition of the compounds, and therefore indicate an immediate toxic effect.

The development of a truly long-acting (i.e., >24 h) local anesthetic could be very useful for clinical practice. In the outpatient setting, in particular, the ability to obtain long-lasting analgesia with a single injection, whether locally around the surgical wound or for nerve blockade, would be expected to have a significant impact on patient recovery. Antidepressants are one of several classes of drugs investigated for this purpose. They have been shown to provide profound and prolonged nerve blockade in animal models,5,13 and provide long-lasting cutaneous analgesia as well.14,15 Amitriptyline has been investigated in humans, where it has been used for ulnar nerve blockade16 and topical analgesia17 in volunteers.

Unfortunately, in higher concentrations, the TCAs show a consistent toxicity that is not observed with the classical local anesthetics. Neuronal toxicity of amitriptyline and derivatives was demonstrated in the rat13 at concentrations as low as 10^–7M,8 and in our previous studies we showed amitriptyline, as well as several other antidepressants, to be damaging to isolated neutrophils.10 At concentrations above 10^–4M, the compounds induced disintegration of human neutrophils. In a different model, oocytes from the frog Xenopus laevis, which were used for mechanistic studies of antidepressant actions on receptor signaling, we also found the compounds to induce cellular death.7 These findings were confirmed in the present study, where neutrophil damage was observed consistently at millimolar antidepressant concentrations. This indicates that, unfortunately, the whole-blood environment does not provide protection against these detrimental effects. However, there seemed to be a shift in the concentration– response relationship. In our previous study, neutrophil toxicity was caused by amitriptyline at 10^–3 M and by nortriptyline and fluoxetine at 10^–4 M. In the present study, we observed the same effect, but at one order of magnitude larger concentration. The presence of plasma proteins18 in the whole blood model might have decreased the free antidepressant concentrations and may have been responsible for this shift.

In micromolar concentrations, we did not observe any effects on phagocytosis and oxidative burst. These findings did not confirm our hypothesis that inhibitory effects, in analogy to the effects of local anesthetics, can be observed in concentrations of one or two magnitudes lower than where toxic effects occur. Instead, the results of our present study demonstrate that the effects of antidepressants are different from the antiinflammatory actions of local anesthetics. Antidepressants do not gradually impair host-defense functions; instead, an abrupt toxicity occurs at millimolar concentrations. Furthermore, we did not observe any time-dependent increase in sensitivity after prolonged exposure to antidepressants, as we observed previously with local anesthetics.12,19 Prolonged incubation times did not affect neutrophil functions or toxicity. The toxic action of TCAs was observed immediately after addition of the compounds to the blood samples. Kitagawa et al. proposed a direct detergent effect of amitriptyline to cause the reported neurotoxicity.20 Concentrations of 10^–5 M amitriptyline or above induced membrane disruption of artificial lipid membranes and hemolysis. This suggested mechanism is an excellent candidate for the toxic effects observed in the present study, since our findings are consistent with membrane disruption: in nortriptyline- and fluoxetine-treated samples cells were destroyed and hemolysis could be observed during sample preparation.

In a previous study, Struemper et al. reported an inhibition of neutrophil priming of oxidative burst by antidepressants in micromolar concentrations.10 This effect is considered to be beneficial in diseases with excessive leukocyte hyperactivation. The lack of effect in the present study can be explained with the differences between the models used. Struemper et al. used a sequential exposure of isolated neutrophils to platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine10; whereas, in the present study, live S. aureus in whole blood were used. The PAF/formyl-methionyl-leucyl-phenylalanine model is optimized to demonstrate a priming effect by PAF, an endogenous mediator. In contrast, the application of live S. aureus is a well established model to mimic the response to an infection with the clinically most common pathogen S. aureus; however, a sequential stimulation of neutrophils does not occur after exposure to bacteria. Furthermore, the intracellular signaling pathways in the two models are different. Struemper et al. observed an inhibition of the extracellular release of superoxide, whereas in the present study the generation of intracellular reactive oxygen species, necessary for an undisturbed host defense, was determined.

Of course our in vitro model is associated with some limitations. First, blood samples from healthy volunteers do not correctly reflect the immune status of patients, i.e., those with infectious complications. Second, our in vitro environment does not reflect the scenario of a local wound infection. Antidepressant concentrations in the wound have not clearly been defined, and concentrations have been extrapolated from local amitriptyline concentrations or local anesthetic administration. The lack of interaction with other tissue bound immunocompetent cells as well as the lack of systemic metabolism might overstate the toxic effect of the antidepressants.

	CONCLUSIONS

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Our findings indicate that amitriptyline, nortriptyline, and fluoxetine, in micromolar concentrations, do not adversely affect several key neutrophil functions that are of importance for defending the host against bacterial invasion. Previous investigations demonstrated that these compounds, at similar low concentrations, might be able to prevent hyperactivation of neutrophils and excessive extracellular superoxide concentrations. Taken together, these data suggest that TCAs might be able to attenuate inflammatory host injury while still allowing an adequate defense against bacteria. Millimolar concentrations of the drugs, however, are profoundly toxic to neutrophils. It seems unlikely that such concentrations can be used in the clinical setting. In practical terms, this means that approaches such as nerve blockade16 or topical application,17 which have been attempted in volunteers, should not be used in patients. In contrast, administration of systemic, low doses of amitriptyline might offer several benefits in the perioperative setting, including analgesic effects and beneficial inflammatory modulation.3 Therefore, the safety and efficacy of this approach should probably be further investigated in phase I and II clinical trials.

	Footnotes

 
Accepted for publication April 30, 2008.

Marcel E. Durieux is editor of Anesthetic Pre-Clinical Pharmacology for the Journal. This manuscript was handled by Steven L. Shafer, Editor-in-Chief, and Dr. Durieux was not involved in any way with the editorial process or decision.

Supported by a Carl Koller award from the American Society for Regional Anesthesia, and by institutional scientific budgets.

Reprints will not be available from the author.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ploppa, A. Articles by Durieux, M. E. Search for Related Content PubMed PubMed Citation Articles by Ploppa, A. Articles by Durieux, M. E. Related Collections Mechanisms Preclinical Pharmacology Pharmacology