Histidine Decarboxylase Knockout Mice Production and Signal

Nitric Oxide Mediates T Cell Cytokine Production and Signal Transduction in Histidine Decarboxylase Knockout Mice

This information is current as Agnes Koncz, Maria Pasztoi, Mercedesz Mazan, Ferenc of October 1, 2021. Fazakas, Edit Buzas, Andras Falus and Gyorgy Nagy J Immunol 2007; 179:6613-6619; ; doi: 10.4049/jimmunol.179.10.6613 http://www.jimmunol.org/content/179/10/6613 Downloaded from

References This article cites 41 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/179/10/6613.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 1, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Nitric Oxide Mediates T Cell Cytokine Production and Signal Transduction in Histidine Decarboxylase Knockout Mice1

Agnes Koncz,*† Maria Pasztoi,* Mercedesz Mazan,* Ferenc Fazakas,‡ Edit Buzas,* Andras Falus,*§ and Gyorgy Nagy2,3*¶

Histamine is a key regulator of the immune system. Several lines of evidence suggest the role of histamine in T cell activation and accelerated Th1 immune response is a hallmark of histidine decarboxylase knockout (HDC-KO) mice, with a complete lack of endogenously produced histamine. According to our previous work, T lymphocytes produce NO upon activation, and NO is necessary for effective T cell activation. To study the role of histamine in T cell activation, we investigated cytokine production and T cell signal transduction in HDC-KO and wild-type (WT) mice. In the absence of histamine, an elevated IFN-␥ mRNA and (0.0009 ؍ respectively) were associated with a markedly increased (2.5-fold, p ,0.001 ؍ protein levels of splenocytes (p < 0.001; p NO production, compared with WT animals. Furthermore, histamine treatment decreased the NO production of splenocytes from Downloaded from (respectively). NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl ,0.0004 ؍ p ;0.001 ؍ both WT and HDC-KO mice (p -whereas NO synthase inhibitors NG ,(0.0002 ؍ amino] diazen-1-ium-1,2-diolate-diethylenetriamine elicited IFN-␥ production (p respectively), suggesting the ,0.01 ؍ and p 0.002 ؍ monomethyl-L-arginine and nitronidazole both inhibited IFN-␥ production (p role of NO in regulating IFN-␥ synthesis. Cytoplasmic Ca2؉ concentration of unstimulated T cells was increased in the HDC-KO whereas T cell activation-induced ␦ Ca2؉-signal was similar in both HDC-KO and WT animals. Our present data ,(0.02 ؍ mice (p indicate that, in addition to its direct effects on T lymphocyte function, histamine regulates cytokine production and T cell signal http://www.jimmunol.org/ transduction through regulating NO production. The Journal of Immunology, 2007, 179: 6613–6619.

istamine, released from effector cells (mast cells and ba- est activity of HDC and histamine content were detected in mast sophils) during inflammatory reactions, can influence cells and basophils, the expression of HDC and histamine was H the immune responses (1). Mast cells, the primary ef- observed in nearly all of the proliferative tissues studied, suggest- fectors of immediate-type allergic reactions, reside in peripheral ing a possible further biological relevance of histamine (3). In tissue that directly interface with the external environment. addition to its effect on effector cells, histamine also presents im- Crosslinking of surface IgE by cognate Ags or direct binding of munoregulatory properties as it modulates cytokine production by microbial products or complement components to cellular recep- different cell types. Histamine exerts its effects through four types by guest on October 1, 2021 tors induce mast cells to release large amounts of inflammatory of histamine receptors (H1, H2, H3, and H4), signaling via differ- mediators, such as histamine, proteases, prostaglandins, and leu- ent signal-transduction pathways (4, 5). kotrienes (2). Histamine accounts for ϳ10% of mast cell granule Engagement of TCR leads to the activation of multiple protein 4 content. It is exclusively the histidine decarboxylase (HDC) en- tyrosine and serine kinases, resulting in the phosphorylation of zyme that generates histamine from L-histidine. Although the high- intracellular substrates. The subsequent hydrolysis of phospholip- ids and elevation of cytoplasmic Ca2ϩ ultimately leads to clonal *Department of Genetics, Cell and Immunbiology, Semmelweis University, Medical expansion of Ag-specific T cells (6). Once activated, CD4 T cells † School, Budapest, Hungary; Heim Pal Children’s Hospital, Budapest, Hungary; proliferate and differentiate into two main subsets of primary ef- ‡Clinical Research Center, Thrombosis, Haemostasis and Vascular Biology Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and fector cells, Th1 or Th2 cells, characterized by their specific cy- Health Science Center, Debrecen, Hungary; and §Inflammation Biology and Immu- tokine expression pattern (7). Th1 cells promote cellular immunity nogenomics Research Group, Hungarian Academy of Siences-Semmelweis Univer- sity and ¶Department of Rheumatology, Semmelweis University, Medical School, and macrophage activation largely through the production of IL-2 Budapest, Hungary and IFN-␥ (8). Th2 cells, through the expression of IL-4, IL-5, and Received for publication April 18, 2007. Accepted for publication September IL-13, induce IgE production by B cells and eosinophil-mediated 13, 2007. and mast-cell-mediated immune responses, and orchestrate the de- The costs of publication of this article were defrayed in part by the payment of page fense against extracellular parasites (9). Th2 cells have a central charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. role in driving the immune response in asthma and atopic diseases 1 This work was supported by Grants OTKA F 61030 and OTKA T 046468. (10). The Th1/Th2 balance is therefore considered to be pivotal in chronic inflammatory diseases. Recently, Th17 cells have been 2 G.N. is a Bolyai research fellow. identified as cells induced by IL-6 and TGF-␤ and expanded by 3 Address correspondence and reprint requests to Dr. Gyorgy Nagy, Department of Rheumatology, Semmelweis University, Medical School, Budapest, Hungary. E-mail: IL-23 (11). Th17 cells have been strongly implicated as well in [email protected] allergic diseases (12). Histamine selectively enhances the secretion 4 Abbreviations used in this paper used in this paper: HDC, histidine decarboxylase; of Th2 cytokines, such as IL-4, IL-5, IL-10, and IL-13, and inhibits ROI, reactive oxygen intermediate; HDC-KO, HDC gene knockout; WT, wild type; the production of Th1 cytokines IL-2 and IFN-␥ (13) and mono- NOC-18, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate-diethylenetriamine; DAF-FM, 4-amino-5-methylamino-2Ј,7Ј-difluorofluorescein kine IL-12 (10, 14). The crucial role of histamine in the early diacetate; NOS, NO synthase; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, events of the pathogenesis of atopic asthma is associated with the endothelial NOS; DC, dendritic cell. increased production of Th2 and decreased production of Th1 cy- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 tokines (10). www.jimmunol.org 6614 NO MEDIATES T CELL SIGNAL TRANSDUCTION IN HDC-KO MICE

Crosslinking of the TCR has been associated with elevation of ELISPOT 2ϩ the cytosolic Ca , reactive oxygen intermediate (ROI), NO pro- Splenocytes were plated in duplicates in 96-well plates (MultisScreen; Mil- duction, and mitochondrial hyperpolarization (15). In contrast to lipore) (105 cells/plate) precoated with an anti-IFN-␥, anti-IL-10, or anti- histamine, NO selectively enhances Th1 cell proliferation (16) and IL-4 capture mAb (mouse IFN-␥, IL-10, and IL-4 Duoset; R& D Systems), represents an additional signal for the induction of T cell subset blocked in the presence of 1% FCS-containing PBS for1hat37°C, and ␮ response (17). Although there were several pharmacological ap- were stimulated with 2 g/ml Con-A (Sigma-Aldrich) for 24 h at 37°C, 5% CO2. After washing with PBS-Tween 20 ten times, the cells were incu- proaches to study the biological role of histamine in allergic and bated with biotinylated anti-IFN-␥, anti IL-10, or anti IL-4 detection mAb autoimmune diseases, its exact role in immune regulation is far (mouse IFN-␥, IL-4, and IL-10 Duoset, R&D Systems) for 2 h followed by from being uncovered. It has been shown that both histamine and 10 washes and incubation with streptavidin-HRP conjugate for 1 h, and NO can modulate the cytokine network in multiple ways, however, then, after ten further washes by the chromogenic substrate (aminoethyl carbazole, Sigma-Aldrich and H2O2) for 20 min, the plates were washed the possible role of NO in the immunoregulatory functions of his- with distilled water. The number of IFN-␥, IL-10, or IL-4 producing spot tamine is not known (18). In our present study, we investigated the forming cells was counted by ELISPOT reader (CTL). In some experi- effect of genetically induced histamine deficiency on T lymphocyte ments, ELISPOT assays were performed in the presence of NO donor cytokine expression and T cell activation, using HDC gene knock- (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2- G out (HDC-KO) and congenic wild-type (WT) mice. Our results diolate diethylenetriamine (NOC-18) or NOS inhibitor N -monomethyl-L- arginine (Molecular Probes), or nitronidazole (Calbiochem). indicate that histamine deficiency profoundly alters T lymphocyte cytokine expression and T cell activation. Furthermore, our data Measurement of intracellular NO levels show that, in addition to its direct effects on T cells, histamine Production of NO was assessed by using 4-amino-5-methylamino-2Ј,7Ј- modulates immune responses through regulating NO production. difluorofluorescein diacetate (DAF-FM; Molecular Probes) (15, 20). DAF-FM passively diffuses across cellular membranes; once inside cells, it Downloaded from is deacetylated by intracellular esterases and caged in the cell. DAF-FM is Materials and Methods essentially nonfluorescent until it reacts with NO to form a fluorescent Animals benzotriazole. Measurement of NO was calibrated by incubating splenocytes with NO donors NOC-18 (60 ␮M to 1.8 mM). Following testing, The strategy to generate HDC-KO mice has been described previously 1–10 ␮M dye concentration during incubation times ranging from 10 min Ϫ Ϫ (19). HDC / CD1 mice were backcrossed onto the BALB/c background to 3 h, maximum NO sensitivity was achieved by loading cells with 1 ␮M

over nine generations. Female mice were 6–8 wk of age at the beginning DAF-FM for2hin37°C. NO production within T cell subsets were con- http://www.jimmunol.org/ of the sensitization. All mice were maintained on a histamine-free diet currently analyzed by parallel staining with DAF-FM and PE conjugated ϩ Ϫ (Altromin). The WT and HDC-KO mice were littermates of HDC / ϫ mAb UCHT1 recognizing the CD3 chain (BD Biosciences). Excitation and ϩ Ϫ HDC / crosses. The study was approved by an Institutional Review emission maximum of DAF-FM are 495 and 515 nm, respectively. Committee. In some experiments, WT and HDC-KO mice were injected with CFA and splenocytes were isolated 9 days later. Splenocytes were Serum nitrite and nitrate measurements resuspended at 106 cells/ml in RPMI 1640 medium supplemented with 1% Serum nitrate/nitrite levels (stabile in vivo markers of NO production) FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 ␮g/ml gentamicin were measured by using High-Sensitivity Nitrite Assay kit (Molecular in 12-well plates at 37°C in a humidified atmosphere with 5% CO . 2 Probes) (21). Cell viability assays Measurement of cytoplasmic calcium level and mitochondrial mass by guest on October 1, 2021 Apoptosis was monitored by observing cell shrinkage and nuclear frag- 2ϩ mentation and was quantified by flow cytometry after concurrent staining Cytoplasmic calcium levels ([Ca ]c) were assessed by loading the cells with fluorescein-conjugated annexin V [Annexin VFITC;R&DSystems; with 1 ␮M Fluo-3-acetoxymethyl ester (excitation, 506 nm; emission, 526 fluorescence channel 1 (FL-1)] and propidium iodide (FL-2), as described nm recorded in FL-1; Molecular Probes) (15, 20). After entering the cell, earlier (15). acetoxymethyl ester hydrolysis occurs and, thereafter, the dye is trapped in the cytosol. Fluo-3 elicits a large increase of fluorescence intensity on 2ϩ RT-PCR binding calcium. To investigate early Ca fluxing, cells were prelabeled by Fluo-3 and stimulated with Con-A 2 ␮g/ml, whereas Fluo-3 fluo- Total RNA was isolated from spleens of CFA-injected HDC KO and WT rescence of CD3ϩ T cells was continuously recorded by flow cytometry. mice using TRI-reagent (Sigma-Aldrich). First strand cDNA was produced In some experiments, cell permeable BAPTA-AM (Molecular Probes) using random hexamers (Promega). In the RT-PCR, the following primers was used to chelate intracellular Ca2ϩ. Samples were analyzed using a were used: mouse IFN-␥ sense: CCT CAG ACT CTT TGA AGT CT and FACSCalibur flow cytometer (BD Biosciences) equipped with 20-mW antisense: CAG CGA CTC CTT TTC CTC TT, (annealing temperature argon (emission at 488 nm) and 16-mW helium-neon lasers (emission at was 54°C, 35 cycles) and mouse GAPDH sense: CTG GTG CTG AGT 634 nm). Dead cells and debris were excluded from the analysis by ATG TCG TG, antisense: CAG TCT TCT GAG TGG CAG TG (57°C, 30 electronic gating of forward and side scatter measurements. Each mea- cycles). The PCR were conducted using a Perkin-Elmer thermal cycler. surement was conducted on 10,000 cells. PCR products were investigated on 3% agarose gels. Flow cytometric analysis of mitochondrial mass and ROI Quantitative RT-PCR production T cells were isolated by using magnetic beads (negative selection) (Milte- Mitochondrial mass was monitored by staining with 50 nM nonyl acridine nyi Biotech), total RNA was extracted from T cells using RNeasy Mini Kit orange (NAO, excitation, 490 nm; emission, 540 nm recorded in FL-1) (15, 20). Fluorescent probe was obtained from Molecular Probes. ROI was (Qiagen). First strand cDNA was produced using random hexamers (Pro- ␮ mega). Relative quantification of neuronal NO synthase (NOS (nNOS)), monitored by 1 M hydroethidine (Molecular Probes). Fluorescence emis- inducible NOS (iNOS), and endothelial NOS (eNOS) mRNAs was per- sion from oxidized hydroethidine was detected at a wavelength of 560 nm. formed with a TaqMan real-time RT-PCR assay on an ABI Prism 7000 Dead cells and debris were excluded from the analysis by electronic gating Sequence Detector (Applied Biosystems) using standard protocols. In of forward- and side-scatter measurements. brief, 2 ␮l of cDNA in water was added to 12.5 ␮l of the 2ϫ PCR mas- Intracellular flow cytometry termix (Applied Biosystems), primers, and probe in 1.3 ␮l and water to 25 ␮l. The reactions were amplified for 15 s at 95°C and 1 min at 60°C for 45 Splenocytes form HDC-KO and WT mice were stimulated with 2 ␮g/ml cycles followed by the thermal denaturation protocol. All reactions were ConA for 48 h, treated with GolgiPlug Protein Transport Inhibitor (con- run in duplicate and included no template and no reverse transcription tains brefeldin A) (BD Biosciences) and were incubated for5hat37°C in ␮ controls for each gene. Analyses of real-time quantitative PCR data were a humidified CO2 incubator. Then 0.5 g/ml fluorochrome-conjugated performed using the comparative threshold cycle method. The relative mAb specific for cell surface Ags (CD3-PE, CD4-PerCP, CD8-PerCP, amount of mRNA was referred to the one of hypoxanthine phosphoribosyl CD45-PerCP, or CD25-PerCP-CY5, all from BD Biosciences) was added transferase. and incubated for 30 min at 4°C in the dark. Cells were washed in PBS, and The Journal of Immunology 6615

FIGURE 1. Splenocytes of HDC-KO mice produce increased amount of IFN-␥ both at the mRNA and protein levels. A, Splenocyte IFN-␥ mRNA levels were assessed by RT-PCR method. HDC-KO splenocytes displayed signiﬁcantly higher levels of IFN-␥. B, IFN-␥ production of HDC-KO splenocytes following in vitro stimulation with 2 ␮g/ml. Con-A was sig- niﬁcantly higher, measured by ELISPOT method. Data represent relative Ϯ spot number SD of three independent experiments. FIGURE 2. Expression of NOS isoforms in HDC-KO and WT T lymphocytes. Relative amounts of NOS mRNA was referred to hypoxanthine- phosphoribosyl transferase (HGPRT).

then ﬁxed and permeabilized by using Fixation/Permeabilization solution Downloaded from (BD Biosciences) for 20 min. at 4°C. Cells were washed two times in Perm/Wash buffer (BD Biosciences), then incubated 0.5 ␮g/ml FITC-conjugated anti-IFN-␥ Ab (BD Biosciences) for 30 min at 4°C in the dark. The increased IFN-␥ production seems to represent a shift in the Samples were washed in PBS and analyzed by three or four color analysis entire T cell population. on a FACSCalibur ﬂow cytometer (BD Biosciences) using CellQuest soft- ware version 3.1. Histamine regulates NO production in T cells

Mass spectrometry NO is an important physiological regulator of T cell function (15), http://www.jimmunol.org/ thus, we next studied NO synthase (NOS) expression and NO pro- One hundred milliliters of cold methanol containing 2 mM stable isotope- labeled (U-13C6, 98%; U-D5; U-15N3) l-histidine (Cambridge Isotope duction of T lymphocytes derived from HDC-KO and WT mice. Laboratories) was added to 5 ml serum, and then samples were filtrated in Following the isolation of T cells from splenocytes, NOS expres- a Millipore Multiscreen Solvinert filter plate. The filtrates were evaporated sion of the HDC-KO and WT T lymphocytes was measured by to dryness under nitrogen, and 70 ml 3 N butanolic HCl was added to each quantitative real time RT-PCR. Our data indicate the highest ex- sample. After esterification at 60°C for 20 min, butanol was evaporated and samples were reconstituted in 100 ml 50% acetonitrile/water, 0.02% formic pression of the nNOS isoform, whereas lower levels of iNOS and acid. For quantitative analysis 10 ml of reconstituted sample was delivered eNOS isoforms were detected (Fig. 2). Although all three isoforms to an API 2000 (Applied Biosystems) triple quadruple mass spectrometer. were expressed predominantly in the HDC-KO T cells, there was by guest on October 1, 2021 The intensities of ions representing nonlabeled (212.2/110.2) and isotope- no significant difference in the expression of NOS isoforms be- labeled (226.3/123.2) histidine butil esters were measured and the ratios of tween the WT and HDC-KO T cells. ion intensities derived from nonlabeled and labeled histidine were used for the calculation of serum histidine concentration. The NO production of T lymphocytes was measured by flow cytometry. T cells from HDC-KO mice produced higher amounts Statistics of NO than those from the WT (Fig. 3A; p ϭ 0.0009). T cell Results were analyzed by a Student’s t test or Mann-Whitney U rank sum activation is associated with ROI and NO production (15). In ad- test for nonparametric data. Changes were considered significant at dition to higher baseline NO production of the T lymphocytes of p Ͻ 0.05. HDC-KO animals, T cell activation elicited accelerated NO signal ( p ϭ 0.00024, Fig. 3B). To further investigate the role of hista- Results mine in the regulation of NO production, HDC-KO and WT ␥ Ϫ HDC-KO splenocytes produce increased IFN- at both mRNA splenocytes were costimulated with 2 ␮g/ml ConA and 10 6 M and protein levels histamine for 24 h, and the NO production of T cells was measured Previous data from our laboratory reveal that HDC-KO mice are by flow cytometry. According to our data, histamine down-regu- characterized by a strong Th1-biased cytokine pattern (13, 14). lates NO production of both HDC-KO and WT T lymphocytes Because IFN-␥ is a prototype of Th1 cytokines, we first studied (Fig. 3, C and D, p ϭ 0.0004 and p Ͻ 0.001, respectively). IFN-␥ production of HDC-KO and WT splenocytes. Splenocyte Nitrite and nitrate are stable end products of NO production. Our IFN-␥ mRNA levels were measured by RT-PCR. In comparison data indicate that T cells from HDC-KO mice produce higher with WT splenocytes, HDC-KO splenocytes displayed signifi- amounts of NO than control T cells, but a substantial amount of cantly higher levels of IFN-␥ mRNA ( p Ͻ 0.001, Fig. 1A). To NO can be generated by many other cell types as well (22). To further study the cytokine production of HDC-KO and WT spleno- estimate total NO production, the serum nitrite and nitrate concen- cytes, IFN-␥, IL-4, and IL-10 protein levels were measured by trations of HDC-KO and WT mice were measured. Nitrite and ELISPOT method, following in vitro stimulation with 2 ␮g/ml nitrate levels were measured both following in vivo CFA treatment Con-A. In accordance with the PCR data, the IFN-␥ production of and without in vivo stimulation. There was no significant differ- splenocytes from HDC-KO mice was higher than that of the WT ence in the nitrite and nitrate productions between the HDC-KO animals (Fig. 1B; p Ͻ 0.001). IL-4 and IL-10 protein levels were and WT animals in either conditions (Fig. 3E). ϭ ϭ similar in both HDC-KO and WT splenocytes ( p 0.23 and p 2ϩ 0.4, respectively, data not shown). The IFN-␥ production of the T cell activation-induced rapid Ca signal is accelerated in CD4, CD4/CD25, CD8, and CD45 positive T cell subsets were HDC-KO T cells measured by intracellular flow cytometry. IFN-␥ production was Our previous data show that NO regulates the activation and signal similar in all these subsets of both the HDC-KO and WT T cells. transduction of T cells (15, 20), therefore, we next investigated the 6616 NO MEDIATES T CELL SIGNAL TRANSDUCTION IN HDC-KO MICE

FIGURE 3. Histamine regulates NO production of splenocytes. A, NO production of HDC-KO and WT T lymphocytes B, ConA induced NO signal in T lymphocytes from HDC-KO and WT animals. Cells were stimulated with 2 ␮g/ml ConA for 4 and 24h. C and D, Effect of histamine treatment on the NO production of HDC-KO (C), and WT (D) T cells. Cells were stimulated with 2 ␮g/ml ConA for 24h (Ⅺ) or with ConA ϩ 10Ϫ6 M histamine for 24h (u). NO production was monitored by DAF-FM fluorescence and values were expressed as relative DAF-FM fluorescence intensity. E, Serum nitrite, nitrate levels were measured by fluorescent nitrite assay. Downloaded from http://www.jimmunol.org/

T cell activation-induced Ca2ϩ-signal in HDC-KO and WT T lym- and T cell signal transduction. Because NO may modulate gene phocytes. Activation of T cells through the TCR initiates a bipha- transcription and cytokine production (15, 22, 23), next we inves- ϩ sic elevation in the cytosolic-free Ca2 concentration, a rapid ini- tigated if NO directly regulates IFN-␥ production. NO precursor tial peak observed within 5–10 min, and a plateau phase lasting 4 NOC-18 releases NO in a dose dependent manner. Treatment of 2ϩ to 48 h (6). Cytoplasmic Ca concentration of unstimulated T splenocytes with NO precursor NOC-18 (60 ␮M for 24h) in- 2ϩ cells and T cell activation-induced rapid Ca signal are both creased 2 ␮g/ml ConA-induced IFN-␥ production (Fig. 5A; 1.38 Ϯ markedly increased in HDC-KO T cells (Fig. 4A; p ϭ 0.02; p ϭ ϭ ␮ ϩ 0.13-fold; p 0.0002). Six hundred M nitronidazole and 100 2 by guest on October 1, 2021 G 0.04, respectively). Indeed, although the basal Ca level is higher ␮MN -Monomethyl-L-arginine both inhibited IFN-␥ production in the HDC-KO T cells, there is no difference in the stimulation- of HDC-KO splenocytes (Fig. 5B; p ϭ 0.01; p ϭ 0.002, respec- induced ␦ Ca2ϩ 2ϩ levels. T cell activation-induced sustained Ca tively). In parallel, NO production, monitored by DAF-FM signal was similar both in HDC-KO and WT T lymphocytes (data fluorescence, was inhibited by Ͼ60%, following both NG-Mono- not shown). To investigate the role intracellular Ca2ϩ on IFN-␥ ϩ methyl-L-arginine and nitronidazole treatment. Moreover, pharmo- production, the effect of cell permeable intracellular Ca2 chelator cological inhibition of NO production in HDC- KO splenocytes BAPTA-AM was studied. Con-A treatment induced IFN-␥ pro- ␥ duction was inhibited by 10 ␮M BAPTA-AM cotreatment (Fig. decreased IFN- synthesis, further supporting the role of NO in ␥ 4B; p ϭ 0.001). regulating IFN- production. By contrast, treatment of splenocytes with 10Ϫ6 M histamine ␥ NO regulates IFN- production did not significantly alter 2 ␮g/ml ConA-induced IFN-␥ produc- Our present data indicate that the increased NO production of tion (13) (data not shown). There was no significant difference in HDC-KO T cells is associated with altered cytokine production the L-histidine serum levels of the HDC-KO and WT mice (73 Ϯ

FIGURE 4. A, T cell activation induced rapid Ca2ϩ-signal is increased in HDC-KO T cells. T lymphocytes of HDC-KO and WT mice were stimulated with ConA. Cytoplasmic Ca2ϩ concentration was measured by flow cytometer by using Fluo-3 fluorescent dye. Values were expressed as relative Fluo-3 fluorescence. B, ConA-induced IFN-␥ production was inhibited by using 10 ␮M cell permeable intracellular Ca2ϩ chelator BAPTA- AM. Results were expressed as relative number of IFN-␥ secreting spot forming cells. The Journal of Immunology 6617

FIGURE 5. Effect of NO donor and NOS inhibitors on the IFN-␥ production of splenocytes. A, Spleno- cytes from WT mice were stimulated with 2 ␮g/ml ConA for 24h (Ⅺ)or were treated with 2 ␮g/ml ConA ϩ 60 ␮M NOC-18 for 24h. (f). IFN-␥ production of splenocytes was measured by ELISPOT. Results were expressed as relative number of IFN-␥-secreting spot forming cells. B, Representative ELISPOT assays, Downloaded from indicating the effect of NOS inhibition on IFN-␥ production. Spleno- cytes from HDC-KO mice were left untreated or stimulated with 2 ␮g/ml ConA, with or without 100 G ␮MN-monomethyl-L-arginine, or ␮

600 M nitronidasole. http://www.jimmunol.org/ by guest on October 1, 2021

18 ␮M; 64 Ϯ 13 ␮M, respectively, p ϭ 0.39). Furthermore, L- Mitochondrial biogenesis in HDC-KO and WT T cells ␥ histidine treatment did not alter Con-A-induced IFN- production NO has recently been recognized as a key signal of mitochondrial ϭ ( p 0.3), measured by ELISPOT assay. biogenesis (15, 20). Mitochondria can take up, store, and release Ca2ϩ thus altered mitochondrial mass have a role in shaping Ca2ϩ signal in many cell types (15, 20), including T lymphocytes. Be- cause HDC-KO T cells produce higher amounts of NO, mitochondrial mass was measured in both HDC-KO and WT T cells. Our data indicate that there is no signiﬁcant difference between the mitochondrial mass of HDC-KO and WT T cells (Fig. 6; p ϭ 0.1).

FIGURE 7. T cell activation-induced ROI production of HDC-KO and FIGURE 6. Mitochondrial mass of T lymphocytes from HDC-KO and WT T lymphocytes. HDC-KO and WT T cells were stimulated with ConA. WT mice. Mitochondrial mass of HDC-KO and WT mice were monitored Basal, 4 and 24 h ConA stimulation-induced ROI production of spleno- by NAO fluorescent dye. Values were expressed as relative fluorescence. cytes was monitored by 1 ␮M hydroethidine. Values were expressed as ns, Not significant. relative fluorescence. ns, Not significant. 6618 NO MEDIATES T CELL SIGNAL TRANSDUCTION IN HDC-KO MICE

ROI production and CD3 internalization in HDC-KO and tion (33). NO has newly been recognized as a key signal for mi- WT T cells tochondrial biogenesis. This operates through the cGMP-depen- ␭ ␣ Because ROI production is associated with T cell activation (15), dent peroxisome proliferator-activating receptor coactivator-1 , and both Ca2ϩ flux and NO production are different in HDC-KO a master regulator of mitochondrial biogenesis. NO was shown to T cells, we next investigated constitutive and activation-induced induce mitochondrial biogenesis in brown adipocytes, U937 and ROI production. Our data indicate that basal ROI production and HeLa cells, and human lymphocytes (34). According to our 4 h Con-A stimulation-induced ROI production are similar both in present data, increased NO production of HDC-deficient mice is the HDC-KO and the WT T cells (Fig. 7). However, the 24 h not associated with increased mitochondrial biogenesis. Con-A treatment-induced ROI signal was smaller in the HDC-KO Earlier studies (35) showed that mice deficient in iNOS devel- T cells (Figure 7). This is in accordance with our previous data, oped an enhanced Th1 response, following infection with protozoa indicating that NO regulates T cell activation-induced ROI signal parasite Leishmania major, suggesting a role of NO in regulating (15). The T cell activation-induced CD3 internalization was sim- Th1/Th2 phenotypes. NO was reported to inhibit (36, 37) Th1-type ␥ ilar in both HDC-KO and WT T lymphocytes (data no shown). responses (IL-2, IFN- ) but not Th2-type responses (IL-4 and IL- 10). By contrast, other findings suggest that NO preferentially promotes IFN-␥ synthesis (38, 39) and type 1 Th cell differentiation Discussion by selective induction of IL-12R␤2 via cGMP (16). Elevated lev- Histamine is an important mediator of allergic diseases and in- els of cGMP, corresponding to enhanced Th1 cell activation, were flammation (24). In addition, involvement of histamine in the mo- detected in CD4ϩ T cells 30 min after the cells were exposed to lecular machinery of melanoma progression have been reported NO. Naive T cells express IL-12R upon TCR engagement. The recently (25, 26). The development of some allergic reactions, in- expression of IL-12R appears to be transient and is down-regulated Downloaded from fection, and tumors are associated with excessive histamine pro- in committed memory Th1 cells, which can be maintained and duction. Histamine modulates the cytokine production of immu- expanded by other factors including IL-15 (40). NO is an addi- nocompetent cells, including T lymphocytes, by binding to tional factor for enhancing IL-12R expression and, thus, promoting histamine receptors on their cell surface. T cells express both type type 1 cell differentiation (38). Thereafter, the regulatory role of 1 and type 2 histamine receptors, histamine inhibits the production NO on Th1/Th2 polarization appears to be concetration dependent, of Th1 cytokines such as IL-2 and IFN-␥ and enhances the secre- low NO concentrations selectively enhance the induction of Th1 http://www.jimmunol.org/ tion of Th2 cytokines such as IL-4, IL-5, IL-10, and IL-13. His- cells, while higher concetrations may inhibit Th1 response. The tamine shifts the Th1/Th2 balance toward Th2, via regulation of polarization of naive T cells toward Th1 or Th2 phenotypes is JAK-STAT signal transduction pathway (27). Decreased produc- mediated primarily by dendritic cells (DCs). Both mast cells and tion of IFN-␥ following histamine treatment, was reported in ac- iDCs are located in the periphery, often in close proximity to each tivated human blood mononuclear cells (13, 28, 29). Our present other. Both the cytokines that are expressed by DCs and the ca- data confirm and extend the observations of others, regarding the pacity of DCs to polarize naive T cells are regulated by histamine immunoregulatory role of histamine. In the present study, we show (41). Our previous data revealed that histamine has a negative for the first time that T lymphocytes in vivo, in the absence of effect on the Ag presentation and the adaptive immune response by by guest on October 1, 2021 histamine, exhibit Th1-type cytokine dominance, as they produce altering the cytokine profile of DCs (14). Complete and long-last- higher levels of IFN-␥ both at mRNA and protein levels. Our data ing elimination of histamine in the experimental animals in vivo indicate that histamine deficiency is associated with a markedly cannot be achieved by the currently available pharmacological in- increased T cell NO production, and histamine directly regulates hibitors. Due to the overlapping and sometimes antagonistic func- NO production. Thereafter, NO may contribute to the shifted cy- tion of the receptors in the presence of endogenous histamine, tokine profile of HDC-KO T cells. receptor blocking alone cannot achieve complete blockade of the NO is a diffusible, multifunctional, transcellular messenger that histamine system. Therefore, knockout mice with defective hista- has been implicated in numerous physiological and pathological mine synthesis were used in our work to investigate the role of conditions (22). NO is synthetized from L-arginine by NOS. Three histamine in T cell signal transduction. distinct isoforms of NOS are known, including nNOS, iNOS, and Both histamine and NO are well-known regulators of the im- eNOS enzymes. According to our present data, mouse T cells pre- mune responses. Collectively, our present data indicate that hista- dominantly express the nNOS isoform, while lower level of iNOS mine can modulate the T cell-mediated immune response through and eNOS isoforms were detected (15, 30). Although the iNOS regulating NO production. These data contribute to the understand- activity depends on transcription, the eNOS and the nNOS are ing of the increasingly complex network of immune regulation constitutively expressed and are activated upon elevated intracel- essential for health and disease. lular Ca2ϩ (30). Cytoplasmic Ca2ϩ level is higher ( p ϭ 0.002) in HDC-KO T cells which may activate eNOS and nNOS enzymes Acknowledgements and induce NO production. According to our previous data, NO We thank Andras Simon for his technical help. donor treatment increases both cytoplasmic Ca2ϩ concentration and T cell activation induced Ca2ϩ signal (20), which observation Disclosures is in accordance with our present data. Furthermore, the NO pro- The authors have no financial conflict of interest. duction and the T cell activation-induced NO signal are both References higher in HDC-KO T cells. Our data suggest that histamine is an 1. Metcalfe, D. D., D. Baram, and Y. A Mekori. 1997. Mast cells. Physiol. Rev. 77: additional factor that regulates NO production. This is in accor- 1033–1079. dance with our previous observations, confirming an important 2. Hart, P. H. 2001. Regulation of the inflammatory response in asthma by mast cell role of NO in T cell activation. products. Immunol. Cell. Biol. 79: 149–153. 3. Laszlo, V., and A. Falus. 2000. Yet unrevealed aspects of histamine: questions NO promotes mitochondrial hyperpolarization, ATP depletion, from outside: answers at inside? Semin. Cancer Biol. 10: 1–2. and relative resistance to apoptotic stimuli in astrocytes, lympho- 4. Jutel, M., T. Watanabe, S. Klunker, M. Akdis, O. A. Thomet, J. Malolepszy, T. Zak-Nejmark, R. Koga, T. Kobayashi, K. Blaser, and C. A. Akdis. 2001. cytes, and Jurkat cells (23, 31, 32). Low concentrations of NO may Histamine regulates T-cell and antibody responses by differential expression of specifically inhibit cytochrome-c oxidase, leading to ATP deple- H1 and H2 receptors. Nature 413: 420–425. The Journal of Immunology 6619

5. Zhang, M., J. D. Venable, and R. L. Thurmond. 2006. The histamine H4 receptor ciency in gene-targeted mice strongly reduces antigen-induced airway hyper- in autoimmune disease. Expert Opin. Investig. Drugs 15: 1443–1452. responsiveness, eosinophilia and allergen-specific IgE. Int. Immunol. 15: 6. Lanzaveccia, A., G. Lezzi, and A. Viola. 1999. From TcR engagement to T cell 963–973. activation: a kinetic view of T cell behavior. Cell 96: 1–4. 25. Falus, A., H. Hegyesi, E. Lazar-Molnar, Z. Pos, V. Laszlo, and Z. Darvas. 2001. 7. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. Paracrine and autocrine interactions in melanoma: histamine is a relevant player 1986. Two types of murine helper T cell clone, I: definition according to profiles in local regulation. Trends Immunol. 22: 648–652. of lymphokine activities and secreted proteins. J. Immunol. 136: 2348–2357. 26. Pos, Z., G. Safrany, K. Muller, S. Toth, A. Falus, and H. Hegyesi. 2005. Phe- 8. Skapenko, A., J. Leipe, P. E. Lipsky, and H. Schulze-Koops. 2005. The role of notypic profiling of engineered mouse melanomas with manipulated histamine the T cell in autoimmune inflammation. Arthritis Res. Ther. 7 (Suppl. 2) :S4–S14. production identifies histamine H2 receptor and rho-C as histamine-regulated 9. Kay, A. B. 2001. Allergy and allergic diseases: first of two parts. N. Engl. J. Med. melanoma progression markers. Cancer Res. 65: 4458–4466. 344: 30–37. 27. Osna, N., K. Elliott, O. Chaika, E. Patterson, R. E. Lewis, and M. M. Khan. 2001. 10. Meiler, F., M. Zimmermann, K. Blaser, C. A. Akdis, and M. Akdis. 2006. T-cell Histamine utilizes JAK-STAT pathway in regulating cytokine production. Int. subsets in the pathogenesis of human asthma. Curr. Allergy Asthma Rep. 6: Immunopharmacol. 1: 759–762. 91–96. 28. Carlsson, R., M. Dohlsten, and H. O. Sjogren. 1985. Histamine modulates the 11. Laurence, A., C. M. Tato, T. S. Davidson, Y. Kanno, Z. Chen, Z. Yao, production of interferon-␥ and interleukin-2 by mitogen-activated human mono- R. B. Blank, F. Meylan, R. Siegel, L. Hennighausen, et al. 2007 Interleukin-2 nuclear blood cells. Cell. Immunol. 96: 104–112. signaling via STAT5 constrains T helper 17 cell generation. Immunity 26: 29. Dohlsten, M., H. O. Sjogren, and R. Carlsson. 1986. Histamine inhibits interfer- 371–381. on-␥ production via suppression of interleukin 2 synthesis. Cell. Immunol. 101: 12. Bettelli, E., M. Oukka, and V. K. Kuchroo. 2007. Th-17 cells in the circle of 493–501. immunity and autoimmunity: Th-17 cells in the circle of immunity and autoim- 30. Reiling, N., R. Kroncke, A. J. Ulmer, J. Gerdes, H. D. Flad, and S. Hauschildt. munity. Nat. Immunol. 8: 345–350. 1996. Nitric oxide synthase: expression of the endothelial Ca2ϩ/calmodulin-de- 13. Horvath, B., C. S. Szalai, Y. Mandi, V. Laszlo, Z. S. Radvanyi, Z. S. Darvas, and pendent isoform in human B and T lymphocytes. Eur. J. Immunol. 3: 511–516. A. Falus. 1999. Histamine and histamine-receptor antagonists modify gene ex- 31. Almeida, A., J. Almeida, J. P. Bolanos, and S. Moncada. 2001. Different re- pression and biosynthesis of interferon a˜in priferial human blood mononuclear sponses of astrocytes and neurons to nitric oxide: the role of glycolitycally gen- cells and in CD19-depleted cell subsets. Immunol. Lett. 70: 95–99. erated ATP in astrocyte protection. Proc. Natl. Acad. Sci. USA 26: 15294–15299. 14. Jelinek, I., V. Laszlo, E. Buzas, E. Pallinger, B. Hangya, Z. Horvath, and 32. Beltran, B., M. Quintero, E. Gracia-Zaragoza, E. O’Connor, J. V. Esplugues, and A. Falus. 2007. Increased antigen presentation and Th1 polarization in genetically S. Moncada. 2002. Inhibition of mitochondrial respiration by endogenous nitric Downloaded from histamine-free mice. Int. Immunol. 19: 51–58. oxide: a critical step in Fas signalling. Proc. Natl. Acad. Sci. USA 13: 15. Nagy, G., A. Koncz, and A. Perl. 2003. T cell activation-induced mitochondrial 8892–88997. hyperpolarization is mediated by Ca2ϩ- and redox-dependent production of nitric 33. Brown, C. G. 1999. Nitric oxide and mitochondrial respiration. Biochem. Bio- oxide. J. Immunol. 171: 5188–5197. phys. Acta 1411: 351–369. 16. Niedbala, W., X. Q. Wei, C. Campbell, D. Thomson, M. Komai-Koma, and 34. Nisoli, E., E. Clementi, and C. Paolucci. 2003. Mitochondrial biogenesis in mam- F. Y. Liew. 2002. Nitric oxide preferentially induces type 1 T cell differentiation mals: the role of endogenous nitric oxide. Science 299: 896–899. by selectively up-regulating IL-12 receptor ␤ 2 expression via cGMP. Proc. Natl. 35. Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu,

Acad. Sci. USA 99: 16186–16191. W. Muller, S. Moncada, and F. Y. Liew. 1995. Altered immune responses in mice http://www.jimmunol.org/ 17. Niedbala, W., B. Cai, and F. Y. Liew. 2006. Role of nitric oxide in the regulation lacking inducible nitric oxide synthase. Nature 375: 408–411. of T cell functions. Ann. Rheum. Dis. 65 (Suppl. 3): iii37–iii40. 36. Eun, J. S., Y. H. Suh, D. K. Kim, and H. Jeon. 2000. Regulation of cytokine 18. Packard, K. A., and M. M. Khan. 2003. Effects of histamine on Th1/Th2 cytokine production by exogenous nitric oxide in murine splenocyte and peritoneal mac- balance. Int. Immunopharmacol. 3: 909–920. rophage. Arch. Pharm. Res. 23: 531–534. 19. Ohtsu, H., S. Tanaka, T. Terui, Y. Hori, Y. Makabe-Kobayashi, G. Pejler, 37. Daniel, T., M. Alexander, W. J. Hubbard, I. H. Chaudry, M. A. Choudhry, and E. Tchougounova, L. Hellman, M. Gertsenstein, N. Hirasawa, et al. 2001. Mice M. G. Schwacha. 2006. Nitric oxide contributes to the development of a post- lacking histidine decarboxylase exhibit abnormal mast cells. FEBS Lett. 502: injury Th2 T-cell phenotype and immune dysfunction. J. Cell. Physiol. 208: 53–56. 418–427. 20. Nagy, G., M. Barcza, N. Gonchoroff, P.E. Phillips, and A. Perl. 2004. Nitric 38. Niedbala, W., X. Q. Wei, D. Piedraﬁta, D. Xu, and F. Y. Liew. 1999. Effects of oxide-dependent mitochondrial biogenesis generates Ca2ϩ signaling proﬁle of nitric oxide on the induction and differentiation of Th1 cells. Eur. J. Immunol. 29: lupus T cells. J. Immunol. 173: 3676–3683. 2498–2505.

21. Gree, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and 39. Ibiza, S., V. M. Vıćtor, I. Bosca´, A. Ortega, A. Urzainqui, J. E. O’Connor, by guest on October 1, 2021 S. R. Tannenbaum. 1982. Analysis of nitrat, nitrite and 15N nitrate in biological F. Sańchez-Madrid, J. V. Esplugues, and J. M. Serrador. 2006. Endothelial nitric fluids. Anal. Biochem. 126: 131–138. oxide synthase regulates T cell receptor signaling at the immunological synapse. 22. Bredt, D. S. 1999. Endogenous nitrice oxide synthesis: biological functions and Immunity 24: 753–765. pathophysiology. Free Radical Res. 31: 577–596. 40. Geginat, J., F. Sallusto, and A. Lanzavecchia. 2001. Cytokine-driven proliferation 23. Beltran, B., A. Mathur, M. R. Duchen, J. D. Erusalimsky, and S. Moncada. 2000. and differentiation of human naive, central memory, and effector memory CD4ϩ The effect of nitric oxide on cell respiration: a key to understanding its role in cell T cells. J. Exp. Med. 194: 1711–1719. survival, or death. Proc. Natl. Acad. Sci. USA 26: 14602–14607. 41. Mazzoni, A., H. A. Young, J. H. Spitzer, A. Visintin, and D. M. Segal. 2001. 24. Kozma, G. T., G. Losonczy, M. Keszei, Z. Komlosi, E. Buzas, E. Pallinger, Histamine regulates cytokine production in maturing dendritic cells, resulting in J. Appel, T. Szabo, P. Magyar, A. Falus, and C. Szalai. 2003. Histamine defi- altered T cell polarization. J. Clin. Invest. 108: 1865–1873.