International Immunopharmacology 23 (2014) 550–557

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Regulation of neutrophil phagocytosis of Escherichia coli by antipsychotic drugs Mao-Liang Chen a, Semon Wu a,b, Tzung-Chieh Tsai c, Lu-Kai Wang d, Fu-Ming Tsai a,⁎ a

Department of Research, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation, New Taipei City, Taiwan Department of Life Science, Chinese Culture University, Shih Lin, Taipei, Taiwan Department of Microbiology, Immunology and Biopharmaceuticals, National Chiayi University, Chiayi, Taiwan d Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan b c

a r t i c l e

i n f o

Article history: Received 6 August 2014 Received in revised form 24 September 2014 Accepted 26 September 2014 Available online 18 October 2014 Keywords: Clozapine Haloperidol Neutrophils HL-60 Phagocytosis

a b s t r a c t Antipsychotic drugs (APDs) have been used to ease the symptoms of schizophrenia. APDs have recently been reported to regulate the immune response. Our previous studies revealed that the atypical APDs risperidone and clozapine and the typical APD haloperidol can inhibit the phagocytic ability of macrophages. Our research next determined the effects of APDs on the phagocytic ability of neutrophils, which are the most abundant type of white blood cells in mammals. Here we provide evidence that clozapine and haloperidol can induce increased phagocytic uptake of Escherichia coli by differentiated HL-60 cells and by purified human neutrophils. Furthermore, clozapine and haloperidol can increase the myeloperoxidase activity and IL-8 production in neutrophils. Our results also show that clozapine can inhibit E. coli survival within differentiated HL-60 cells. Furthermore, clozapine and haloperidol are shown to enhance cell surface Mac-1 expression and the activated AKT signaling pathway in purified neutrophils exposed to E. coli. These results indicate that clozapine and haloperidol can increase the phagocytic ability of neutrophils by increasing AKT activation when cells are exposed to bacteria. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Antipsychotic drugs (APDs) are a group of psychiatric medications used to ease the symptoms of psychotic disorders. First-generation antipsychotics, known as typical antipsychotics, such as chlorpromazine and haloperidol, bind mainly to dopamine D2 receptors [1–3] and were discovered in the 1950s. Most second-generation drugs, known as atypical antipsychotics, such as risperidone and clozapine, bind not only to dopamine D2 receptors, but also to type II serotonin receptors (5-HTRs), dopamine D3 receptor, and dopamine D4 receptor [4–8] and were developed later. Over the years, APDs have been shown to regulate the immune response, and this is involved in the side effects of the medications. Changes in the serum levels of interleukin (IL)-1β, IL-2, IL-6, IL-8, IL-10, and interferon (IFN)-γ as well as regulation of the expression of the receptors for IL-2 and IL-6 have been observed in patients receiving APDs [9–13]. APDs have also been shown to affect immune cell responses. Clozapine and risperidone can inhibit Th1 differentiation, which decreases IFN-γ production by T cells [14,15]. Risperidone has been shown to modulate chemokine/cytokine release by dendritic cells, which regulate Th1/Th2 differentiation [16]. Decreased phagocytic

⁎ Corresponding author. E-mail address: [email protected] (F.-M. Tsai).

http://dx.doi.org/10.1016/j.intimp.2014.09.030 1567-5769/© 2014 Elsevier B.V. All rights reserved.

ability of macrophages has been reported for cells treated with risperidone, clozapine, or haloperidol [17]. Neutrophils are the most abundant white blood cells in the circulation and act as the first line of host defense against invasion by microbial pathogens [18,19]. Many receptors on neutrophils mediate neutrophil phagocytosis. For example, toll-like receptor 4 (TLR4), Fc-γ receptors (CD16), complement receptors CR1 (CD35), and Mac-1 (CD11b/CD18) play important roles in neutrophil-mediated phagocytic uptake of pathogens [20,21]. Previous studies have demonstrated that the intracellular PI3K/AKT and MAPK signaling pathways are involved in phagocytosis by neutrophils [22–26]. Drug-induced neutropenia (neutrophil count b1.5 × 109/L) or agranulocytosis (neutrophil count b5.0 × 108/L) has been observed in patients treated with APDs [27–35]. Previous studies have emphasized the possible mechanism of side effects observed in APD-treated patients. Formation of immature neutrophils has been reported in patients who are on APD medications [36,37]. Also, our recent study indicated that the treatment of dendritic cells with risperidone may induce their production of TNF-α, which subsequently promotes death of neutrophils [16]. Both of these phenomena may be causes of the neutropenia or agranulocytosis induced by APDs. In addition to functions of T cells and macrophages, neutrophil functions may also be regulated by APDs. Due to the short lifespan of purified neutrophils [38,39], it is difficult to study the effect of APDs on purified neutrophils. Many studies use reagents, such as DMSO or all-trans retinoic

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acid (ATRA), to induce differentiation of HL-60 cells [40,41], which can be used as a neutrophil model [42–44]. Comparisons of differentiated HL-60 cells and purified neutrophils have also been investigated [42,45]. To study the effects of APDs on neutrophils, we first examined the effects of a typical APD (haloperidol) and of atypical APDs (risperidone and clozapine) on the phagocytic uptake of Escherichia coli (E. coli) by ATRA-treated differentiated HL-60 cells and purified neutrophils, which were obtained from participants who had no history of APD treatment. We also examined the effects of APDs on cell surface markers and on the activation of AKT/MAPK signaling cascades in purified neutrophils. We found that purified neutrophils treated in vitro with clozapine or haloperidol show enhanced phagocytic uptake of bacteria. Furthermore, increased Mac-1 expression and activation of AKT signaling were also observed in neutrophils treated with clozapine and haloperidol. 2. Materials and methods 2.1. Reagents Clozapine and haloperidol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Risperidone was obtained from Janssen-Pharmaceutica (Beerse, Belgium). 2.2. Preparation of ATRA-treated HL-60 cells HL-60 cells, a human promyelocytic leukemia cell line, were maintained in growth medium consisting of RPMI-1640 supplemented with 10 mM HEPES, 1.5 g/L NaHCO3, 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS), and also 1 μM ATRA for 5 days to induce HL-60 cell differentiation. On the second day of ATRA treatment, APD was added to the culture medium and was replenished daily during HL-60 cell differentiation. 2.3. RNA isolation and quantitative real-time reverse transcription PCR The levels of gene expression in control HL-60 cells, ATRA-treated HL-60 cells, and purified human neutrophils were measured using real-time quantitative PCR (Q-PCR). Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA was then reverse-transcribed to cDNA by incubation at 37 °C for 1 h in 20 μL of a mixture containing 5 μg of total RNA, 1 U of Moloney murine leukemia virus (MuLV) reverse transcriptase (Invitrogen), 0.5 μg of oligo-dT12–18, 4 μL of 5× RT buffer, 0.5 mM dNTP, and 1 U of RNaseout recombinant RNase inhibitor. Q-PCR was performed in triplicate using 25 μL of a reaction mixture containing 12.5 μL of Fast SYBR Green Master Mix (ABI, Applied-Biosystems, Foster City, CA, USA), 50 ng of cDNA, and gene-specific forward and reverse primers at 1 μM final concentration in a thermal cycler (7900HT Fast Real-Time PCR System, ABI). The PCR cycling had an initial incubation at 95 °C for 3 min followed by 45 cycles of denaturation at 95 °C for 15 s and reaction for annealing and extension at 60 °C for 1 min. The PCR primers used for amplification included the following: β-actin (sense, 5′-TCCCTGGAGAAGAGCTACG-3′ and antisense, 5′-GTAGTTTC GTGGATGCCACA-3′), CD66a (sense, 5′-CTCTCCTGCTATGCAGCCTC-3′ and antisense, 5′-ACTGTGGTCTTGCTGGCTTT-3′), CD66b (sense, 5′GAAACAGTGGATGCCAACCG-3′ and antisense, 5′-GAGTCTCCGGATGT ACGCTG-3′), CD66c (sense, 5′-ACCGTCCAGGGGAAAATCTG-3′ and antisense, 5′-AGGAGCACTTCCAGAGACTGT-3′), CD66d (sense, 5′-GACACA AACATTTACTGCCGGA-3′ and antisense, 5′-GAGAGGCCTTTGTCCTGA CC-3′), HTR2A (sense, 5′-AGGGTGCCTCTCACCGTCGT-3′ and antisense, 5′-AAGCTTGCTCGGCAGAGGCC-3′), HTR2B (sense, 5′-ACTGCACTGGGC AGCTCTTCTG-3′ and antisense, 5′-GTGGGAGGGGCCACATAGCCT-3′), and HTR2C (sense, 5′-ACGTGCGTGCTCAACGACC-3′ and antisense, 5′TCGGCGTGCGTTCTGGTCTT-3′). DRD1 (sense, 5′-GGGACTGGGCTGGT GGTGGA-3′ and antisense, 5′-CAGCCACTGCCTTCCAGGGC-3′), DRD2

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(sense, 5′-TGTGCACGGCGAGCATCCTG-3′ and antisense, 5′-GACCACGA AGGCCGGGTTGG-3′), DRD3 (sense, 5′-GCCAGGACACTGCCTTGGGTG3′ and antisense, 5′-CCCGAAGTGGCACTCCCCGA-3′), DRD4 (sense, 5′CGCCCTCCCCACTCCTTGGT-3′ and antisense, 5′-TGGGCTACGTCAACAG CGCC-3′), DRD5 (sense, 5′-TACCCGGGGCAGTTCGCTCT-3′ and antisense, 5′-CTGCGCACACCAGCACGTTG-3′). The relative expression levels of the target cDNA were calculated after normalizing the relative intensity of target cDNA to the intensity of β-actin. 2.4. Phagocytosis Laboratory strain E. coli DH5α bacteria were grown in LB broth (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, and 31.24 mg/L 2,2′dipyridyl). E. coli were stained with 5 μM Oregon Green 488-X succinimidyl ester (Molecular Probes, Eugene, OR, USA) and 1 mg/mL human IgG (Sigma) for 30 min in the dark at room temperature. After washing three times to remove excess fluorescent dyes, bacteria were kept on ice until use. Cells and bacteria were prepared and suspended in Na-medium (5.6 mM glucose, 127 mM NaCl, 10.8 mM KCl, 2.4 mM KH2PO4, 1.6 mM MgSO4, 10 mM HEPES, 1.8 mM CaCl2, pH 7.3). Phagocytosis of bacteria was performed in Na-medium at 37 °C for 5 min at a bacteria/cell ratio of 2:1. The incubation was stopped by the addition of 2 mL of ice-cold phosphate buffered saline (PBS), and the cells were then washed three times with ice-cold PBS. After fixing the cells with 4% paraformaldehyde, phagocytic uptake was analyzed using a FACS flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA). 2.5. Intracellular survival assays APD-treated, ATRA-treated HL-60 cells were prepared, and phagocytic uptake of E. coli was performed at 37 °C for 1 h at a bacteria/cell ratio of 10:1. The phagocytic process was stopped by placing the samples on ice. Subsequently, gentamicin (final concentration 200 μg/mL) was added to kill extracellular bacteria, and the tubes were incubated for an additional 60 min on ice. The neutrophils were then lysed by incubating the samples for 20 min in 2% saponin. The number of E. coli colony-forming units (CFU) was determined. 2.6. Isolation of human neutrophils Peripheral blood samples were obtained from normal adult donors. Following erythrocyte sedimentation with dextran and lysis of contaminating erythrocytes with buffered potassium chloride, the neutrophils were separated from the leukocyte fraction over a discontinuous gradient of Ficoll-Paque PLUS as previously described [46]. The purity of isolated neutrophils was routinely greater than 95% determined by light microscopy based on nuclear morphology. The purified neutrophils were resuspended in a round-bottom polypropylene tube in RPMI medium supplemented with 10% FBS and antibiotics. 2.7. Measurement of myeloperoxidase (MPO) activity of stimulated neutrophils Purified human neutrophils were each seeded at 3 × 106 cells/well in 24-well plates and were treated with various APDs for 4 h. Cells were further treated with 1 μM N-Formyl-Met-Leu-Phe (fMLP, SigmaAldrich) and 5 μg/mL cytochalasin B (CB, Sigma-Aldrich) for another 30 min. The supernatants of cells were collected and levels of MPO in the supernatants were determined using a neutrophil myeloperoxidase activity assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's instructions. 2.8. Measurement of IL-8 production of stimulated neutrophils Purified human neutrophils were each seeded at 3 × 106 cells/well in 24-well plates and treated with various APDs for 4 h. Cells were further

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treated with 100 ng/mL lipopolysaccharide (LPS, Sigma-Aldrich) for another 2 h. The supernatants of cells were collected and the IL-8 production was measured using an ELISA kit (Peprotech, Rock Hill, NJ, USA). 2.9. Measurement of neutrophil surface receptor expression Human neutrophils were prepared and then incubated for 4 h in complete medium containing APDs. The phagocytic uptake of E. coli was performed at 37 °C for 1 h at a bacteria/cell ratio of 10:1. Neutrophils were washed three times with ice-cold PBS and then stained with mouse anti-human TLR4, CD16, CD11b, and Mac-1 antibodies. For TLR4 staining, mouse anti-human TLR4 (BD Biosciences, San Jose, CA, USA) was used to determine TLR4 cell surface expression of neutrophils by indirect immunofluorescence staining. The negative controls were prepared by staining neutrophils with appropriate secondary antibodies alone. Cell surface expression of Mac-1, CD16, and CD11b on neutrophils was assessed by direct immunofluorescence staining using phycoerythrin (PE)-conjugated mouse anti-human Mac-1 (BD Biosciences), CD16 (BD Biosciences), and CD11b (BD Biosciences) antibodies. Concentration-matched and isotype-matched mouse PE-IgG1 was used as the negative control for Mac-1, CD16, and TLR4. Samples were analyzed using a FACS flow cytometer. 2.10. Western blot analysis APD-treated neutrophils were prepared and stimulated with E. coli for 10 min. Cells were then collected by centrifugation followed by resuspension in lysis buffer (25 mM HEPES [pH 7.5], 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, and 10% glycerol) containing protease inhibitors (20 μg/mL aprotinin and 20 μg/mL phenylmethylsulfonyl fluoride) and phosphatase inhibitors (2 mM NaF and 1 mM Na3VO4). Sample aliquots (40 μg) were separated on 10–12% polyacrylamide gels containing sodium dodecyl sulfate and were then transferred to polyvinylidene fluoride membranes. After blocking, the membranes were incubated with antiphospho-p44/42 ERK1/2 (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-AKT (Cell Signaling), anti-p44/p42 ERK1/2 (Cell Signaling), anti-AKT (Cell Signaling), or anti-actin (Sigma-Aldrich) antibodies at 4 °C for 12 h. The blots were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Calbiochem, Darmstadt, Germany) at room temperature for 1 h. Specific protein bands were developed using Amersham ECL (Amersham, Bucks, UK). Relative protein expression was quantified following normalization to the protein levels of actin. 2.11. Statistics Data are represented as mean ± SD of at least triplicated studies. Statistical analyses were performed using one-way ANOVA with Dunnett's post hoc test. A p-value b 0.05 was considered as statistically significant. 3. Results 3.1. mRNA expression of 5-HTR and DR subtypes in control or ATRAinduced differentiated HL-60 cells In order to examine the effects of each APD on neutrophils, we used ATRA-induced differentiated HL-60 cells as neutrophils based on previous studies [40,47]. Fig. 1A shows that expression of CD66d (which is expressed on mature granulocytes) dramatically increased in ATRAtreated HL-60 cells. This indicates that ATRA-treated HL-60 cells represent an appropriate model for granulocyte differentiation. In addition to CD66d, CD66a and CD66b were also increased in ATRA-treated HL60 cells. These results are similar to those observed in a previous study [40]. Because APDs are receptor agonists/antagonists, and their main targets are type II 5-HT or dopamine receptors, we also examined

Fig. 1. Control and ATRA-induced differentiated HL-60 cells express mRNA for CD66, 5HTR, and DR subtypes. HL-60 cells were incubated for 5 days with DMSO (control) or ATRA (ATRA-induced differentiated cells). Total mRNA was isolated, and mRNA expression for CD66 subtypes (A) or for 5-HTR and DR subtypes (B) was analyzed by Q-PCR (n = 3). Shown are the ΔCt values for the indicated receptors of control or ATRA-induced differentiated HL-60 cells after normalization to β-actin.

gene expression of related receptors in HL-60 cells and ATRA-induced differentiated HL-60 cells. In ATRA-treated HL-60 cells, the expression of 5HTR2B, 5-HTR2C, DRD1, DRD2, DRD3, and DRD5 were significantly increased compared to that of control HL-60 cells (Fig. 1B). The expression of the rest of the receptors that were tested was not significantly changed (less than 2-fold) during ATRA-induced HL-60 cell differentiation. 3.2. Clozapine and haloperidol enhance the phagocytic ability of ATRAinduced differentiated HL-60 cells To discern the effect of each APD on neutrophil function, we first determined whether APDs affect the growth of HL-60 cells. Treatment HL-60 cells with ATRA for 5 days resulted in a decrease in cell growth. Cell viability of control and ATRA-treated HL-60 cells was decreased by 19.4–63.3% in cells treated with 10−5 M haloperidol (Fig. 2A). Risperidone, clozapine, and lower doses of haloperidol had no effect on cell viability of HL-60 cells. In addition to cell viability, CD66d expression was also examined. Our results indicate that the APD treatment of ATRAinduced HL-60 cells did not affect CD66d expression (data not shown). This suggests that APDs do not affect differentiation of HL-60 cells. We next evaluated the effects of APDs on the phagocytic capability of HL-60 cells by examining their uptake of fluorescent dye-labeled E. coli as previously described [42]. The method was modified in our study. The intensity of fluorescent in HL-60 cells was considered as the control. The increased intensities of the fluorescence in HL-60 cells after incubating with dye-labeled E. coli were proposed as the uptake of E. coli by HL60 cells (Fig. 2B). We found that there is a dose-dependent increase in phagocytic uptake of E. coli in clozapine- and haloperidol-treated ATRA-induced differentiated HL-60 cells (Fig. 2B and C). In addition, the treatment of control HL-60 cells with 10−5 M clozapine and haloperidol enhanced their phagocytic uptake. Our data show that risperidone did not change the phagocytic uptake of labeled E. coli in control or ATRA-induced differentiated HL-60 cells. 3.3. Clozapine inhibits intracellular survival of E. coli in ATRA-treated HL-60 cells Since clozapine and haloperidol can enhance phagocytic uptake of E. coli in differentiated HL-60 cells, we also examined whether these

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Fig. 3. Clozapine inhibits intracellular survival of E. coli. ATRA-induced differentiated HL-60 cells were incubated with the indicated concentrations of APDs for 72 h. Cells were harvested and allowed to interact with E. coli at 37 °C for 1 h. After incubation with gentamicin, cells were lysed with saponin, and the intracellular survival of bacteria was determined by counting the number of colonies formed after overnight growth. Data shown are expressed as CFU capacity (n = 3).

3.5. Clozapine and haloperidol enhance the phagocytic ability, MPO activity, and IL-8 production of purified human neutrophils

Fig. 2. Clozapine and haloperidol enhance phagocytic uptake of E. coli by ATRA-induced differentiated HL-60 cells. HL-60 cells (3 × 105 cells/mL) were seeded in triplicate in 24well plates and cultured for 5 days with or without ATRA. APDs were then added on the second day at the indicated concentrations. Cell viability was measured by trypan blue exclusion (A). APD-treated ATRA-induced differentiated HL-60 cells were harvested and allowed to interact with Oregon Green-labeled and IgG-opsonized (1 mg/mL) E. coli. The phagocytic capability was determined by flow cytometric analysis. Shown is a representative result obtained from one of three replicate studies (n = 3) (B). The experimental results are summarized as the mean (±SD) of the percentage of HL-60 cells that showed phagocytosis (C). *Indicates p value b 0.05.

Cell viability of neutrophils treated with various APDs for 4 h was measured by trypan blue exclusion. No significant change in neutrophil viability was observed for cells treated with various APDs (data not shown). The effect of each APD on the phagocytic ability of neutrophils was determined by uptake of fluorescent dye-labeled E. coli. Fig. 5 shows that only 10−5 M clozapine and 10−5 M haloperidol have slight potency to enhance neutrophil phagocytic uptake of E. coli by 6.4– 8.1%. Lower doses of clozapine, haloperidol, or risperidone have no effect on phagocytic uptake of E. coli by neutrophils. In addition to the phagocytic ability, we also examined the effect of APDs on MPO activity, which is most abundantly expressed in neutrophil granulocytes. The treatment of neutrophils with APDs did not significantly change the release of MPO. Upon fMLP and CB addition, the activity of MPO was enhanced, indicated the mimic effect of bacterial peptides on neutrophil surface (Fig. 5B). MPO activity was increased when fMLP and CB-stimulated neutrophils were treated with a high dose of clozapine (10−6–10−5 M) and haloperidol (10−5 M) (Fig. 5B). Similar to MPO activity, the IL-8 production of LPS-stimulated neutrophils increased by 15.2–43.6% in neutrophils treated with clozapine (10−5 M) and haloperidol (10−6–10−5 M) (Fig. 5C). Since clozapine and haloperidol increase the phagocytic ability, MPO release, and IL-8 production of neutrophil, in vitro cell migration assay

drugs can influence E. coli survival in differentiated HL-60 cells by determining the intracellular E. coli number using gentamicin to kill all extracellular bacteria [48]. We observed that only clozapine reduced E. coli survival inside ATRA-treated HL-60 cells in a dose-dependent manner (Fig. 3). Risperidone and haloperidol had no effect on E. coli survival in differentiated HL-60 cells.

3.4. Neutrophils express different type II 5-HTR and DR mRNA Previous results suggest that clozapine and haloperidol increase phagocytic uptake by neutrophil cell lines. Therefore, we next investigated the effects of each APD on phagocytosis by purified human neutrophils. We first examined gene expression of related receptors in neutrophils. mRNA from neutrophils was isolated and analyzed by Q-PCR. Our results show that expression of DRD2 in neutrophils is the lowest of all the receptors examined (Fig. 4) (the ΔCt for DRD2 is 17.35 when normalized to β-actin). This result suggests that the effects of APDs on neutrophils may be related to their agonist/antagonist effects on 5-HTR or another DR instead of their antagonist effect on DRD2.

Fig. 4. Neutrophils express mRNA for 5-HTR and DR subtypes. Human neutrophils were freshly prepared, and total RNA was isolated. mRNA expression for 5-HTR and DR subtypes was detected by Q-PCR (n = 3). Shown are the ΔCt values for the indicated neutrophil receptors after normalization to the mRNA of β-actin.

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regulated in the phagocytosing neutrophils (Fig. 6A and B). Increased Mac-1 expression was also observed in a previous study [24]. Neutrophils treated with 10−5 M clozapine and 10−5 M haloperidol significantly increased their expression of Mac-1 (Fig. 6C and D), whereas there was no autofluorescence effect observed in cells treated with APDs (data not shown). Risperidone had no effect on Mac-1 expression by neutrophils. 3.7. Clozapine and haloperidol enhance AKT signaling in purified human neutrophils in response to E. coli stimulation Both PI3K/AKT and MAPK, especially the AKT pathway, are the most extensively studied intracellular signaling cascades involved in phagocytosis by neutrophils [22,24,49]. Our data show that exposure of neutrophils to E. coli stimulated phosphorylation of AKT and increased the p42/p44 MAPK signaling cascades. Pre-treating cells with 10−5 M clozapine significantly resulted in increased AKT phosphorylation by 2.17fold (Fig. 7A and B). Although there was a trend towards increase of AKT phosphorylation in cell treated with haloperidol, the difference was not statistically significant (p = 0.072). No significant difference in p42/p44 MAPK phosphorylation was observed in cells treated with these APDs. 4. Discussion

Fig. 5. Clozapine and haloperidol enhance phagocytic uptake of E. coli, MPO activity, and IL8 production of neutrophils. Purified neutrophils (3 × 106 cells/mL) were incubated with the indicated concentrations of APDs for 4 h. Cells were harvested and allowed to interact with Oregon Green-labeled and IgG-opsonized (1 mg/mL) E. coli. The phagocytic capability was determined by flow cytometric analysis (A). Purified neutrophils (3 × 106 cells/mL) were incubated with the indicated concentrations of APDs for 4 h. Cells were further treated with 1 μM fMLP and 5 μg/mL CB for another 30 min (B) or treated with LPS for another 2 h (C). The supernatant of cultured neutrophils was collected, and the MPO (B) and IL-8 (C) release were measured as described in Materials and methods. The experimental results are summarized as the mean (±SD) of the percentage of neutrophils that showed phagocytosis (n = 3). *Indicates p value b 0.05 when cell treated with APDs followed by E. coli (A), fMLP and CB (B) or LPS (C) stimulation compared to cells stimulated with E. coli, fMLP and CB or LPS only.

was also performed using a modified Boyden chamber. However, our result indicated that APDs had no significant effect on the neutrophils migration upon fMLP stimulation (data not shown). 3.6. Clozapine and haloperidol enhance Mac-1 activation in purified human neutrophils in response to E. coli stimulation Since clozapine and haloperidol can enhance the phagocytic ability of neutrophils, we next determined the effect of each APD on modulating cell surface receptors that regulate phagocytosis by the cells. The increased expression of Mac-1 but not CD11b, CD16, or TLR4 in neutrophils exposed to E. coli indicated that Mac-1 has been up-

Although APDs were developed for blocking D2 dopamine receptors, many receptors on cells may be targets for APDs. Risperidone is classified as an atypical antipsychotic agent, and it acts as a binding antagonist for 5-HT2A/dopamine D2 receptors and as a binding agonist for other receptors [50]. Clozapine is also classified as an atypical APD. It interferes with binding by dopamine receptors (having a high affinity for the D4 receptor), and also interferes with binding by GABAB and 5-HT receptors, but acts as an agonist for NMDA or 5-HT1A receptors [51–53]. Haloperidol, even though it is a typical APD that exhibits high affinity dopamine D2 receptor antagonism, acts as an inverse agonist on other dopamine receptors and binds to sigma-1, adrenergic, Nmethyl-D-aspartate, and 5-HT receptors [54–56]. Elucidation of APD modulation of immunoregulation is difficult due to the complex binding profile of APDs. Clozapine and risperidone are both considered as atypical antipsychotics and only clozapine can affect the neutrophils phagocytic ability. The various drug effects on phagocytic ability of neutrophils might be caused by different receptor binding profile of two APDs. To date, the receptors networks signal transductions underlying the various therapeutic properties of most atypical agents are still not known. Regardless the pharmacological mechanism, clozapine is the most effective APD in treating therapy-resistant schizophrenia [4,57]. Both clozapine and haloperidol would enhance phagocytic ability of neutrophils. Also, clozapine might induce higher MPO and AKT activity of neutrophils than haloperidol. (Figs. 5B and 7) This observation might explain that clozapine is more effective than haloperidol in killing intracellular E. coli. The therapeutic concentration of APDs in the serum of patients is about 1 × 10−7 M for risperidone, 1 × 10−6 M for clozapine, and 2 × 10−8 M for haloperidol [58–62]. Cell viability for HL-60 cells and neutrophils was not affected by APD treatment within the range of clinical doses. This suggests that APDs do not directly cause neutrophil death, which correlates with our previous study [16]. APD-induced neutropenia or agranulocytosis may be caused by APD alteration of neutrophil differentiation or by APD-induced release of cytokines by other cells. Dopamine has been shown to enhance the phagocytic ability of neutrophils [63]. We found that the treatment of differentiated HL-60 cells with clozapine or haloperidol leads to increased phagocytic uptake of E. coli. Also, neutrophils show low expression of DRD2 (Fig. 4). These results suggest that APD inhibition of DRD2-mediated signaling may not play a prominent role in the increased phagocytic ability of neutrophils that is induced by clozapine and haloperidol. In addition to dopamine

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Fig. 6. Clozapine and haloperidol enhance neutrophil Mac-1 cell surface receptors. Purified neutrophils were freshly prepared and stimulated for 1 h with opsonized E. coli. Cells were harvested and then stained with anti-TLR4, anti-CD11b, anti-CD16, or anti-Mac-1 antibody. Representative results are shown from one of three replicate experiments (A) and experimental results are summarized as MFI values for neutrophils (B). Purified neutrophils were treated for 4 h with the indicated concentrations of APDs and stimulated for 1 h with opsonized E. coli. Cells were then harvested and stained with anti-TLR4, anti-CD11b, anti-CD16, or anti-Mac-1 antibody. Shown are flow cytometric data from one of three replicate experiments (n = 3) for Mac-1 expression by neutrophils treated with 10−5 M APDs (C). Experimental results are summarized as MFI values for neutrophils (D). *Indicates p value b 0.05 when cell treated with APDs followed by E. coli stimulation compared to cells stimulated with E. coli only.

receptors, 5-HT receptors have also been shown to modulate phagocytosis by murine macrophages [64]. Whether there is a similar effect of 5-HT receptors on phagocytosis by neutrophils is still unclear, but the results of this study provide information to help discern the effects of APDs on neutrophil death and phagocytic ability. Previous studies reported APD effects on cell function after culture for at least 3 days [14–17,65]. Neutrophils are short-lived cells that spontaneously undergo apoptosis within 24 to 36 h after introduction into culture with active caspase 8 detected at 6 h [66]. To avoid interference of cell death on the observation of APD effects on neutrophil phagocytosis, a short-term experiment is necessary. The short-term culture conditions for APD-treated neutrophils lead to an underestimation of the effects of APDs on neutrophil phagocytic ability, surface markers, and intracellular signaling. Results with APD-treated ATRA-induced differentiated HL-60 cells show that clozapine and haloperidol (10−8 to 10−5 M) increased phagocytic uptake of E. coli. A similar result was observed for neutrophils treated with clozapine and haloperidol at 10−5 M. The clozapine concentration in this study is similar to therapeutic drug concentrations in the serum of patients (about 1 × 10−6 M), whereas a higher dose of haloperidol was used than that for clinical treatment (2 × 10−8 M). These results suggest that a therapeutic dose of clozapine can affect the phagocytic ability of neutrophils. Further studies in APDs-treated animal model or in APD-treated patients should be preceded to verify if therapeutic concentration of clozapine can affect the neutrophil activity. Both neutrophil and macrophage are responsible for phagocytosis and killing microorganisms. In contrast to the inhibitory effect of APDs on macrophage activities, both clozapine and haloperidol increase

neutrophil phagocytic ability, Mac-1 expression, and AKT activation. Neutrophil and macrophage are produced by different progenitors, this might be the cause that differential expression of dopamine receptors, 5-HTR receptors and other APDs-bound receptors between neutrophil and macrophage. In addition to the presence of related receptors, the synergistic action of receptors due to APD binding may also be a factor in the effects of APDs on cells. In addition to acting as a binding antagonist for 5-HT2A/dopamine D2 receptors, many studies showed that APDs may act as binding agonists for other receptors such as 5HT5, 5-HT6, or other dopamine receptors [67–70]. Therefore, the different results for macrophages and neutrophils may result from the specific APD-binding receptors that are present on these cells and the combined effects of these receptors after binding of the APDs. The inhibitory effects on phagocytic activity of macrophage caused by APDs will be a minor issue in vivo because the differentiated macrophage would only appear after cytokines or LPS induction in livings. For those neutrophils presented in circulation system, whether long-term exposure of neutrophils to APDs will affect phagocytic activity of neutrophils should be further studied in animal models or in clinical observation. In conclusion, our study demonstrates that the treatment of ATRAinduced differentiated HL-60 cells and neutrophils with clozapine or haloperidol increases phagocytic uptake of E. coli. Also, clozapine and haloperidol increase cell surface Mac-1 expression by neutrophils exposed to bacteria. Furthermore, activation of AKT signaling pathways may be the primary reason for clozapine- and haloperidol-induced modulation of neutrophil phagocytic uptake.

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Fig. 7. Clozapine and haloperidol increase AKT phosphorylation in neutrophils. Neutrophils were treated with 10−5 M APDs for 4 h. Cells were harvested and stimulated with E. coli for 10 min. Cells were lysed with cell lysis buffer, and aliquots of the cell lysates were resolved by SDS-PAGE. The indicated protein expression levels were determined by immunoblotting (A). Bar graphs quantify the phosphorylation of AKT and p42/p44 proteins with each sample normalized to the level of actin protein in three replicates (B). *Indicates p value b 0.05 when cell treated with APDs followed by E. coli stimulation compared to cells stimulated with E. coli only.

Acknowledgments This work was supported by a grant (TCRD-TPE-103-037) from the Taipei Tzuchi Hospital, the Buddhist Tzuchi Medical Foundation, Taiwan, Republic of China. We thank the Core Laboratory of Taipei Tzuchi Hospital, the Buddhist Tzuchi Medical Foundation for their technical support.

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