Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;71:531-537.)
© 2002 by Society for Leukocyte Biology

ß3-Mediated engulfment of apoptotic tumor cells by dendritic cells is dependent on CAMKII: inhibition by HIV-1 Tat

Alessandro Poggi*, Roberta Carosio*, Anna Rubartelli{ddagger} and Maria Raffaella Zocchi{dagger},{ddagger}

* Laboratory of Immunology and
{ddagger} Unit of Protein Biology, National Cancer Research Institute, Genoa, Italy; and
{dagger} Laboratory of Tumor Immunology, Scientific Institute San Raffaele, Milan, Italy

Correspondence: Maria Raffaella Zocchi, Laboratory of Tumor Immunology, Scientific Institute San Raffaele, Via Olgettina 60 Milan, Italy. E-mail: zocchi.maria{at}hsr.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this paper, we show that the engulfment of apoptotic tumor cells by DC requires the activation of the calcium-calmodulin kinase II (CAMKII). Indeed, DC phagocytosis of apoptotic lymphoma cells is consistently inhibited by KN62 and KN93, two blockers of CAMKII, but not by the inactive compound KN92. Wortmannin and LY294002, two inhibitors of the phosphatidyl-inositol-3 kinase, slightly decrease the phagocytosis of apoptotic cells, at variance with PD98059, an inhibitor of the mitogen-activated protein kinase. It is interesting that the addition of synthetic HIV-1 Tat, which we demonstrated to inhibit phagocytosis and calcium influx in DC, blocks the activation of CAMKII elicited via ß3 integrin, which is involved in apoptotic body engulfment by DC. Experiments performed with Tat-derived peptides showed that this inhibition is mediated by the C-terminal domain of Tat. Finally, pertussis toxin can prevent HIV-1 Tat-mediated inhibition, suggesting the involvement of a guanosine triphosphate-binding (G) protein in DC-mediated phagocytosis.

Key Words: AIDS • antigen-presenting cells • calcium • G proteins • phagocytosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are the professional antigen-presenting cells (APC) able to endocytose soluble antigens and corpuscolated particles, including apoptotic tumor cells, and to initiate the immune response [1 2 3 4 ]. Integrin receptors, mainly {alpha}vß3 and {alpha}vß5, are crucial for the phagocytic process, mediating internalization of apoptotic bodies and triggering of signal transduction pathways, which precede antigen processing [2 3 4 ]. Previously, we have shown that the phagocytosis of apoptotic tumor cells, as other DC functions [5 , 6 ], is dependent on extracellular calcium entry; in turn, apoptotic body engulfment mediated by {alpha}vß3 integrin elicits a calcium influx in DC [2 ]. Phagocytosis and calcium entry are inhibited by HIV-1 Tat protein [7 ], which is known to contribute to immunosuppression in AIDS [8 ], and this inhibition is apparently related to the inactivation of L-type calcium channels [9 ]. With a similar mechanism, Tat impairs the secretion by DC of interleukin-12 (IL-12), a cytokine that amplifies the immune response [9 , 10 ], thus interfering with multiple functions of the APC.

In the present work, we define a biochemical mechanism that follows extracellular calcium entry in DC, leading to the phagocytosis of apoptotic tumor cells. We show that apoptotic body engulfment is mainly dependent on the activation of calcium-calmodulin kinase II (CAMKII); the phosphoinositol-3-kinase (PI-3K) is partially involved; synthetic Tat blocks the activation of CAMKII elicited in DC by ß3 integrin; and pertussis toxin can prevent Tat-mediated inhibition, suggesting the involvement of an inhibitory guanosine monophosphate-binding (Gi) protein in DC-mediated phagocytosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and culture of DC
Peripheral blood mononuclear cells were isolated by density-gradient centrifugation and monocytes obtained after 1 h of adhesion to plastic. Adherent cells were then cultured in RPMI 1640 with 2mM L-glutamine, 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin (Biochrom, Berlin, Germany), and 40 ng/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF; Shering-Plough, Milan, Italy). Recombinant (r)IL-4 (PreproTech, London; 1000 U/ml) was used only during the first 4 days of culture and then removed to allow DC to adhere firmly to the plastic substrate (needed for the engulfment assay). The phenotype of DC obtained under these culture conditions is demonstrated in Table 1 (see also ref. [2 ]). Media were endotoxin-free, as assessed by the Limulus lysate colorimetric assay (PBI, Milan, Italy). DC were used after 8–12 days of culture, as described [2 ].


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  		Table 1. Monocyte and Monocyte-Derived DC Phenotype by Flow Cytometry

 
DC phenotype by flow cytometry
Surface phenotype of monocytes (freshly isolated adherent cells) and monocyte-derived DC were analyzed at days 1, 4, and 8 of culture, by indirect immunofluorescence and flow cytometry using the following monoclonal antibodies (mAb): anti-human leukocyte antigen (HLA)-DR [D1.12, immunoglobulin (Ig)G2a; a kind gift from R. Accolla, IST-CBA, Genoa], anti-CD14 (IgG1), anti-CD80 (B7.1, IgG1), and anti-CD86 (B7.2, IgG1), purchased from Becton Dickinson (San Jose, CA); anti-CD61 (ß3 integrin, IgG1) and anti {alpha}v (CD51, IgG1) from Serotec (Kidlingtone, Oxford, UK); anti-CD83 (IgG2a) from Immunotech (Luminy Marseille, France); and anti-ß5 integrin rabbit antiserum obtained from Chemicon International (Hofheim, Germany). On day 4, cells were checked for cytoplasmic expression of CD83 (mostly surface-negative). Permeabilization was performed with 0.01% Triton-X 100 after fixation with2% paraforlmaldehyde. Cells (105 cells/sample) were stained with the various antibodies for 30 min at 4°C, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (GAM) or goat anti-rabbit (GAR) Ig. Samples were run on a FACSort (Becton Dickinson), gated to exclude nonviable cells. At least 10,000 cells per sample were analyzed, and results were plotted as percentage of positive cells and mean fluorescence intensity [arbitrary units (a.u.)].

Engulfment of apoptotic bodies by DC
Apoptosis was induced by sublethal irradiation (4000 rad) of the human Jurkat lymphoma cell line (clone JA3), purchased from the American Type Culture Collection (Manassas, VA). To check apoptosis, JA3 cells were stained with 50 µg/ml propidium iodide [2 ]. Engulfment assay was performed as described [2 ]: Briefly, apoptotic bodies were labeled with 51Cr-Na (sodium chromate; NEN-DuPont, Cologno Monzese, Italy) for 1 h at 37°C, washed, and cocultured with adherent DC at the ratio of 3:1 at 37°C for 60 min. Noningested apoptotic cells were removed by extensive washing, and DC-associated radioactivity was measured in a {gamma}-counter (Beckman Instruments, Irvine, CA) after cell lysis. Phagocytosis of opsonized yeasts (not mediated by {alpha}vß3 integrin) or phagocytosis of apoptotic cells at 4°C or in the presence of microtubule poisons was also assayed (ref. [2 ], and not shown in this paper). In particular, DC could not phagocytose efficiently opsonized yeasts at variance with activated monocytes [2 ]. Results are expressed as percentage of engulfment calculated as described previously [2 ]. In some experiments, DC were pretreated for 15 min with 100 nM HIV-1 Tat or Tat peptides (see below; Technogen, Piana di Monteverna, Italy). In other experiments, cells were exposed to the vitronectin peptide (Vn-pep; 100 nM) DYMEQCKPQVTRGDF (PRIMM, Milan, Italy), which binds to {alpha}vß3 integrin, or the RGD-containing fibronectin peptide (Fn-pep; 100 nM; Sigma Chemical Co., St. Louis, MO), which binds to {alpha}3ß1 or {alpha}5ß1 integrins or other fibronectin receptors [7 ]. Some samples were pretreated with 10-6–10-9 M cholera toxin (CTX) or pertussis toxin (PTX), 10-8 M adenylate-cyclase activator forskolin (FSK), and/or 10-8 M phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX; Sigma Chemical Co.) [11 ]. In other experiments, DC were exposed to 10 µ–0.1 µM CAMKII inhibitors KN62 or KN93 or the inactive KN92 compund, the PI-3K inhibitors wortmannin (100 nM–1 nM) or LY294002 (10 µM–0.1 µM), the mitogen-activated protein kinase (MAPK) blocker PD98059 (10 µM–0.1 µM), or 30–3 ng/ml rapamycin (Calbiochem-MERCK, KgaA, Darmstadt, Germany).

CAMKII and Akt activation
CAMKII and Akt activation were measured in DC with the respective assay kit, using the specific substrates and 32{gamma}-adenosine 5'-triphosphate (ATP) after immunoprecipitation with the specific anti-CAMKII or anti-Akt antibody (Upstate Biotechnology, Lake Placid, NY). CAMKII and Akt activity were tested 4 min after oligomerization of ß3 integrin with 5 µg/ml anti-CD61 mAb, 20 min at 4°C, followed by 10 µg/ml purified F(ab')2 GAM Ig (Zymed Laboratories, San Francisco, CA) as described [7 ]. Control experiments using an isotype-matched control antibody were also performed (ref. [7 ], and not shown in this paper). The timepoint of maximal activation of either enzyme was assessed by experiments of kinetics (not shown). As a control, cells were exposed to GAM F(ab')2 alone and checked for kinase activation. Some experiments were carried out in the presence of the 100 nM HIV-1 Tat or of the overlapping Tat-derived peptides: Tat20–39, Tat24–51, Tat46–60, Tat56–70, and Tat65–80 (Technogen) [12 ]. In other experiments, cells were pretreated with the Vn-pep or Fn-pep or with the specific kinase inhibitors KN62 or LY294002, as above. Results are expressed as cpm x 10-3 and are the mean ± SD of four independent experiments.

Intracellular cyclic adenosine monophosphate (cAMP) measurement
Aliquots of 2 x 106 DC were resuspended in assay buffer and incubated with CTX or PTX (10-6 M, 10-9 M) for 2 h at 37°C in shaking water bath; then, cells were lysed by boiling for 5 min and centrifuged at 15,000 g for 10 min at 4°C. The cAMP content of each sample was determined using a commercial kit (NEN-Dupont) [11 ].

Results are expressed as pmoles/2 x 106 cells and are the mean ± SD of four independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Engulfment of apoptotic bodies by DC is dependent on CAMKII
We have obtained DC by culturing peripheral blood adherent monocytes in the presence of GM-CSF and IL-4 (during the first 4 days of culture to avoid macrophage differentiation). On day 8, these DC displayed the morphology [7 ] and phenotype (Table 1) of immature DC. In particular, they lost the CD14 monocyte marker and acquired the CD80 and CD86 costimulatory molecules; moreover, HLA-DR and {alpha}vß3 integrin were up-regulated, and CD83 began to be detectable in the cytoplasm on days 4–5 and at the surface of 40–50% of the cells after 8 days (Table 1) . Of interest, these DC lacked the CD36 and CD68 antigens [2 , 7 ], both markers of macrophages. At this day of culture, DC were able to phagocytose apoptotic cells but not other meals such as necrotic cells or opsonized yeasts [2 ]. Phagocytosis of apoptotic tumor cells by DC is dependent on extracellular calcium entry, possibly through calcium channels [2 , 13 ]; in particular, calcium interaction with calmodulin induces conformational changes, which allow the binding of this protein to CAMKII and its activation [14 ]. Figure 1 shows that the CAMKII inhibitors KN62 and KN93, but not the inactive compound KN92, reduced apoptotic body engulfment in a dose-dependent way, from 90% at 10 µM concentration to 50% at 1 µM concentration ( Fig. 1A ). It is interesting that an inhibitory effect was also observed, although to a lesser extent, using two blockers of PI-3K [15 , 16 ]: wortmannin (20% inhibition at 100 nM concentration; Fig. 1B ) and LY294002 (40% and 20% inhibition at 10 µM and 1 µM concentration, respectively; Fig. 1C ). On the contrary, the MAPK blocker PD98059 did not affect apoptotic body engulfment (Fig. 1C) ; likewise, rapamycin, which inactivates the PI-3K substrate S6K but not Akt [15 , 17 ], did not exhert any inhibition (Fig. 1D) . These results indicate that CAMKII is the major kinase involved in the phagocytosis of apoptotic tumor cells by DC.



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  		Figure 1. Apoptotic body engulfment by DC is reduced by CAMKII and PI-3K inhibitors. Apoptosis was induced by sublethal irradiation (4000 rad) of the human Jurkat lymphoma cell line. 51Cr-labeled apoptotic bodies were cocultured with adherent DC at the ratio of 3:1 at 37°C for 60 min. DC were pretreated (15 min) with the CAMKII inhibitors KN62 and KN93 or the inactive compound KN92 (A), the PI-3K inhibitor wortmannin (B), LY294002 or the MAPK inhibitor PD98059 (C), or rapamycin (D) at the indicated concentrations. Noningested apoptotic cells were removed, and DC-associated radioactivity was measured in a {gamma}-counter. Results are expressed as percentage of radioactivity calculated as described previously [2 ]. Mean ± SD of six independent experiments. *, P < 0.05.

 
The C-terminal domain of HIV-1 Tat inhibits CAMKII activation in DC
We have shown that Tat inhibits the extracellular calcium entry elicited via the ß3 integrin, a receptor involved in the phagocytosis of apoptotic cells by DC [2 , 7 ]. ß3 Integrin-mediated signaling is also strictly dependent on CAMKII, which in turn, is activated by calcium influx [14 , 18 ]; furthermore, PI-3K-dependent Akt activation via {alpha}vß3 has been described [19 , 20 ]. Thus, we investigated whether synthetic Tat or Tat peptides led to an impairment in CAMKII or PI-3K activation upon ß3 engagement. Figure 2 shows that the addition of synthetic Tat to DC blocks the activation of CAMKII elicited via ß3 integrin, which is also impaired by the use of the specific inhibitor KN62 but not by the PI-3K blocker LY294002 (Fig. 2A) . This would indicate that engagement of ß3 integrin during phagocytosis triggers a CAMKII pathway of activation. Along this line, synthetic Tat could only slightly inhibit (<20%) Akt activation triggered via ß3 integrins (Fig. 2B) , which in turn, is blocked by the PI-3K inhibitor LY294002.



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  		Figure 2. ß3 Integrin-mediated activation of CAMKII and Akt is regulated by HIV-1 Tat C-terminal domain. DC were incubated with saturating amounts of ß3 integrin, followed by GAM; as a control, cells were exposed to GAM alone. Some samples were pretreated with synthetic Tat (100 nM), KN62 (10 µM), or LY294002 (10 µM) or with Fn-pep, Vn-pep, or Tat peptides (100 nM; C), as indicated. Nil, activation of CAMKII (A and C) or Akt (B) 4 min after ß3 integrin engagement in the absence of Tat. CAMKII and Akt activation was measured in DC with the respective assay kit, using the specific substrates and 32{gamma}-ATP after immunoprecipitation with the specific anti-CAMKII or anti-Akt antibody. Results are expressed as cpm x 10-3 and are the mean ± SD of four independent experiments. *, P < 0.05.

 
Experiments performed with Tat-derived overlapping synthetic peptides provided evidence that this inhibition is mediated by the C-terminal domain of Tat and in particular, to the RGD sequence, which binds ß integrins [12 , 21 ]. Indeed, Tat65–80 peptide, spanning the C-terminal domain and containing the RGD amino acids, exerted a strong inhibitory effect (>60% of inhibition; Fig. 2C ), whereas Tat56–70 peptide, which also belongs to the C-terminal region of Tat but does not include the RGD sequence, was almost ineffective in decreasing CAMKII activation (<10% of inhibition; Fig. 2C ). The cysteine-rich Tat20–39 peptide, the core Tat24–52 region, or the basic domain Tat46–60 could not affect the activity of CAMKII (Fig. 2C) . It is interesting that the Vn-pep could also interfere with the induction of the enzyme elicited via ß3 integrin at variance with the Fn-pep (Fig. 2C) , supporting that in this system, Tat inhibits the {alpha}vß3-mediated signal transduction.

The inhibition of apoptotic body engulfment by HIV-1 Tat involves a pertussis toxin-sensitive G protein
Because CAMKII activation and calcium-channel function are regulated by G proteins [13 , 14 ], we investigated the effect of PTX and CTX, two toxins able to ADP-ribosylate distinct G-protein {alpha} subunits [11 , 22 ], on apoptotic body engulfment and on the inhibitory effect of HIV-1 Tat. As shown in Figure 3 A , treatment of DC with PTX, but not with CTX, enhanced the phagocytosis of apoptotic cells by 20%. It is interesting that PTX, at variance with CTX, could prevent the inhibition of apoptotic body engulfment mediated by Tat (Fig. 3B) or Tat C-terminal domain (Fig. 3C) . It is also interesting that PTX was able to interfere with the inhibition of engulfment exerted by the Vn-pep but not by the Fn-pep, further supporting that the pathway inhibited by Tat is dependent on {alpha}vß3 integrin engagement. This effect is unlikely because of a rise in intracellular cAMP concentrations: Indeed, CTX and PTX can increase intracellular cAMP (Fig. 4 A ) [11 ], and only PTX was able to prevent Tat inhibition (Fig. 3B) . Moreover, exposure of DC to FSK, which increases cAMP levels through the activation of adenylate cyclase, or FSK plus IBMX, an inhibitor of phosphodiesterase, did not affect apoptotic body engulfment or the inhibitory effect of Tat (Fig. 4B) . These data suggest that DC-mediated phagocytosis is regulated by the recruitment of a PTX-sensitive G protein.



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  		Figure 3. A putative PTX-sensitive G protein is involved in apoptotic body engulfment by DC: interference by HIV-1 Tat. Phagocytosis of apoptotic Jurkat cells by DC was evaluated as in Figure 1 . DC were pretreated (1 h) with the indicated concentrations of PTX or CTX alone (A) or before exposure to synthetic Tat (100 nM; B) or to the indicated Tat peptides or Vn-pep or Fn-pep (C). Noningested apoptotic cells were removed, and DC-associated radioactivity was measured in a {gamma}-counter. Nil, percentage of engulfment by untreated DC. Results are expressed as percentage of engulfment calculated as described previously [2 ] and are the mean ± SD of six independent experiments. *, P < 0.05.

 


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  		Figure 4. Intracellular cAMP increase is not involved in apoptotic body engulfment. (A) DC were incubated with CTX or PTX (10-6 M), 10-8 M FSK, 10-8 M FSK, plus 10-8 M IBMX for 1 h at 37°C, lysed by boiling for 5 min, and centrifuged at 15,000 g for 10 min at 4°C. The cAMP content of each sample was determined using a commercial kit [13 ]. Nil, intracellular cAMP content in untreated DC. Results are expressed as pmoles/2 x 106 cells and are the mean ± SD of four independent experiments. (B) Phagocytosis of apoptotic Jurkat cells by DC. 51Cr-labeled Jurkat cells, induced to apoptosis by sublethal irradiation (4000 rad), were cocultured with adherent DC at the ratio of 3:1 at 37°C for 60 min. DC were untreated (Nil) or pretreated (1 h) with 10-8 M FSK or 10-8 M FSK plus 10-8 M IBMX in the presence or absence of 100 nM Tat. Noningested apoptotic cells were removed, and DC-associated radioactivity was measured in a {gamma}-counter. Results are expressed as percentage of engulfment calculated as described previously [2 ] and are the mean ± SD of four independent experiments. *, P < 0.05.

 
PTX prevents Tat-mediated inhibition of ß3 integrin-induced CAMKII activation
To further investigate the involvement of PTX-sensitive G protein in the integrin-mediated signal transduction inhibited by Tat, CAMKII activation upon engagement of ß3 integrin was analyzed in DC pretreated with the toxin. As shown in Figure 5 , PTX but not CTX could partially prevent the inhibition of ß3-induced CAMKII activation exerted by synthetic Tat, further supporting the involvement of a PTX-sensitive G protein.



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  		Figure 5. PTX prevents Tat-mediated inhibition of ß3 integrin-induced CAMKII activation. Engagement of ß3 integrin was performed as in Figure 2 . Some samples were pretreated (15 min) with PTX or CTX at the indicated concentrations before exposure to 100 nM synthetic Tat. Nil, activation of CAMKII 4 min after ß3 integrin engagement in the absence of Tat. CAMKII activation was measured using the specific substrate and 32{gamma}-ATP after immunoprecipitation with the specific anti-CAMKII. Results are expressed as cpm x 10-3 and are the mean ± SD of four independent experiments. *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present paper, we describe that the phagocytosis of apoptotic tumor cells by DC is dependent on CAMKII activation and is regulated by a PTX-sensitive G protein. Moreover, HIV-1 Tat inhibits apoptotic body engulfment by blocking CAMKII activation via ß3 integrin.

Phagocytosis of apoptotic cells by DC is now a well-known phenomenon that contributes to the clearance of neoplastic or infected cells and to antigen presentation to T lymphocytes [1 2 3 4 ]. This is an active process that needs the engagement of specific surface receptors and an intracellular [Ca2+]i rise [2 3 4 ]. At least two integrins contribute to apoptotic body engulfment, {alpha}vß3 and {alpha}vß5, mediating endocytosis and signal transduction. In particular, the cytoplasmic tail of the ß5 chain triggers Rac1 activation and phagosome formation [3 ], and ß3 elicits an intracellular [Ca2+]i rise needed for the phagocytic process [4 ]. We found that phagocytosis of apoptotic tumor cells by DC is dependent on the induction of CAMKII, which is also triggered by ß3 engagement. Calcium interaction with calmodulin elicits conformational changes, which allow its binding to CAMKII and the activation of this enzyme [13 ]. In turn, CAMKII induces the opening of L-type Ca2+ channels [23 ], thus creating a positive feedback, which possibly favors the phagocytic process. This is in keeping with the observation that ß3 integrin-mediated signaling is strictly dependent on CAMKII activity [18 ] and with our previous findings that {alpha}vß3 complex mediated phagocytosis and calcium mobilization in DC [2 ]. So far, there is no evidence that ß5 signaling leads to CAMKII activation. Nevertheless, it has been shown that ß1, ß3, and ß5 integrin-intracellular domains share some common feature in signal transduction, raising the possibility that CAMKII may be activated by any of these integrins, which are able to elicit an extracellular calcium influx [24 , 25 ]. Along this line, it has been shown recently that {alpha}5ß1 is regulated by {alpha}vß3 through the modulation of CAMKII [26 ]. Conversely, an endocytic route triggered via {alpha}vß;5 integrin, which in turn activates PI-3K, has been described recently [27 ], thus supporting the existence of a cross-talk between the various integrin-dependent pathways of kinase activation.

Previously, we have demonstrated that exogenous HIV-1 Tat inhibits the engulfment of apoptotic bodies by interfering with extracellular calcium entry elicited via ß3 integrin in DC [7 ]. We have also shown that Tat acts as a blocker of L-type calcium channels that mediate calcium influx in DC and lymphocytes [9 , 10 ]. Herein, we provide evidence that inhibition of apoptotic body engulfment is a result of the block of CAMKII activation via ß3 integrin exerted by the C-terminal, RGD-containing domain of Tat, which binds to ß integrins [21 ]. Because CAMKII contributes to the up-regulation of L-type calcium-channel function [23 ], it is now clear why Tat inhibited the ß3 integrin-dependent calcium mobilization [7 ]. PI-3K-dependent Akt activation via {alpha}vß3 has been described as well [19 , 20 ]. Along this line, we found that PI-3K is also involved in the phagocytosis of apoptotic cells by DC, although to a lower extent than CAMKII. However, synthetic Tat or Tat peptides led only to a slight inhibition of PI-3K activation upon ß3 ligation. This is not surprising, because this kinase is triggered by the engagement of the integrin through a binding domain on the ß3 chain distinct from the RGD recognition site [20 ]. Moreover, at variance with CAMKII, PI-3K activation is dependent on cytoplasmic calcium rises as a result of not only extracellular calcium entry but also calcium mobilization from intracellular stores, thus being less sensitive to a selective impairment of extracellular calcium influx [15 , 17 ].

In addition, we found that PTX could prevent the inhibition exerted by HIV-1 Tat on apoptotic body engulfment by DC. This effect was not simply because of a PTX-induced rise in intracellular cAMP, due to CTX strongly increased cAMP levels without affecting phagocytosis or Tat-mediated inhibition. Alternatively, a G protein might be involved. This is of interest, because CAMKII activation and calcium-channel function are regulated by different G proteins that maintain the channels in the open (G{alpha}) or closed (Gi) state [13 , 14 ]. One possible explanation for our findings (see a model for this hypothesis in in Fig. 6 ) is that PTX displaces a Gi protein involved in calcium-channel inhibition [28 ], thus allowing the opening of these channels. The consequent rise in intracellular calcium might activate CAMKII [18 ], which in turn up-regulates ß3 integrin function. Indeed, it has been demonstrated that {alpha}vß3-dependent cell migration, inhibited by cyclic RGD peptides in vascular smooth muscle cells, can be restored by maintaining elevated CAMKII activity, raising the intracellular calcium levels with ionophores [18 ]. A role for Gi protein in down-regulating IL-12 production by murine splenocytes has also been proposed: secretion of the cytokine was restored by blocking Gi with PTX [29 ]. It is interesting that we demonstrated that Tat inhibits IL-12 secretion by blocking L-type calcium channels in DC, and this inhibition is not RGD-mediated. It is tempting to speculate that a non-RGD domain located in the C-terminal portion of the protein interacts with L-type calcium channels in the close state, e.g., when they are blocked by a Gi protein. Another possibility is that a PTX-sentive G{alpha} subunit is engaged, allowing the displacement of Gß{gamma} complexes, which can contribute to maintaining calcium channels in the open state. Indeed, calcium and potassium channels are activated by the Gß{gamma} subunits of heterotrimeric G proteins [30 ]. In particular, engagement of G-protein-coupled receptors displaces Gß{gamma} from the G{alpha} subunit [31 ]. Thus, Gß{gamma} complexes can exert their biological effects, including opening and regulation of calcium channels. In turn, Gß{gamma} dimers have been shown to stimulate vascular L-type calcium channels via PI-3K [32 ], thus providing an additional explanation to our findings of a partial involvement of PI-3K in apoptotic body engulfment by DC. Finally, we rule out a potential activation of G-protein-coupled receptors by Tat in our system, because the protein domains responsible for chemokine-receptor binding did not exert any effect.



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  		Figure 6. Model of the interplay among Tat, G protein(s), and CAMKII. Engagement of ß3 integrin during phagocytosis leads to CAMKII activation, which in turn, contributes to the opening of calcium channels and enhances the extracellular calcium entry, creating a positive feedback. Occupancy of {alpha}vß3 by RGD-containing Vn-pep or Tat peptides would block this activation pathway, which could be rescued by PTX as a result of the displacement of a Gi protein involved in calcium-channel inhibition [28 ], thus allowing the opening of these channels. The consequent rise in intracellular calcium might activate CAMKII, which in turn, up-regulates ß3 integrin function [18 ]. Alternatively, a PTX-sensitive G{alpha} subunit is engaged, allowing the displacement of Gß{gamma} complexes, which can contribute to maintaining calcium channels in the open state [30 ], possibly through the engagement of PI-3K [32 ]. When the {alpha}vß3-integrin pathway is blocked (e.g., by Tat), PI-3K might be activated alternatively via {alpha}vß5. Finally, the non-RGD domain, located in the C-terminal portion of Tat, might interact with L-type calcium channels in the close state, e.g., when they are blocked by a Gi protein.

 
Taken together, our results point to an essential role for CAMKII in mediating L-type calcium channel-regulated functions in DC and highlight a biochemical mechanism whereby HIV-1 Tat is able to paralize these cells, thus contributing to a delay in the development of protective immune responses before HIV-1 spreading. In asymptomatic infection, a reduced antigen-presenting function of DC, before evidence for T-cell abnormalities, has been found and related to a decline in the production of CD4 memory T cells [33 , 34 ]. It is interesting that it has been shown recently that transfection of a human macrophage cell line with a Tat expression vector results in a down-regulation of mannore receptor-mediated phagocytosis, possibly leading to a decreased pathogen/antigen capture [35 ]. We also show that exogenous Tat may impair this mechanism, potentially involved in the clearance of infected cells and in the initiation of antigen-specific antiviral immunity. Moreover, we provide evidence for a possible pharmacological approach to prevent Tat inhibitory effects by acting on a subset of G proteins. Treatments that prevent the defect in antigen uptake, processing, and presentation by DC might improve cell-mediated immunity.


   ACKNOWLEDGEMENTS
 
This work was supported by ISS (Special Project AIDS 1999).

Received August 17, 2001; revised October 20, 2001; accepted October 25, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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