Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2014/10/01/jpet.114.218107.DC1.html 1521-0103/351/3/519–526$25.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

http://dx.doi.org/10.1124/jpet.114.218107 J Pharmacol Exp Ther 351:519–526, December 2014

No Evidence for Histamine H4 Receptor in Human Monocytes s Kristin Werner, Detlef Neumann, Armin Buschauer, and Roland Seifert Institute of Pharmacology, Hannover Medical School, Hannover, Germany (K.W., D.N., R.S.); and Department of Pharmaceutical and Medicinal Chemistry II, University of Regensburg, Regensburg, Germany (A.B.) Received July 6, 2014; accepted September 29, 2014

Introduction Histamine (HA) plays an important role in the pathogenesis of pruritus (Thurmond et al., 2014a). HA exerts its effects via four histamine receptor subtypes (Seifert et al., 2013). HA H4 receptor (H4R) is of clinical importance because an H4R antagonist alleviates HA-induced pruritus in humans (Kollmeier et al., 2014; Seifert, 2014). Several independent research groups have rigorously established the presence of H4R in human eosinophils (Raible et al., 1994; O’Reilly et al., 2002; Buckland et al., 2003; Ling et al., 2004; Reher et al., 2012; Thurmond et al., 2014b). H4R couples to pertussis toxin (PTX)– sensitive Gi proteins to mediate increases in intracellular calcium concentration ([Ca21]i), shape change, chemotaxis, and inhibition of cAMP-dependent luciferase expression (Oda et al., 2000; Hofstra et al., 2003; Buckland et al., 2003). In Sf9 insect cells, the hH4R effectively couples to Gi proteins but not Gq proteins (Schneider et al., 2009). It has also been suggested that H4R is expressed in human monocytes to mediate increases in [Ca21]i (Dijkstra et al., 2007). However, the suggested H4R expression in monocytes hinges in part on H4R antibodies with questionable specificity (Neumann et al., 2012; Seifert et al., 2013). This situation

This work was supported by a fellowship of the Studienstiftung des deutschen Volkes (to K.W.) and the Research Training Group 1910 of the Deutsche Forschungsgemeinschaft [GRK1910] (to A.B. and R.S.). dx.doi.org/10.1124/jpet.114.218107. s This article has supplemental material available at jpet.aspetjournals.org.

UR-PI376 (2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio) ethyl]guanidine) induced pertussis toxin–sensitive [Ca21]i increases. However, in quantitative real-time polymerase chain reaction studies we failed to detect hH4R mRNA in human monocytes and U937 promonocytes. In human monocytes, ATP and N-formyl-L-methionyl-L-leucyl-L-phenylalanine increased [Ca21]i, but HA, UR-PI376, and 5-methylhistamine (a dual H4R/H2 receptor agonist) did not. In U937 promonocytes and differentiated U937 cells, HA increased [Ca21]i, but this increase was mediated via HA H1 receptor. In conclusion, there is no evidence for the presence of H4R in human monocytes.

prompted us to reinvestigate H4R expression in human monocytes, focusing on the analysis of Gi protein–mediated Ca21 signaling.

Materials and Methods HA, JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine; herein referred to as JNJ), mepyramine maleate (MEP), 5-methylhistamine dihydrochloride (5-MHA), and Fura-2 AM were purchased from Tocris Bioscience (Bristol, UK). N-Formyl-L-methionyl-L-leucylL-phenylalanine (fMLP), famotidine (FAM), ATP, Triton X-100, RPMI-1640 medium, Dulbecco’s modified Eagle’s medium, bovine calf serum, L-glutamine, penicillin-streptomycin (10,000 U/ml; 10 mg/ml), nickel(II) chloride (NiCl2), Pluronic F-127 and propidium iodide were obtained from Sigma-Aldrich (St. Louis, MO). UR-PI376 (2-cyano1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine) was synthesized as described elsewhere (Igel et al., 2009). Stock solutions (10 mM) of HA, 5-MHA, FAM, and ATP were prepared in distilled water and stock solutions (10 mM) of JNJ, MEP, UR-PI376, and fMLP in dimethylsulfoxide. PTX (lot no. 181218A1) was obtained from List Biologic Laboratories (Campbell, CA). PTX (50 mg) was reconstituted with 100 ml of double-distilled sterile water and used immediately. Geneticin (G418) was obtained from InvivoGen (San Diego, CA). EGTA was purchased from Fluka (Deisenhofen, Germany). Minimum Eagle’s medium nonessential amino acids solution (100
) (NEA) and 10
Dulbecco’s phosphate-buffered saline without Ca21 and Mg21 were purchased from PAA Laboratories (Pasching, Austria). Biocoll separation solution was obtained from Biochrom (Berlin, Germany). CD14 MicroBeads human, autoMACS Pro Running Buffer and MACSQuant Running Buffer were supplied by Miltenyi Biotec

ABBREVIATIONS: ACTB, b-actin; [Ca21]i, intracellular calcium concentration; CT, cycle threshold; FAM, famotidine; fMLP, N-formyl-L-methionyl-Lleucyl-L-phenylalanine; GUSB, b-glucuronidase; h, human; HA, histamine; HEK293T, human embryonic kidney 293T cells; H1R, histamine H1 receptor; H2R, histamine H2 receptor; H4R, histamine H4 receptor; HxR, any histamine receptor subtype; JNJ7777120/JNJ, 1-[(5-chloro-1H-indol-2yl)carbonyl]-4-methylpiperazine; MEP, mepyramine maleate; 5-MHA, 5-methylhistamine; PCR, polymerase chain reaction; PTX, pertussis toxin; UR-PI376, 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine. 519

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ABSTRACT The histamine H4 receptor (H4R) is a classic pertussis toxinsensitive Gi protein–coupled receptor that mediates increases in intracellular calcium concentration ([Ca21]i). The presence of H4R in human eosinophils has been rigorously documented by several independent groups. It has also been suggested that H4R is expressed in human monocytes, but this suggestion hinges in part on H4R antibodies with questionable specificity. This situation prompted us to reinvestigate H4R expression in human monocytes. As positive control, we studied human embryonic kidney 293T cells stably expressing the human H4R (hH4R). In these cells, histamine (HA) and the H4R agonist

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Werner et al. containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.25 mM CaCl2, 10 mM D-glucose, and 20 mM HEPES. Fura2 AM as well as Pluronic were added at final concentrations of 10 mM and 0.2% (m/v), respectively. Subsequently, cells were incubated in the dark under rotation at room temperature for 35 minutes followed by a 1:2 dilution with Krebs HEPES buffer and incubation for further 15 minutes. Afterward, cells were centrifuged at 300g for 10 minutes at 20°C. 1
107 cells/ml were then resuspended in Krebs HEPES buffer and kept under rotation until measurement of [Ca21]i. Immediately before use, 2.5
105 cells were diluted to a final volume of 2 ml with Krebs HEPES buffer. Fluorescence was analyzed at 37°C under constant stirring using a Shimadzu RF 5301 fluorescence spectrometer (Shimadzu, Duisburg, Germany). Excitation and emission wavelengths were 304 and 508 nm, respectively. Cells were incubated for 2 minutes at 37°C in the presence or absence of various substances (any histamine receptor subtype [HxR] antagonists, EGTA, or NiCl2). Experiments with 100 mM EGTA were performed in the absence of extracellular calcium. Basal fluorescence was recorded for 1 minute followed by the addition of a stimulus to the sample. The fluorescence signal was then measured for 2 minutes. Triton X-100 was added at a final concentration of 0.5% (v/v) to lyse the cells and to obtain the maximum fluorescence signal (Fmax). After 1 minute, EGTA was added at a final concentration of 12 mM to chelate Ca21 and to determine the minimum fluorescence signal (Fmin), which was recorded for 1 minute as well. [Ca21]i was calculated using the following equation:  21   Ca 5 Kd
½ðF 2 Fmin Þ ðFmax 2 FÞ: i Kd is the dissociation constant of Fura-2 with 224 nM (Grynkiewicz et al., 1985). Measurement of [Ca21]i in Human Monocytes and U937 Promonocytes. Monocytes (1
107 cells/ml) and U937 promonocytes (5
106 cells/ml) were resuspended in assay buffer (pH 7.4) consisting of 138 mM NaCl, 6 mM KCl, 1 mM MgSO4,1 mM Na2HPO4, 5 mM NaHCO3, 5.5 mM D-glucose, 20 mM HEPES, and 0.1% (v/v) bovine serum albumin. Fura-2 AM was added at a final concentration of 4 mM, and the cells were incubated at 37°C and 5% (v/v) CO2 for 10 minutes. Thereafter, the suspension was diluted 1:2 with assay buffer and incubated for additional 45 minutes. Subsequently, cells were washed and centrifuged at 300g for 10 minutes at 20°C and resuspended to a final volume of 2 ml per 1
106 cells (monocytes) and 5
105 cells (U937 promonocytes) in assay buffer containing 1 mM CaCl2. Fluorescence was measured as described previously for HEK293T cells. While we studied the human monocytes, the basal fluorescence values were so close to Fmin, despite using 1
106 cells per cuvette, that we were not able to calculate [Ca21]i precisely. Apparently, the basal [Ca21]i in our cell preparations was very low, indicating that the cells were not preactivated despite the positive selection with CD14. Consequently, only relative changes in fluorescence intensity are shown for monocytes.

Results As positive control we studied HEK293T cells stably expressing the hH4R (Nordemann et al., 2013). In these cells, the endogenous ligand HA and the selective H4R antagonist UR-PI376 (Igel et al., 2009) induced increases in [Ca21]i that were abrogated by the selective H4R antagonist JNJ7777120 (Thurmond et al., 2004) (Fig. 1). JNJ or the solvent dimethylsulfoxide alone had no stimulatory effect on [Ca21]i. PTX ADP-ribosylates Gi protein a-subunits and thereby blocks interaction of receptors with Gi proteins (Ui and Katada, 1990; Klinker et al., 1996). Because Gi proteins are very abundant in cells (Gilman, 1987; Birnbaumer et al., 1990), it is not trivial to

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(Bergisch-Gladbach, Germany). The fluorescein isothiocyanate mouse anti-human CD14 antibody was obtained from Becton Dickinson (Franklin Lakes, NJ). RevertAid M-MuLV reverse transcriptase (200 U/ml), oligo(dT)18 primer, random hexamer primer, dNTP mix (10 mM), and 5
reaction buffer were supplied by Fermentas (St. LeonRot, Germany). RiboLock RNAse Inhibitor (40 U/ml) was purchased from Thermo Scientific (Wilmington, DE). TaqMan probes Hs99999903_m1 (ACTB [b-actin]; lot no. 1048618), Hs00939627_m1 (GUSB [b-glucuronidase]; lot no. 1191192), Hs01010880_m1 (hH4R; lot no. 595445), and TaqMan Gene Expression Master Mix were purchased from Applied Biosystems (Carlsbad, CA). All other chemicals were obtained from standard sources. Culture of Human Embryonic Kidney 293T Cells. Human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (Manassas, VA). Stably transfected HEK293T cells expressing the hH4R were prepared as described elsewhere (Nordemann et al., 2013). Cells were grown to 80% confluency at 37°C in a humidified atmosphere with 5% (v/v) CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) bovine calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Medium for hH4R-transfected HEK293T cells was additionally supplemented with 1 mg/ml G418 (geneticin). For experiments with PTXtreated cells, hH4R-transfected HEK293T cells were treated with 1 mg/ml PTX for 24 hours. Culture of U937 Promonocytes. U937 cells were purchased from the American Type Culture Collection. Cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) bovine calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 1% (v/v) nonessential amino acids solution at 37°C in a humidified atmosphere with 5% (v/v) CO2. Cells were maintained at a density of 0.5–1.0
106 cells/ml. Isolation of Human Monocytes. The present study was approved by the ethics committee of the Hannover Medical School. Monocytes were purified as described elsewhere using positive CD14 selection (Werner et al., 2014). We studied two healthy female Caucasians (25 and 31 years old) and a male Caucasian (37 years old). Monocyte purity amounted to 92.0 6 5.0% (mean 6 S.D.), and viability was 98.2 6 0.4% (mean 6 S.D.). Quantitative Real-Time Polymerase Chain Reaction. For RNA isolation, cells were sedimented by centrifugation, and the total RNA was isolated using NucleoSpin RNA II Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. The RNA concentrations were measured with a NanoDrop 1000 spectrophotometer (ThermoScientific, Wilmington, DE). First-strand cDNA was synthesized via reverse transcriptase. We mixed 1 mg of total RNA with 1 ml of oligo-(dT)18 primer, 1 ml of random hexamer primer, 2 ml of 10 mM dNTP mix, 0.5 ml of RiboLock RNAse Inhibitor, 4 ml of 5
reaction buffer, and 1.5 ml of RevertAid M-MuLV reverse transcriptase. Distilled water was added to a final volume of 40 ml. Samples were then incubated at 25°C for 10 minutes followed by an incubation step at 37°C for 60 minutes and at 72°C for 10 minutes. For quantitative real-time polymerase chain reaction (PCR), 2 ml of cDNA, 0.4 ml of TaqMan probe (hH4R, ACTB, and GUSB), 10 ml of TaqMan gene expression master mix and distilled water added to a final volume of 20 ml were pipetted into each well of a 96-well plate. Quantitative real-time PCR was performed using a StepOnePlus thermocycler (Applied Biosystems). To start the reactions, samples were heated to 95°C for 10 minutes followed by 40 amplification cycles with a 15-second step at 95°C and a 60-second step at 60°C. Human b-actin (ACTB) and b-glucuronidase (GUSB) served as housekeeping genes. To assess mRNA expression of hH4R, DCT (CT 5 cycle threshold) values were calculated according to following equation: DC T 5 C T (hH 4 R) 2 C T (housekeeping gene). Measurement of [Ca21]i in hH4R-Transfected HEK293T Cells. The Fura-2 AM method was performed to determine changes in [Ca21]i. Essentially, we followed the protocol by Seifert et al. (1992a) with modifications. Specifically, hH4R-transfected HEK293T cells (1
107 cells/ml) were resuspended in Krebs HEPES buffer (pH 7.4)

No Evidence for H4R in Human Monocytes

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completely ADP-ribosylate all available Gi proteins; that is, long incubation times with high toxin concentrations are required to properly interpret PTX-insensitive effects relative to PTXsensitive effects (Cowen et al., 1990; Wenzel-Seifert and Seifert, 1990). Pretreatment of HEK293T cells expressing hH4R with PTX at a high concentration (1 mg/ml) and for a long period of time (24 hours) abrogated HA- and UR-PI376–induced increases in [Ca21]i, whereas ATP-induced [Ca21]i increases were unaffected (Fig. 2). These data show that, in accordance with the literature (Oda et al., 2000; Buckland et al., 2003; Hofstra et al., 2003; Schneider et al., 2009), hH 4 R couples to Gi proteins, whereas ATP receptors bypass Gi proteins. The HA-induced increase in [Ca21]i depended on the presence of extracellular Ca21 and was blocked by NiCl2 (Supplemental Fig. 1), indicative for Ca21 influx via cation channels (Nilius and Droogmans, 2001). In untransfected HEK293T cells, no PTX-sensitive HA-induced increase in [Ca21]i was observed (data not shown). We assessed hH4R expression in various cell types using quantitative real-time PCR with Taqman probes (Bustin, 2000). As expected from the [Ca21]i experiments shown in Figs. 1 and 2, we detected large quantities of hH4R mRNA in HEK293T cells stably expressing hH4R, the housekeeping genes ACTB and GUSB serving as references (Fig. 3; Table 1).

In marked contrast, nontransfected HEK293T cells did not contain hH4R mRNA. Likewise, we did not detect hH4R mRNA in human monocytes or U937 promonocytes. In human monocytes, ATP and the formyl peptide fMLP induced prolonged and large increases in [Ca21]i (Fig. 4). These data are in accordance with the literature (Cowen et al., 1989). In contrast, at concentrations ranging from 10 nM to 100 mM, HA, 5-MHA (a dual H4R/HA H2 receptor [H2R] agonist) (Seifert et al., 2013), and UR-PI376 did not increase [Ca21]i in these cells (Fig. 4). Similar results were obtained with monocytes from two independent donors (Supplemental Figs. 2 and 3). In human U937 promonocytes, ATP effectively increased [Ca21]i (Fig. 5), a result in accordance with the literature (Cowen et al., 1989). In these cells, fMLP is ineffective because the formyl peptide receptor is not expressed (Cowen et al., 1989). HA induced a transient increase in [Ca21]i in these cells, which was abrogated by the HA H1 receptor (H1R) antagonist MEP but not by the H2R antagonist FAM or the H4R antagonist JNJ. Similar results regarding the HA effect on U937 promonocytes were obtained with monocytoid-differentiated HL-60 cells (Seifert et al., 1994), the only difference being that the dual HA H3 receptor/H4R antagonist thioperamide (Seifert et al., 2013) was used instead of JNJ. The latter ligand did not

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Fig. 1. Effects of HA, UR-PI376, and JNJ on [Ca2+]i in hH4R-transfected HEK293T cells. The hH4R-transfected HEK293T cells were loaded with Fura-2 AM, and the changes in [Ca2+]i were assessed. Representative time courses are shown of [Ca2+]i after stimulation with (A and B) HA (10 mM) and (C and D) UR-PI376 (10 mM) in the presence or absence of JNJ (1 mM). Cells were preincubated with JNJ for 3 minutes before HA or UR-PI376 was added. In addition, control measurements were performed with (E) JNJ (1 mM) and (F) dimethylsulfoxide (DMSO; 1%, v/v). Results were confirmed in three independent experiments.

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become available to the research community until 2004 (Thurmond et al., 2004). In the early 1990s, one of the authors (R.S.) examined HA-induced increases in [Ca21]i in U937 cells. The the data were not published because they simply confirmed the data obtained with monocytoid-differentiated HL-60 cells (Seifert et al., 1994). However, in the context of our current discussion on the assumed expression of H4R in monocytes, those previous data have become again relevant. In U937 promonocytes and U937 cells differentiated with dibutyryl-cAMP, interferon-g or 1,25-dihydroxyvitamin D3, HA-induced increases in [Ca21]i were attributable to H1R as assessed by blockade with clemastine (H1R antagonist) (Seifert et al., 2013) and lack of effect of FAM (Supplemental Figs. 4 and 5). As was the case for monocytoiddifferentiated HL-60 cells (Seifert et al., 1994), the HA-induced increases in [Ca21]i in U937 promonocytes were mediated by cation channels (Supplemental Fig. 6). These channels were blocked by gadolinium Gd31.

(Raible et al., 1994; Buckland et al., 2003; Reher et al., 2012). These data indicate that receptor expression levels and stoichiometries of signaling partners are similar in these systems. Unfortunately, one cannot directly compare H4R expression levels in native and recombinant systems because suitable receptor antibodies are not available (Neumann et al., 2012).

Discussion In HEK293T cells stably transfected with hH4R, HA induced PTX-sensitive [Ca21]i increases (Fig. 2). These data corroborate the concept developed by several independent groups that H4R couples to Gi proteins (Oda et al., 2000; Buckland et al., 2003; Hofstra et al., 2003; Schneider et al., 2009). In accordance with the presence of functional H4R protein (Figs. 1 and 2), hH4R mRNA was present in transfected but not in nontransfected HEK293T cells as assessed by TaqMan PCR (Fig. 3; Table 1) (Bustin, 2000). H4R-mediated rises in [Ca21]i in transfected HEK293T cells and human eosinophils are transient and relatively small (Figs. 1 and 2)

Fig. 3. Expression of hH4R on mRNA level in various cells. Quantitative real-time PCR was conducted to evaluate expression of hH4R in hH4Rtransfected HEK293T cells, HEK293T cells, human monocytes, and U937 promonocytes. Results were calculated as DCT values normalized to human GUSB and are from three independent experiments (three different individuals in the case of monocytes) performed in duplicate. ***P , 0.001, statistical significance calculated using one-way analysis of variance followed by Dunnett’s multiple comparison test.

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Fig. 2. Effect of PTX on changes in [Ca2+]i in hH4R-transfected HEK293T cells caused by ATP, HA, and UR-PI376. Cells were treated with 1 mg/ml PTX for 24 hours. [Ca2+]i was determined using the Fura-2 AM method. Representative time courses are shown of [Ca2+]i in control cells stimulated with (A) ATP (10 mM), (B) HA (10 mM), (C) UR-PI376 (10 mM), and in PTX-treated cells stimulated with (D) ATP (10 mM), (E) HA (10 mM), and (F) UR-PI376 (10 mM). Results were replicated in three independent experiments.

No Evidence for H4R in Human Monocytes TABLE 1 DCT values of hH4R expression on mRNA level in various cells Quantitative real-time PCR was conducted to assess hH4R expression on the mRNA level. Data were calculated as DCT values normalized to the housekeeping genes ACTB and GUSB. Data are expressed as mean 6 S.E.M. from three independent experiments (three different individuals in case of monocytes) performed in duplicate. Statistical significance was calculated using one-way analysis of variance followed by Dunnett’s multiple comparison test. Significance was calculated compared with HEK293T cells. Cell Type

hH4R-HEK293T cells HEK293T cells Monocytes U937 promonocytes

DCT (hH4R–ACTB)

22.02 11.92 14.49 13.53

6 6 6 6

0.51* 0.96 0.27** 0.30

DCT (hH4R–GUSB)

25.52 9.10 9.65 9.87

6 6 6 6

0.21* 0.74 0.17 0.31

*P , 0.001; **P , 0.05.

Supplemental Figs. 2 and 3) (Dubyak et al., 1988; Cowen et al., 1989, 1990; Wenzel-Seifert and Seifert, 1990). As predicted from the mRNA data, there was no evidence for H4R-mediated [Ca21]i increases in human monocytes, U937 promonocytes, and untransfected HEK293T cells although control receptors worked (Figs. 4 and 5; Supplemental Figs. 2 and 3). Experiments with hH4R-transfected HEK293T cells confirmed that HA, UR-PI376, and JNJ functioned (Fig. 1). In agreement with our data, Triggiani et al. (2007) reported that HA did not increase [Ca21]i in human monocytes, whereas a reference stimulus did. Dijkstra et al. (2007) reported that 5-MHA and clobenpropit, another H4R agonist (Seifert et al., 2013), increased signals of the Ca21 sensor Fluo-4 in adherent human monocytes. However, the shapes of the fluorescence signals recorded by Dijkstra et al. (2007, see their figure 2) are very different from the Ca21 signals recorded by us. Dijkstra et al. (2007) also reported that HA induced a Ca21 signal, but it is not clear where the traces for HA are shown. The baseline of recordings declined after addition of medium (control) or ligands, whereas in our studies, the baseline remained stable after solvent addition (Fig. 1, E and F). Moreover, fluorescence signals sharply decreased shortly after stimulus addition in the study by Dijkstra et al. (2007), whereas in our studies the fluorescence signals declined gradually (Figs. 1, 2, 4, and 5; Supplemental Figs. 1–6). Stable baselines and gradual decreases of fluorescence signals in

Fig. 4. Effects of HA, 5-MHA, and UR-PI376 on [Ca2+]i in human monocytes. Monocytes were loaded with Fura-2 AM, and the changes in [Ca2+]i were assessed. Representative time-courses are shown of fluorescence intensities from one individual after stimulation with (A) ATP (10 mM), (B) fMLP (1 mM), (C–G) HA (10 nM–100 mM), (H–L) 5-MHA (10 nM–100 mM), and (M–Q) UR-PI376 (10 nM–100 mM). Results were replicated with two additional individuals (see Supplemental Figs. 2 and 3).

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In contrast with transfected HEK293T cells, no hH 4R mRNA was detected in monocytes and U937 promonocytes, two housekeeping genes, and untransfected HEK293T cells serving as control (Fig. 3; Table 1). In monocytic and neutrophilic cells, Gi and Gq protein–coupled receptors mediate phospholipase C activation and increases in [Ca21]i (Dubyak et al., 1988; Cowen et al., 1989, 1990; Wenzel-Seifert and Seifert, 1990). Additionally, fMLP, a ligand for the Gi protein–coupled formyl peptide receptor, and ATP, a ligand for predominantly Gq protein–coupled P2Y-receptors, induced the expected increases in [Ca21]i in human monocytes (Fig. 4;

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monocytes were also reported by Cowen et al. (1989). Perhaps in the study of Dijkstra et al. (2007) the recordings were impacted nonhomogenous distribution of assay constituents. Our studies showed that in HEK293T cells hH4R mediates Ca21 influx from the extracellular space, using removal of extracellular Ca21 with EGTA and the cation channel blocker Ni21 (Nilius and Droogmans et al., 2001) as experimental tools (Supplemental Fig. 1). We did not observe Ca21 mobilization from intracellular stores with the hH4R expressed in HEK293T cells. In the study by Dijkstra et al. (2007), the terms Ca21 mobilization and Ca21 influx are used, but the investigators did not use the experimental tools to discriminate between the two processes. We observed large [Ca21]i increases in human monocytes by ATP and fMLP (Fig. 4; Supplemental Figs. 2 and 3) and in U937 promonocytes by ATP (Fig. 5) as did Cowen et al. (1989), but positive controls were not presented by Dijkstra et al. (2007). Hence, it is very difficult to interpret the Fluo-4 recordings presented in figure 2 of Dijkstra et al. (2007). Dijkstra et al. (2007) did not study the effect of PTX on [Ca21]i increases. In a follow-up study, Gschwandtner et al. (2013, their figure 3C) reported that PTX at a rather low concentration (100 ng/ml) and after a very short incubation time (just 20 minutes) strongly reduced interferon-g– and lipopolysaccharide-induced interleukin-12p70 secretion, rendering assessment of the effect of PTX on H4R agonist actions difficult (see figure 3C in their article). PTX is taken up into target cells and activated before the ADP-ribosyltransferase becomes effective (Ui and Katada, 1990). A pioneering publication on PTX recommended that myeloid cells be incubated for 4 hours with 100 ng/ml PTX to inactivate all Gi proteins (Okajima and Ui, 1984). We used PTX at a high concentration (1 mg/ml) and even much longer incubation time (24 hours). Under these

conditions, hH4R was completely uncoupled from Gi proteins in hH4R-transfected HEK293T cells (compare Fig. 2, B and E, and C and F) with no signs of nonspecific effects (compare Fig. 2, A and D). Other studies also yielded no evidence for nonspecific PTX toxicity in human myeloid cells (Seifert et al., 1992b). Oda et al. (2000) used PTX at 100 ng/ml for 12 hours, Hofstra et al. (2003) used PTX at 50 ng/ml for 16 hours, and Buckland et al. (2003) used PTX for 90 minutes at 1 mg/ml to uncouple the hH4R from Gi proteins. Taken together, the unusually short PTX incubation used by Gschwandtner et al. (2013) was most likely insufficient to ADP-ribosylate all Gi proteins in monocytes, and the strong inhibitory effect of PTX on interferon-g– and lipopolysaccharide-induced interleukin-12p70 secretion remains unexplained. Dijkstra et al. (2007) studied adherent monocytes isolated from buffy coat, whereas we examined monocytes in suspension from three individuals (see Materials and Methods). Age, sex, and ethnicity have only a relatively small quantitative impact on monocyte function (Antonaci et al., 1984; Sondell et al., 1990; Seidler et al., 2010; Appleby et al., 2013). We can exclude that our monocyte preparations were nonresponsive to HA because the cells express H2R positively coupled to adenylyl cyclase and negatively to NADPH oxidase (Werner et al., 2014). There is no evidence for a role of H4R in the regulation of cAMP levels or NADPH oxidase in human monocytes (Werner et al., 2014). In accordance with our data (Fig. 3; Table 1), Ling et al. (2004) did not detect H4R mRNA in monocytes, but Morse et al. (2001) detected H4R mRNA in activated monocytes. However, in the study of Morse et al. (2001), H4R mRNA levels varied considerably among different monocyte populations. Compared with the levels of the housekeeping gene hypoxanthinephosphoribosyl-transferase (HPRT), H4R mRNA levels in the

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Fig. 5. Effects of HxR antagonists on HA-induced [Ca2+]i increases in U937 promonocytes. Changes in [Ca2+]i were determined using the Fura-2 AM method. Representative time-courses are shown of [Ca2+]i after stimulation with (A) ATP (10 mM), (B) HA (10 mM), (C) HA (10 mM) + MEP (1 mM), (D) HA (10 mM) + FAM (10 mM), and (E) HA (10 mM) + JNJ (1 mM). Cells were preincubated with HxR antagonists for 3 minutes before HA was added. Results were confirmed in three independent experiments.

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TABLE 2 Synopsis of the stimulatory effects of HA on [Ca2+]i in various human myeloid cell types and the HxR subtypes and G proteins involved Summary of the stimulatory effects of HA on [Ca2+]i in human myeloid cells. For some cell types, such as dimethylsulfoxide-differentiated HL-60 cells and U937 promonocytes, stimulatory effects of HA were not observed in all studies (indicated by yes/no). Subtle cell strain differences or differences in cell culture conditions may account for these discrepancies. Most studies on cells other than eosinophils focused on H1R and H2R antagonists because these antagonists were fully sufficient to explain the observed effects of HA. In parentheses, the differentiation-inducing compound is given. In all cell types studied, appropriate control ligands, such as ATP, fMLP, or platelet-activating factor, were used to demonstrate functionality of cells. Information about the G proteins involved in signaling is provided as far as possible. Partially PTX-insensitive effects are always difficult to interpret because complete ADP-ribosylation of Gi proteins must be checked (Cowen et al., 1990; Wenzel-Seifert and Seifert, 1990). Cell Type

Stimulatory Effect of HA

G Proteins Involved

References

Seifert et al., 1992c Raible et al., 1994; Buckland et al., 2003; Reher et al., 2012 Triggiani et al., 2007; This study Seifert et al., 1992a Mitsuhashi et al., 1989 Burde and Seifert, 1996 Seifert et al., 1992c Seifert et al., 1994 Seifert et al., 1992a; This study This study This study This study Seifert et al., 1992a Seifert et al., 1992a

Neutrophils Eosinophils

No Yes

NA H4R, H1R excluded

NA Gi, no evidence for Gq

Monocytes

No

NA

NA

HL-60 promyelocytes HL-60 neutrophils (DMSO) HL-60 neutrophils (retinoic acid) HL60 neutrophils (dibutyryl-cAMP) HL-60 monocytes (vitamin D3) U937 promonocytes

Yes Yes/No Yes Yes Yes Yes/No

H1R/H2R H2R H1R/H2R H1R H1R, H4R excluded H1R, H4R excluded

Gq Gq Gq Gi and Gq Gi and Gq Gq?

U937 monocytes (vitamin D3) U937 monocytes (interferon-g) U937 monocytes (dibutyryl-cAMP HEL K562

Yes Yes Yes No No

H1R H1R H1R NA NA

Gq? Gq? Gq? NA NA

DMSO, dimethylsulfoxide; NA, not applicable.

study by Morse et al. (2001) were low. Morse et al. (2001) and Ling et al. (2004) used different methods to detect H4R mRNA. The quantitative real-time PCR used in our study is the most precise method to assess an abundance of a given gene transcript (Bustin, 2000). The experiments with stably transfected hH4R-HEK293T cells (Fig. 1) and with hH4R expressed in Sf9 cells (Schneider et al., 2009) and human eosinophils (Buckland et al., 2003) convincingly demonstrated that hH4R couples only to PTXsensitive Gi proteins but not to PTX-insensitive Gq proteins. There is also no evidence for noncanonical coupling of hH4R to b-arrestin in eosinophils or constitutive activity of hH4R in native cells (Reher et al., 2012). Possibly, the effects of H4R ligands reported by Dijkstra et al. (2007) and Gschwandtner et al. (2013) represent H4R-independent off-target effects. Most notably, Dijkstra et al. (2007) reported that clobenpropit inhibits production of CC chemokine ligand 2 (CCL2) with a half-maximal effect of ∼1 pM. This value is at least 10,000fold lower than the EC50 values for clobenpropit reported in other publications for various parameters (Lim et al., 2005; Nordemann et al., 2013). The exceptionally high potency of clobenpropit in the study of Dijkstra et al. (2007) remains unexplained. The search for new HxRs in human myeloid cells beyond the H1R and H2R linked to Ca21 signaling began in the late 1980s (Mitsuhashi et al., 1989). Table 2 provides a summary of the effects of HA on [Ca21]i in human myeloid cells. In human eosinophils, HA increases [Ca21]i via H4R (Raible et al., 1994; Buckland et al., 2003; Reher et al., 2012). In contrast, in human neutrophils and monocytes HA has no stimulatory effect on [Ca21]i (Fig. 4) (Seifert et al., 1992c; Triggiani et al., 2007). In HL-60 neutrophils, the stimulatory effects of HA on [Ca21]i can be attributed to H1R and/or H2R. In HL-60 monocytes, U937 promonocytes, and U937 monocytes the stimulatory effects of HA on [Ca21]i are fully attributable to

H1R. In human erythroleukemia cells (HEL and K562), there is no evidence for HxRs linked to rises in [Ca21]i. In conclusion, we did not obtain evidence for H4R in human monocytes. The absence of hH4R mRNA in monocytes and U937 promonocytes explains the absence of hH4R protein and supports the notion that H4R antibodies lack specificity (Neumann et al., 2012). The discrepancies between our study and those of others call for additional studies on monocytes by independent investigators. Acknowledgments

The authors thank Dr. Erich Schneider for helpful discussions, Mrs. Evelyn Glaß (Institute of Pharmacology, Free University of Berlin) for performing the experiments shown in Supplemental Figs. 4 and 5, and Dr. Ian Musgrave (Institute of Pharmacology, Free University of Berlin) for performing the experiments shown in Supplemental Fig. 6. The authors thank Dr. Sabine Wolter for technical advice with the analysis of the quantitative real-time PCR data. The authors also thank the staff of the Institute of Pharmacology of the Hannover Medical School for donating blood. Lastly, the authors express gratitude to the reviewers of this paper for their helpful suggestions and critique. Authorship Contributions

Participated in research design: Werner, Neumann, Buschauer, Seifert. Conducted experiments: Werner. Performed data analysis: Werner, Neumann, Buschauer, Seifert. Wrote or contributed to the writing of the manuscript: Werner, Neumann, Buschauer, Seifert. References Appleby LJ, Nausch N, Midzi N, Mduluza T, Allen JE, and Mutapi F (2013) Sources of heterogeneity in human monocyte subsets. Immunol Lett 152:32–41. Antonaci S, Jirillo E, Ventura MT, Garofalo AR, and Bonomo L (1984) Non-specific immunity in aging: deficiency of monocyte and polymorphonuclear cell-mediated functions. Mech Ageing Dev 24:367–375. Birnbaumer L, Abramowitz J, and Brown AM (1990) Receptor-effector coupling by G proteins. Biochim Biophys Acta 1031:163–224.

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Address correspondence to: Dr. Roland Seifert, Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany. E-mail: [email protected]

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