The initial step in the activation of the adaptive immune response occurs when antigen-presenting cells present antigen to naive T cells. Dendritic cells (DCs), the most potent antigen-presenting cells in the immune system, play a key role in initiating this primary immune response. DCs exist in a functionally and phenotypically immature state in peripheral tissues. Once immature DCs take up foreign antigens and are activated by cytokines or pathogen-associated molecular patterns such as lipopolysaccharides (LPS), they mature.1 This maturation results in decreased antigen uptake, increased expression of major histocompatibility complex (MHC) class ІІ and costimulatory molecules (CD80 and CD86), and increased production of interleukin (IL)-12.1 Mature DCs allow naive T cells to develop into effector T cells such as T helper 1 (Th1) cells, further augmenting cell-mediated immune responses.
Haloperidol is an antipsychotic drug used to ease the symptoms of psychotic disorders such as schizophrenia and bipolar disorder.2 Sedative drugs such as haloperidol are also used to manage agitated and delirious patients in the intensive care unit. In addition to their effects on the central nervous systems, sedative drugs can have various effects on the immune system, including conferring protective immunity against pathogens and inflammatory processes.3–5
One clinical study has shown that the ratio of interferon (IFN)-γ (Th1) to IL-4 (Th2) in plasma is higher in medication-free patients with schizophrenia than in healthy controls and that the increased levels of IFN-γ/IL-4 were attenuated when antipsychotic drugs, including haloperidol, were given.6 Another study showed a decrease in the levels of the Th1-inducing cytokine IL-12 in plasma after antipsychotic treatment with haloperidol in patients with schizophrenia.7 These reports suggest that haloperidol can attenuate Th1-type immune responses during the management of schizophrenia. Although the mechanism by which haloperidol affects immune regulation is unclear, one possibility is that haloperidol modulates immune function by acting on DCs to attenuate Th1-type immune responses.
In vitro studies have shown that haloperidol also has suppressive effects on the functions of macrophages, another important regulator of antigen-presenting cells.8,9 Haloperidol inhibits production of tumor necrosis factor-α and IL-1β from human whole macrophages.9
We hypothesized that haloperidol affects Th1-type immune responses in part by acting on DCs. To this end, we designed in vitro cell culture experiments using bone marrow–derived DCs (BM-DCs) to clarify the effects of haloperidol on DCs. We also examined the involvement of dopamine receptors on the effects of haloperidol on DCs.
Female C57BL/6 and Balb/C mice (aged 4–6 weeks) were purchased from Japan CLEA (Tokyo, Japan) and housed in groups of 3 per cage with free access to chow and water in a specific pathogen-free central animal facility of Osaka University Medical School. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Use Committee at Osaka University.
Reagents and Antibodies
Recombinant mouse granulocyte macrophage colony-stimulating factor was purchased from R&D Systems (Minneapolis, MN). Haloperidol, SCH23390, L750667, and LPS (from Escherichia coli O55:B5) were obtained from Sigma-Aldrich (St. Louis, MO). The fluorescein isothiocyanate–conjugated, phycoerythrin (PE)-conjugated, and allophycocyanin-conjugated monoclonal antibodies (mAbs) used to detect the expression of CD11c (HL3), MHC class ΙΙ (I-Ab) (AF6-120.1), CD80 (16-10A1), CD86 (GL1), and the isotype-matched control immunoglobulin G (IgG) were purchased from BD Biosciences (San Diego, CA). CD83 (Michel-17) and 7-amino actinomycin D were purchased from eBioscience (San Diego, CA). To evaluate T cell proliferation, we used Alamar Blue® from AbD Serotec (Kidlington, United Kingdom). To obtain purified DCs and enriched CD4-positive and CD8α-positive T cells, we used CD11c microbeads and CD4 (L3T4) and CD8α (Ly-2) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
Culture medium used in the present experiments consisted of RPMI-1640 (Sigma-Aldrich) containing 10% fetal calf serum (Hyclone, South Logan, UT), 5.5 × 10−5 M 2-mercaptoethanol, 1 mM sodium pyruvate, 1 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin. Using a previously described method with minor modifications, we generated DCs from murine bone marrow cells.10,11 Briefly, murine bone marrow was flushed from the tibiae and femurs of C57BL/6 mice, and the red blood cells were removed with ammonium chloride. Bone marrow cells (2.5 × 106) were cultured in 6-cm culture dishes in 5 mL of culture medium supplemented with 20 ng/mL recombinant mouse granulocyte macrophage colony-stimulating factor at 37°C in 5% CO2. On days 3 and 5 of the culture, half of the culture medium was replaced with fresh culture medium. From day 6, to each dish, dimethyl sulfoxide (Sigma-Aldrich) or 5 μg/mL haloperidol dissolved in dimethyl sulfoxide was added for 72 hours. On day 7, loosely adherent cells were harvested and incubated for another 2 days and then stimulated with 20 ng/mL LPS for 24 hours.
To evaluate the effect of a clinical concentration of haloperidol, we conducted experiments in which lower concentrations of haloperidol (50, 100, and 500 ng/mL) were added to DC cultures from day 1 for 9 days.
The viability of cultured cells was assessed by trypan blue exclusion. The cell viability in all experimental cultures used in this study was >90%.
Flow Cytometric Analysis of Surface Molecules
The expression of surface molecules on DCs was analyzed by flow cytometry using a FACS Canto ΙΙ and BD FACS Diva software version 6.1.3 (BD Biosciences). Flow cytometry analysis was performed as previously described.12,13 Samples (5 × 105 cells) were washed with phosphate-buffered saline (PBS; Sigma-Aldrich) and resuspended in 30 μL of PBS containing 2% fetal bovine serum and 0.3 μL of mAb. The cells were then stained with fluorescein isothiocyanate-labeled, PE-labeled, or allophycocyanin-labeled mAbs for the detection of MHC class ΙΙ, CD80, CD86, CD83, and CD11c. After incubation for 20 minutes at 4°C in the dark with saturating amounts of mAbs, the cells were washed once with PBS. Then the cells were resuspended in 200 μL of PBS. These samples were stored at 4°C in the dark before flow cytometric analysis. The expression of cell-surface molecules identified by their respective mAbs was assessed as the mean fluorescence intensity (MFI) per arbitrary unit using flow cytometry. For proper comparison of the MFI values, we measured relative MFI, which is the MFI of cells stained with the PE mAb divided by the MFI of cells stained with the isotype-matched IgG control mAb as described previously.14 Cell debris was excluded by selective gating, and the nonviable cells were excluded by staining with 7-amino actinomycin D; thus, only living cells were phenotypically assessed.
Enzyme-Linked Immunosorbent Assay for IL-12 p40
DCs were cultured in the presence or absence of haloperidol, followed by stimulation with LPS. On day 7 of DC induction, DCs were purified using a magnetic-activated column and CD11c microbeads. On day 9, the culture supernatants were collected and preserved at −30°C until analysis. IL-12 p40 in culture supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (R&D Systems).
Allogeneic Mixed Cell Culture Reaction
To enrich for CD4+ and CD8α+ T cells in the sample, we isolated splenocytes from female 4- to 6-week-old Balb/C mice (haplotype I–Ad) and purified them in a magnetic cell sorting. DCs isolated from C57BL/6 mice (haplotype I–Ab) were incubated with or without 5 μg/mL haloperidol and subsequently stimulated with LPS (20 ng/mL) for 12 hours before they were treated with mitomycin C (50 μg/mL) for 20 minutes. Mitomycin C–treated DCs from a C57BL/6 mouse (haplotype Ι–Ab) were incubated with allogeneic T cells from Balb/C mouse (haplotype Ι–Ad) –induced Balb/C T cells to proliferate and differentiate into IL-2 and IFN-γ–secreting Th1-type T cells. These mixed samples were then cultured in 96-well flat-bottom plates (Corning Incorporated, Corning, NY) for 4 days in RPMI-1640 supplemented with 10% fetal bovine serum at 37°C and 5% CO2. The proliferation of cells was measured using an Alamar Blue assay. Briefly, cultured cells were pulsed with Alamar Blue for the last 12 hours of the culture period, after which the absorbance at 570 and 600 nm of the cell cultures was measured using a microplate reader (SH9000; Corona Electronic Corporation, Ibaraki, Japan). The indicator of proliferation was determined by subtracting the optical density at 600 nm from the optical density at 570 nm.
Culture supernatants were harvested and preserved at −30°C until the concentrations of IFN-γ, IL-2, and IL-4 were determined using an ELISA kit (R&D Systems).
Contact Hypersensitivity Model Using Adoptively Transferred DCs
Contact hypersensitivity (CHS) is a simple in vivo assay of cell-mediated immune function in which 2,4-dinitro-1-fluorobenzene (DNFB) can be used as a contact-sensitizing agent. Briefly, 0.5% DNFB was dissolved in 4:1 acetone/olive oil and was applied to the shaved abdomen of C57BL/6 mice on day 0. On day 5, mice were challenged by epicutaneous application of DNFB on the right ear. After 24 hours, using a spring-loaded micrometer (Mitutoyo Corporation, Kawasaki, Japan), we measured the right (challenged) and left (unchallenged) ear thickness. Increased ear thickness was evaluated by simple subtraction: thickness of the challenged ear − thickness of the unchallenged ear.
For the evaluation of CHS by adoptive transfer of DCs, DCs were prepared as described in the isolation and culture of DCs section above. The CHS model was induced by in vivo inoculation of antigen-loaded DCs.15,16 After culture in the presence or absence of 5 μg/mL haloperidol, 5 × 105 DCs (200 μL RPMI-1640) were pulsed with 100 μg/mL 2,4-dinitrobenzene sulfonic acid (a water-soluble analog of DNFB) and injected subcutaneously on day 0. After 5 days, right and left ear thickness was measured, and each side of the right ear of the mice was epicutaneously treated with 10 μL of 0.2% DNFB diluted in 4:1 acetone/olive oil. The left ear received the vehicle alone. After 24 hours, by using a spring-loaded micrometer, the right and left ear thickness was measured. The ear swelling was calculated as follows: (T − T0 of the right ear) − (T − T0 of the left ear), where T0 and T represent the value of ear thickness before and after the challenge, respectively.
Effects of D1-Like and D2-Like Receptor Antagonists on DCs
We next evaluated which types of dopamine receptors mediated the effect of haloperidol on DCs. To determine whether haloperidol influenced DCs via the dopamine receptor, we cultured BM-DCs for 9 days in the presence or absence of SCH23390, a D1-like receptor antagonist, or L750667, a D2-like receptor antagonist, for the final 3 days. The concentration of SCH23390 and L750667 administered was 3 μg/mL (approximately 10 μM) and 5 μg/mL (approximately 10 μM), respectively. On day 9, DCs were stimulated with 20 ng/mL LPS for 24 hours and were then harvested and analyzed for the expression of CD86 molecules by using flow cytometry.
Statistical analyses were performed using JMP Pro Version 11.2 (SAS Institute, Cary, NC). The sample size was decided as follows. We conducted preliminary tests in each experiment and calculated the effect size. Based on the smallest effect size obtained in each experiment, we determined the sample size. To detect differences between groups with a 2-tailed α error of 0.01, a β error of 0.20, SD of 43, and difference in mean of 92, 7 samples per group were required. Thus, we evaluated each experiment using 8 samples per group. Our study was a prospective, randomized, controlled study using mice and mice-derived DC samples. For in vitro experiments, the samples of BM-DCs were randomly assigned into vehicle-treated and haloperidol-treated groups. For in vivo experiments in CHS models, mice were randomly assigned into groups administered with vehicle-treated DCs or haloperidol-treated DCs. The results are expressed as mean ± SD.
We performed the Welch t test for all analyses except for dose-response experiments between CD86 expression and haloperidol concentrations. Dose-response experiments were evaluated using 1-way analysis of variance followed by Dunnett test. All statistical tests were 2 tailed, with P < 0.01 indicating statistical significance.
Haloperidol Inhibits Maturation of Murine DCs
In initial experiments, we determined whether haloperidol might affect the maturation of DCs. Immature murine DCs were cultured for 9 days and pulsed with or without haloperidol (5 μg/mL) for the final 3 days. After 24 hours of stimulation with LPS, we measured the expression of MHC class ΙΙ, costimulatory molecules (CD80 and CD86), and the maturation marker CD83 on DCs by flow cytometry. Haloperidol suppressed the expression of CD80, CD86, MHC class ΙΙ, and CD83 (Fig. 1). These phenotypic changes in DCs suggested that haloperidol suppressed the maturation of DCs.
Because serum concentrations of haloperidol in patients with schizophrenia may reach 50 ng/mL,17–19 we analyzed the effect of similar haloperidol concentrations (50–500 ng/mL) for 9 days on CD86 expression on DCs. Because the effect of haloperidol on CD80 and MHC class ΙΙ was similar to CD86, we used the effect of haloperidol on CD86 expression as representative of its effect on other surface molecules in terms of concentration dependency. At clinical concentrations, haloperidol suppressed CD86 expression in a dose-dependent manner (Fig. 2, A and B). However, haloperidol concentrations >1000 ng/mL at prolonged durations had toxic effects on DCs and affected DC survival. We also analyzed the dose-dependent reduction of DC maturation at higher concentrations (from 0.5 to 7.5 μg/mL) for a short duration (3 days) (Fig. 3, A and B). Under these conditions, higher concentrations of haloperidol had no toxic effects on DCs. Thus, we chose 5 μg/mL of haloperidol as the concentration at which almost all the experiments in the present study were performed.
Haloperidol Decreases the Production of IL-12 p40
A major function of mature DCs is the synthesis of cytokines that modulate the function and differentiation of T cells. Production of IL-12 is also an important marker for DC maturation and can be used as a method of selecting a Th1-dominant adjuvant. Our results showed that stimulation of DCs with LPS induced the release of IL-12 p40 and that haloperidol attenuated this effect on LPS-induced IL-12 p40 synthesis from DCs (Fig. 4).
Haloperidol Inhibits T Cell Proliferation and Th1-Type Immune Responses
To elucidate the relevance of haloperidol-mediated alteration of DC function to immune function, we assessed the effect of haloperidol on a mixed cell culture reaction of lymphocytes and DCs. From 9-day cultures that had been incubated in the presence or absence of haloperidol for 3 days, DCs matured with LPS were examined for their capacity to stimulate allogeneic T cells. Compared with haloperidol-untreated DCs, haloperidol-treated DCs cocultured with T cells suppressed the proliferative responses by allogeneic T cells, as assessed using Alamar Blue (Fig. 5A). In addition, we harvested the supernatants of cocultured samples with or without haloperidol and analyzed them for the production of IFN-γ and IL-2 (Th1 cytokines) and IL-4 (Th2 cytokine) by using ELISA. The results showed that coculturing DCs with haloperidol effectively inhibited the production of IFN-γ and IL-2 (Fig. 5, B and C). IL-4 secretion was detected in very small amounts in mixed cell cultures in both control and haloperidol-treated groups (Fig. 5D).
Haloperidol-Treated DCs Fail to Elicit Normal Cell-Mediated Immune Responses
The CHS response is a prototype in vivo model of cell-mediated immunity and is induced by Th1-type T cells. To analyze the in vivo effects of phenotypic changes induced by haloperidol on DCs, we used a modified CHS model involving the adoptive transfer of DCs.15 In the original CHS model, hypersensitivity is induced by epicutaneous sensitization with a strong hapten, such as DNFB, on the flanks (sensitization phase) and a subsequent epicutaneous challenge with the same hapten to the ears (elicitation phase), which results in swelling of the challenged ears. CHS also can be induced by in vivo inoculation with DCs that have been loaded with DNFB followed by epicutaneous challenge with DNFB. We used this latter model of CHS whereby DCs were adoptively transferred into mice to analyze the in vivo effects of haloperidol on DC-induced immune responses. Our analysis showed that immunization with haloperidol-treated DCs induced lower CHS responses than did immunization with vehicle-treated DCs (Fig. 6).
An Antagonist to the D2-Like Receptor Suppresses Maturation of DCs
We hypothesized that the suppressive effect of haloperidol on DCs was mediated by the dopamine receptor. To test this hypothesis, DCs were incubated for 9 days and pulsed with the D1-like receptor antagonist SCH23390 or the D2-like receptor antagonist L750667 for the final 3 days. The expression of CD86 molecules after 24 hours of LPS stimulation was then measured. We observed a lower expression of CD86 on DCs treated with the D2-like receptor antagonist L750667 than on untreated DCs. In contrast, we observed a higher expression of CD86 on DCs treated with the D1-like receptor antagonist SCH23390 (Fig. 7, A and B).
The present study showed that haloperidol inhibits maturation and Th1-inducing activity of DCs in vitro. Furthermore, in a protypical cell-mediated immune response model (CHS), we showed that haloperidol suppressed the Th1-type immune response mediated by DCs. To our knowledge, our report is the first to show the effect of haloperidol on BM-DCs. The effects of haloperidol on immune response in vivo have important clinical implications.
Sepsis has been reported to change the Th1/Th2 balance from Th1-dominant to Th2-dominant conditions.20,21 This immunologic shift from Th1 to Th2 is partly responsible for the immunocompromised status of patients with sepsis.21 Data from the present study suggest that the administration of haloperidol in patients with sepsis can augment the immunologic shift from Th1-dominant to Th2-dominant status. This shift can then suppress the host defense to intracellular bacteria such as Mycobacteria and Listeria. Such a shift also may alter host immune defenses against malignant tumors, for which the Th1-type immune response is essential. However, when the excessive activity of the Th1-type immune response is deleterious to the host, such as in autoimmune diseases, the suppressive activity of haloperidol on DCs and DC-mediated immune response may be beneficial.
Besides its antipsychotic effects, the immune effects of haloperidol suggest a potential approach to treating intensive care unit delirium. Elevated levels of cytokines, such as IL-2, IL-6, and IL-8, have been associated with delirium in critically ill patients.22–24 In addition, increased levels of IL-2 and tumor necrosis factor-α are associated with the development of delirium after cardiac surgery.25 IL-2 treatment for cancer has also been associated with delirium, which improved after stopping IL-2 treatment.26–28 Our study showed that haloperidol has a potent ability to regulate DCs, leading to reduced production of potentially delirium-inducing cytokines such as IL-2. Thus, it is possible that the effects of haloperidol on delirium could be partly due to its action on DC-mediated immune responses.
Dopamine receptors include 5 subtypes, from D1 to D5.29,30 D1 and D5 receptors are classified into D1-like receptors and D2, D3, and D4 receptors into D2-like receptors. Haloperidol mainly binds to the dopamine D2 receptor but also can bind to other dopamine receptors, including D1, D3, D4, and D5 receptors with various affinities.29 In the present study, the effects of haloperidol on DCs were mediated by the dopamine D2-like receptor, a result comparable with a recent study showing that D2-like receptors are more important in mediating the suppression of Th1 cell function.31
It was reported that typical and atypical antipsychotics (haloperidol and risperidone, respectively) affect the functions of human monocyte-derived DCs (Mo-DCs).2 In the study, haloperidol did not affect the expression of costimulatory molecules. However, in a culture system consisting of human Mo-DCs, haloperidol affected the viability and survival of Mo-DCs, which might interfere with the accurate analysis of the effects of haloperidol on DCs. In our study, haloperidol did not affect the viability and survival of BM-DCs. Thus, the present study is the first to evaluate the exact effects of haloperidol on DCs. Another possibility for the differences between human Mo-DCs and mouse BM-DCs could be derived from the differences in the human and murine culture systems.
Our study has limitations. We evaluated the effects of haloperidol mainly at the concentration of 5.0 μg/mL. We tested the effect of haloperidol on DC maturation at more clinical concentrations (50, 100, and 500 ng/mL) for a long duration (9 days). Under these conditions, haloperidol suppressed DC maturation in a dose-dependent manner (Fig. 2, A and B). At higher concentrations (>1000 ng/mL), this duration of haloperidol exposure has toxic effects on DCs and affects DC survival. We also showed a dose-dependent reduction in DC maturation at higher concentrations (2.5, 5, and 7.5 μg/mL) for a short duration (3 days) (Fig. 3, A and B). This higher concentration of haloperidol had no toxic effect on DCs. Thus, we chose the concentration of 5 μg/mL for a short period (3 days) as proof of principle supported by the results at the low clinically relevant concentration of haloperidol. Concentrations of approximately 5 μg/mL have frequently been used in previous reports examining the effects of haloperidol on immune cells such as lymphocytes, macrophages, and whole blood.8,32,33
Second, we evaluated only BM-DCs but not other tissue-derived DCs such as DCs residing in thymus, spleen, and lymph nodes. It is possible that the results reported here would differ in other tissue-derived DCs. Therefore, our results need to be confirmed using other tissue-derived DCs. Nevertheless, the results of our in vitro studies are of relevance to in vivo conditions found in whole animals, where conventional DCs, plasmacytoid DCs, and other DCs are present.1 Further investigation of in vivo subsets of DCs is required to clarify the effect of haloperidol on in vivo DCs.
In conclusion, we demonstrated that haloperidol affects DC function and changes the polarity of immune response. Administration of haloperidol may reduce DC-mediated immune functions and possibly exacerbate infection in patients with sepsis. These immune effects of haloperidol on DC-mediated immune response could partly account for the antidelirium effect of haloperidol.
Name: Atsuhiro Matsumoto, MD.
Contribution: This author designed and conducted the study, analyzed the data, and wrote the manuscript.
Attestation: Atsuhiro Matsumoto approved the final manuscript, attests to the integrity of the original data and the analysis reported in this manuscript, and is the archival author.
Name: Noriyuki Ohta, MD, PhD.
Contribution: This author designed and conducted the study, analyzed the data, and wrote the manuscript.
Attestation: Noriyuki Ohta approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Yukiko Goto, MD.
Contribution: This author helped to design the study.
Attestation: Yukiko Goto approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Yozo Kashiwa, MD.
Contribution: This author analyzed the data.
Attestation: Yozo Kashiwa approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Shunsuke Yamamoto, MD.
Contribution: This author analyzed the data.
Attestation: Shunsuke Yamamoto approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Yuji Fujino, MD, PhD.
Contribution: This author helped design the study and analyze the data.
Attestation: Yuji Fujino approved the final manuscript, and attests to the integrity of the original data and the analysis reported in this manuscript.
This manuscript was handled by: Avery Tung, MD.
We thank Dr. Toshimitsu Hamasaki (Chief, Office of Biostatistics and Data Management of the Department of Advanced Medical Technology Development Research and Development Initiative Center, National Cerebral and Cardiovascular Center, Suita, Japan) for reviewing our statistical analyses.
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