Involvement of dihydropyridine-sensitive calcium channels in human dendritic cell function. Competition by HIV-1 Tat.

The entry of extracellular calcium in leukocytes mediates several cellular processes; however, unlike in excitable tissues, the underlying molecular mechanisms are poorly defined. In this paper we provide phenotypical and biochemical evidence that peripheral blood-derived human dendritic cells express dihydropyridine-sensitive calcium channels. Exposure to the dihydropyridine drug nifedipine, which binds L-type calcium channels blocking calcium influx, prevents two dendritic cell functions that are dependent on extracellular calcium entry: apoptotic body engulfment and interleukin-12 production induced by cross-linking of the surface lectin NKRP1A. It is known that exogenous human immunodeficiency virus, type 1 Tat affects several Ca2+-dependent immune cell responses. Here we demonstrate that Tat inhibits apoptotic body engulfment and interleukin-12 production by blocking extracellular calcium influx. This inhibition is prevented by the calcium channel agonist dihydropyridine derivative Bay K 8644, suggesting the involvement of L-type calcium channels. This hypothesis is further supported by the observation that Tat and dihydropyridine drugs compete for binding to dendritic cells. Taken together, these findings indicate that exogenous Tat exerts its inhibitory effects on dendritic cells by blocking dihydropyridine-sensitive L-type calcium channels.

Calcium-linked cellular functions in excitable tissues are mediated by voltage-dependent calcium channels, which participate in the regulation of action potential generation, muscle contraction, and secretion of hormones or neurotransmitters (1). Although in neurons multiple types of calcium channels, which can be distinguished by their pharmacological properties, are expressed, in skeletal and cardiac muscles the principal calcium channels are L-type (1,2). These channels are composed of three transmembrane subunits (␣1C, ␥, and the ␣2␦ complex) and one cytoplasmic chain (the ␤1 chain). A spectrum of compounds, the dihydropyridine (DHP) 1 deriva-tives, which specifically bind with high affinity to the ␣1C chain of L-type channels (3,4), regulating their functional state from blocking to opening, allows both the identification and the functional analysis of this class of molecules (1)(2)(3)(4).
Cytosolic calcium rise is an important signal also in nonexcitable cells, including immune cells, regulating fundamental processes such as activation, growth, and differentiation (5)(6)(7). Increase in free intracellular calcium concentration ([Ca 2ϩ ] i ) may result from calcium mobilization from either intracellular stores or extracellular medium or both (5). Unlike the mechanisms mediating mobilization from intracellular stores, the molecular structures mediating extracellular calcium influxes are still poorly characterized. Recently, the presence of functional calcium channels displaying DHP sensitivity has been observed in B lymphocytes (8), raising the possibility that similar structures are present also in nonexcitable cells. We have previously shown that some functions of dendritic cells (DC) are mediated by [Ca 2ϩ ] i increase. DC are professional antigen presenting cells able to endocytose and process soluble or particulated antigens (9,10) and prime naive T lymphocytes (9). Activated DC produce IL-12, a cytokine that amplifies the immune response promoting the differentiation of the T helper 1 lymphocyte subset, which in turn substains the natural killer (NK) cell activity (11,12). We reported that activation of DC by cross-linking of the surface lectin NKRP1A with consequent IL-12 production is accompanied by extracellular calcium influx (13); similarly, apoptotic body engulfment induces and is dependent on calcium entry in phagocytosing DC (10). Interestingly, the HIV-1 transactivating factor Tat, which can be released by infected cells and play a number of extracellular roles (14), affects several calcium-mediated events in immune cells (15)(16)(17)(18), including the phagocytosis of apoptotic cells by DC (19). We thus investigated the presence of calcium channels on DC and the possible interference by exogenous Tat. Our data indicate that functional DHP-sensitive L-type calcium channels are expressed by DC and regulate both apoptotic body engulfment and NKRP1A-mediated IL-12 production. Interestingly, L-type calcium channels appear to be the molecular target of HIV-1 Tat on DC; indeed, binding of DHP derivatives to these channels is cross-inhibited by Tat. Moreover, the inhibitory effect of HIV-1 Tat on DC function is antagonized by the DHP agonist Bay K 8644 (2).

EXPERIMENTAL PROCEDURES
Reagents-The acetoxymethyl ester of FURA 2 (FURA 2-AM) and all the reagents for electrophoresis were from Sigma. Prestained molecular weight markers were from Bio-Rad. * This work was supported in part by grants from Associazione Italiana Ricerca sul Cancro, Ministero Sanità (Special Project AIDS 1996), and National Council for Research (Special Project Biotech). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Ionomycin, nifedipine (NFP), and (Ϯ) Bay K 8644 (the net functional effect of the racemic mixture is that of the negative enantiomer, which is a L-type Ca 2ϩ channel agonist) were from Calbiochem-Inalco S.p.A (Milan, Italy). Fluorescein DM-BODIPY® DHP was from Molecular Probes Europe (Leiden, the Netherlands). Chemically synthesized and biotinylated HIV-1 Tat protein were provided by Tecnogen (Piana di Monteverna, Cesena, Italy). Synthetic Tat preparations were purified by reverse phase high pressure liquid chromatography yielding a purity of 96%. The biological activities of synthetic Tat were superimposable to those of natural Tat in different assays (20,21). Recombinant fibronectin type III repeat (Fn-III, from amino acids 1086 -1172) was a kind gift of L. Zardi (National Institute for Cancer Research, Genoa, Italy). RPMI 1640, fetal calf serum, L-glutamine, and penicillin-streptomycin were from Biochrom (Berlin, Germany). Recombinant granulocyte-macrophage colony-stimulating factor was from Schering-Plow (Milan, Italy).
Engulfment of Apoptotic Bodies by Dendritic Cells-Peripheral blood monocytes were isolated from healthy donors as described (10,22) and cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin, 10% heat-inactivated fetal calf serum, and 20 ng/ml granulocyte-macrophage colony-stimulating factor for 10 days to obtain monocyte-derived DC. Media were shown to be endotoxin-free using the Limulus lysate colorimetric assay (Procedures and Biological Information International, Milan, Italy). The human Jurkat T cell line (clone JA3) was purchased from the American Type Culture Collection (ATCC, Rockville, MD).
Apoptosis was induced by sublethal irradiation of JA3 cells (4000 radiation absorbed doses) and culture at 37°C for 20 h. Engulfment assay was performed as described previously (10,19,23). Briefly, apoptotic bodies were labeled with 51 Cr (sodium chromate, NEN-DuPont Italiana S.p.A., Cologno Monzese, MI, Italy, 50 Ci/10 6 cells) for 1 h at 37°C, washed, and coincubated with adherent DC (2:1 ratio) at 37°C (4°C for the negative control). After 45 min, noningested apoptotic bodies were removed by four gentle washings and DC-associated radioactivity measured in a ␥-counter (Beckman Instruments Inc., Irvine, CA) after cell lysis. Results are expressed as a percentage of engulfment calculated as described (10,19,23). In some experiments DC were pretreated for 20 min with different concentrations of Tat (from 100 to 1 nM) or with Tat 100 nM plus Bay K 8644 10 M; in other experiments, Bay K 8644 10 M was added immediately prior apoptotic body challenge to untreated or Tat-pretreated cells. Drug concentrations were chosen on the basis of titration experiments (not shown).
Single Cell Analysis of Calcium Fluxes by Video Microscopy and Ratio Imaging-Single cell analysis of calcium fluxes was performed as described (10,19). Briefly, DC cultured on round coverslips were loaded with 1 M FURA 2-AM (1 h at 37°C), placed in a micro-incubator (Medical System Corp., Greenvale, NY) on an inverted epi-fluorescence Axiovert 10 microscope (Zeiss, Oberkochen, Germany), and maintained at 37°C by a temperature controller (TC-202, Medical System). FURA 2 was excited with a high pressure 75 W xenon arc lamp fitted with appropriate filters on a shutter controlled by a Pentium 90 MHz computer. Excitation light was at 334 and 380 nm; emitted light was filtered at 510 nm. Two 334/380 ratio were taken each second, and video images were collected with an intensified charged coupled device (CCD) camera (ATTO Instruments, INC., Rockville, MD) and recorded every 15 s using the image processor program Attofluor RatioVision 6.08 (ATTO Instruments). Results were stored as a ratio of FURA 2 fluorescence at 334 nm divided by the fluorescence at 380 nm excitation.
[Ca 2ϩ ] i was calculated according to Grynkiewicz et al. (24). [Ca 2ϩ ] i increases were measured upon apoptotic body interaction with DC or after DC treatment with either Bay K 8644 (10 M) or ionomycin (1 M). Alternatively, [Ca 2ϩ ] i was measured after cross-linking of the NKRP1A molecule, obtained by incubation of DC with 5 g/ml of the specific F(abЈ) 2 mAb (20 min at 4°C) followed by 10 g/ml of F(abЈ) 2 GAM added during the test at 37°C as described (13). Cross-linking of CD31 or exposure of DC to GAM F(abЈ) 2 alone did not elicit [Ca 2ϩ ] i increases (13). In some experiments, DC were pretreated with NFP (1 or 10 M) or HIV-1 Tat (from 100 to 10 nM) for 20 min, before challenge with apoptotic bodies, Bay K 8644, ionomycin, or NKRP1A cross-linking.
Calcium Channel Detection by Fluorescence-DC (10 6 /sample), untreated or preincubated 20 min with HIV-1 Tat or with Bay K 8644 or with Fn-III as a control (from 0.1 to 1 M), were stained with 3 nM DM-BODIPY R DHP (4) and run on a FACSort (Becton Dickinson, Mountain View, CA). The concentrations of calcium channel antagonists reported to cross-inhibit the binding of 3 nM DM-BODIPY R DHP or other DHP derivatives range 10 -500-fold (4). Alternatively, DC untreated or pretreated for 20 min with HIV-1 Tat or with BayK 8644 or with Fn-III (from 1 to 10 M) were stained with biotinylated HIV-1 Tat Cytokine Production-DC (2 ϫ 10 6 /sample), cultured in 1% Nutridoma (Boehringer Mannheim Italia, Monza, MI, Italy) untreated or treated for 20 min with NFP 10 M or with different concentrations of Tat (from 100 to 1 nM) or with Tat 100 nM plus Bay K 8644 10 M or with Bay K 8644 10 M alone were challenged by cross-linking of the NKRP1A molecule with the F(abЈ) 2 of a specific monoclonal antibody followed by GAM F(abЈ) 2 , as described (13). Cross-linking of CD31 or exposure of DC to GAM F(abЈ) 2 alone did not stimulate IL-12 production (13). Supernatants were collected after 3 h, and secreted IL-12 was measured using the enzyme-linked immunosorbent assay kit for human IL-12 (p40 and p70) purchased from Endogen (Woburn, MA).

The DHP Drug NFP Prevents Calcium Mobilization and Engulfment of Apoptotic Bodies by Dendritic Cells-
We have previously shown that apoptotic body engulfment by DC elicits [Ca 2ϩ ] i rises, mainly due to the entry of extracellular calcium, that are essential for phagocytosis (10,19). The possibility of an involvement of calcium channels in this process has been in- [Ca 2ϩ ] i was monitored with an inverted epi-fluorescence microscope connected to an intensified CCD camera. Results are expressed as [Ca 2ϩ ] i nM. Calcium fluxes were measured during interaction between DC and apoptotic bodies as such or after pretreatment with NFP (10 or 1 M). One representative experiment out of six is shown. B, apoptotic bodies labeled with 51 Cr were co-incubated with adherent DC (2:1 ratio) at 37°C in the absence or presence of NFP (10 or 1 M). Noningested AB were removed by washing and DC-associated radioactivity measured in a ␥-counter after cell lysis. Results are expressed as percentages of engulfment calculated as described (10,19). C, after separation by SDS-polyacrylamide gel electrophoresis (12% gel) under reducing conditions, lysates from DC (50 g) or A431 cells (positive control) were electrotransferred onto nitrocellulose filters, hybridized with anti-calcium channel ␤1 subunit mAb followed by horseradish peroxidase-GAM Ig, and developed by chemiluminescence. vestigated by using the inhibitory DHP derivative NFP, which specifically binds to the ␣1C subunit of L-type calcium channels (1)(2)(3)(4). As shown in Fig. 1A, NFP inhibits in a dose-dependent manner the calcium mobilization that follows the interaction between apoptotic bodies and DC. Likewise, engulfment is prevented by DC exposure to this drug (Fig. 1B). These results suggest that DC express functional L-type calcium channels. This was confirmed by the finding of a specific 58-kDa band detectable by Western blot analysis of DC lysates with a monoclonal antibody recognizing the ␤1 calcium channel subunit (Fig. 1C). Unlike in excitable tissues, these channels are voltage-independent, because exposure of DC to 50 mM KCl failed to induce a calcium influx (not shown).
HIV-1 Tat Prevents the Opening of DHP-sensitive Calcium Channels and Competes with DHP Derivatives for Binding to DC-Exogenous HIV-1 Tat is able to inhibit both apoptotic body engulfment by DC and the [Ca 2ϩ ] i rise induced by apoptotic body-DC interaction (19). We thus investigated whether the inhibitory effect of Tat is due to the block of L-type calcium channels on DC. DC were loaded with apoptotic bodies, and the engulfment was measured under different conditions. Fig. 2A shows that the inhibition of engulfment observed in the presence of HIV-1 Tat is prevented by DC pretreatment with Bay K 8644, a calcium channel agonist that induces Ca 2ϩ entry by opening L-type channels (2,8). In turn, once DC have been pretreated with Tat, Bay K 8644 is unable to revert the inhibition ( Fig. 2A). The calcium influx elicited in DC by exposure to Bay K 8644 is blocked by pretreatment of DC with HIV-1 Tat in a dose-dependent manner (Fig. 2B); in contrast, Tat does not affect the calcium channel-independent calcium rise that follows DC exposure to the ionophore ionomycin (Fig. 2C). These data suggest that HIV-1 Tat competes with Bay K 8644 for binding to DHP-sensitive calcium channels. To further confirm this hypothesis, we performed fluorescence-activated cell sorter analysis of DC stained with DM-BODIPY R DHP, a fluorescent DHP derivative that binds to the ␣1C chain of L-type calcium channels (4), in the presence or absence of Tat or Bay K 8644. As shown in Fig. 3A, DM-BODIPY R DHP stains DC, indicating the presence of L-type channel ␣1C subunit on these cells. Tat antagonizes the DM-BODIPY R DHP binding with the same dose response as the ␣1C ligand Bay K 8644. In turn, the binding of biotinylated Tat to DC is cross-inhibited by pretreatment of the cells with Bay K 8644 (Fig. 3B). In both cases, the unrelated peptide Fn-III, displaying the same length as HIV-1 Tat, has no effects (Fig. 3, A and B).
Both HIV-1 Tat and the DHP Drug NFP Inhibit IL-12 Secretion by DC-An important function of DC is to support T helper cell differentiation by IL-12 production (11,12). Secretion of IL-12 is induced by various stimuli (11,12); one of them is cross-linking of the surface lectin NKRP1A (13). Because this triggering also results in Ca 2ϩ entry (13), we investigated whether L-type calcium channels play a role in IL-12 release. NKRP1A molecules were cross-linked by the specific monoclonal antibody, and the secretion of IL-12 by DC in the presence or absence of NFP or HIV-1 Tat was measured. Fig. 4A shows that the engagement of NKRP1A results in release of IL-12, which is prevented by exposure of DC to NFP. HIV-1 Tat proves to exert the same inhibition of NFP on NKRP1A-induced IL-12 production (Fig. 4A). This inhibition was dose-dependent, being detectable up to 10 nM Tat (Fig. 4A). Again, the calcium channel agonist Bay K 8644 is able to revert the inhibitory effect of HIV-1 Tat (Fig. 4A). In keeping with these results, the calcium rise elicited by NKRP1A cross-linking in DC is inhibited by either the DHP drug NFP (Fig. 4B) or HIV-1 Tat (Fig. 4C), further supporting the hypothesis that both compounds exert their effects by acting on L-type calcium channels.

DISCUSSION
In the present paper we show that molecular structures displaying pharmacological properties of L-type calcium channels (1, 2) mediate extracellular calcium influx in human DC. The presence of functional L-type Ca 2ϩ channels in DC is supported by three lines of evidence: (i) Ca 2ϩ -dependent DC functions, such as apoptotic body engulfment and IL-12 production, are inhibited by L-type Ca 2ϩ channel blockers; (ii) fluorescent DHP drugs, specific for the ␣1C chain of L-type channels (3,4), bind to DC surface; and (iii) the calcium channel ␤1 chain is detectable in DC lysates. In contrast, N-type calcium channels (1,25) are not expressed, because fluorescent -conotoxin failed to stain DC (not shown). Although L-type calcium channels in excitable tissues are voltage-gated (1), DHP-sensitive Ca 2ϩ channels in DC are voltage-independent, suggesting that they lack a membrane voltage sensor. This finding supports the recently reported data on the existence of voltage-independent In some experiments, Bay K 8644 10 M was added immediately prior apoptotic body challenge to untreated (light gray column) or Tat-pretreated cells (dark gray column). Engulfment was performed as described for Fig. 1. Results are expressed as percentages of engulfment calculated as described (10,19). DHP-sensitive channels on B lymphocytes (8), raising the possibility of a common mechanism responsible for Ca 2ϩ entry in immune cells.
An important novel finding of this paper is that HIV-1 Tat blocks two calcium-mediated DC functions by acting on DHPsensitive Ca 2ϩ channels. Indeed, Tat-mediated inhibition of both apoptotic body engulfment and NKRP1A-induced IL-12 production is reverted by the L-type Ca 2ϩ -channel agonist Bay K 8644. In turn, Tat blocks the Ca 2ϩ influx that follows triggering of DC with Bay K 8644. The finding that DHP derivatives and Tat compete for binding to DC strongly supports the hypothesis that L-type calcium channels are the molecular targets of the inhibitory effects of Tat on DC. Several Tat binding molecules have been described on different cell types; Tat binds with high affinity vascular endothelial growth factor receptor (21) and CD26 (26), with low affinity heparan sulfates (27), and with integrins like ␣v␤3 (28) or ␣5␤1 (29). DHP drugs are able to strongly decrease the binding of biotinylated Tat to DC; thus, one major binding site for Tat on these cells seems to be represented by DHP-sensitive channels, although we cannot rule out binding to other receptors, which in turn can be associated to Ca 2ϩ channels.
The role of HIV-1 Tat in AIDS pathogenesis extends beyond its transcriptional activity; indeed, it is secreted by infected cells (14) and affects gene expression and function in many types of infected or noninfected cells (17, 18, 30 -32). Exogenous Tat proved to down-regulate immune cell functions; it inhibits antigen-induced T cell proliferation (15), blocks the phagocytosis of apoptotic cells (19), and modulates cytokine production (16,31,32). Here we show that exogenous Tat inhibits IL-12 production elicited by NKRP1A engagement in DC. IL-12, originally described as NK cell stimulatory factor, is also involved in the differentiation of T helper cell subset 1 cells (11,12). A deficient IL-12 production in AIDS patients may thus play a role in the progression of the disease (33,34), which is associated with decreased NK cell function, loss of T helper cell subset 1 cells, and a corresponding increase in T helper cell subset 2 cells (33,35). In light of these considerations, our data provide evidence for a molecular mechanism possibly underlying T helper cell subsets 1 and 2 embalance during HIV-1 infection, based on the Tat-mediated impairment of IL-12 production. The physiological relevance of our findings relies on the observation that nanomolar concentrations of HIV-1 Tat are detectable in the sera of AIDS patients (36). It is conceivable that the local amount of Tat in the mucosal and lymphoid tissues site of infection is higher due to the concentrating effect of extracellular matrix components, such as heparan sulfates, which bind to Tat (27). This may explain the finding that in vitro, in the absence of matrix components, Tat is usually active at concentrations higher than those found in AIDS patient sera (15,17,29,31,32). Finally, the finding that Tat can act through blocking calcium channels may provide a universal key for understanding the molecular basis of exogenous Tat-mediated immunosuppressive effects during HIV-1 infection.