Published online before print September 12, 2003
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* Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Naples, Italy; and
Istituto di Endocrinologia ed Oncologia Sperimentale, Napoli, Italy
1 Correspondence: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, 5 via S. Pansini, I-80131 Napoli, Italy. E-mail: racioppi{at}unina.it
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but not the engagement of CD4 by monoclonal antibody stimulates the PKA activity and induces a long-lasting hyporesponsivity state in naive CD4+ lymphocytes. The pretreatment of lymphocytes with H89, a cell-permeable PKA inhibitor, prevents the induction of anergy. These findings reveal a novel mechanism by which HIVenv may modulate the processes of clonal expansion, homeostatic proliferation, and terminal differentiation of the naive T lymphocyte subset.
Key Words: CXCR4 • HIVenv • cAMP • CREB
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expression, which appeared to be reversible upon reduction of the virus load with highly active antiretroviral treatment [9
]. A marked decrease in the enzymatic activity of CD45 and altered levels of Lck and Fyn have been observed in lymphocytes isolated from HIV-infected individuals [10
11
12
13
14
15
16
]. Most of these defects can be reproduced in vitro by exposure of normal T lymphocytes to inactivated HIV virions or purified HIV components. In fact, soluble (s)gp120 is able to inhibit IL-2 mRNA expression [17
] and the proliferative response of T lymphocytes when stimulated through the TCR [18
, 19
]. The ability of envelope glycoprotein gp120 (HIVenv) to interfere with TCR-mediated activation of the src-family tyrosine kinases also has been documented [20
21
22
]. We proposed that the effects of gp120 on antigen-dependent proliferation of T lymphocytes are not homogenous and may vary according to their state of activation and differentiation [23
]. Naive and memory CD4 subsets show differential susceptibility, although the exposure of CD4 naive T cells to the envelope glycoprotein results in a marked inhibition of the mitogenic responses, the growth of the memory subset is less severely restrained by gp120 [23
]. This inhibitory effect is associated with alterations in the TCR-mediated signaling such as inhibition of the extracellular-regulated kinase (ERK) and stimulation of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA). PKA is a serine/threonine kinase that regulates a number of cellular processes important for immune activation [24 ]. In lymphocytes, PKA regulates antigen receptor-induced signaling by altering protein–protein interactions and by changing the enzymatic activity of target protein [25 ]. Stimulation of the cAMP/PKA pathway may also increase the level of inducible cAMP early repressor and cAMP-responsive element modulator, two nuclear inhibitory factors involved in modulation of T cell responsiveness by their capacity to transcriptionally attenuate IL-2 gene expression [26 , 27 ]. Hofmann et al. [28 ] suggested the involvement of PKA in the mechanism leading to the HIV-induced defect in T cell function. More recently, this hypothesis has been corroborated by direct measurements of the cAMP intracellular levels in T lymphocytes derived from HIV+ individuals and by the ability of PKA type I antagonist to restore the responsiveness of T cells from HIV-infected patients [29 , 30 ]. Although these reports revealed an involvement of the PKA pathway in the mechanism leading to T cell abnormalities in HIV infection, the molecular base of this phenomenon is still unclear. Here, we provide novel evidences on the ability of the soluble envelope glycoprotein to induce long-lasting effects on naive lymphocytes through a CD4-independent, PKA-mediated pathway. This phenomenon may contribute to the development of immune defects in HIV-1 infected individuals.
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was from Alexis. sh-recombinant CD4 (shrCD4), rgp120 IIIB (HIVenv), and biotinylated rgp120 were from Intracell Corp. (London, UK). sCD4-HIVenv complexes were generated by incubating sCD4 and HIVenv in serum-free medium at a 1:1 ratio for 1 h at 4°C before being added to cells at the final concentration of 2 µg/ml.
Purification of naive CD4 T lymphocytes
T lymphocytes were isolated from peripheral blood of healthy donors by Lymphoprep (Nyegaard and Co., Oslo, Norway) density-gradient centrifugation at 2500 rpm for 30 min and subsequently, by incubating mononuclear cells for 1 h at 37°C in plastic petri dishes. Resting T cells were then purified from nonadherent cells by nylon wool (Robbins Scientific, Sunnyvale, CA) adherence to remove B cells, followed by a two-step (40% and 50%, respectively) Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). Resting lymphocytes were recovered from the high-density cell fraction. The CD4+ and CD8+ CD45RA cells were separated using negative selection with subset-specific monoclonal antibodies (mAb; anti-CD8, -CD19, -DR, -CD45RO, CD16, -CD56, -
TCR, all from Becton Dickinson) and anti-mouse immunoglobulin-coated magnetic beads (Dynabeads, Dynal, Oslo, Norwey). The purity of lymphoid populations was tested by flow cytometry (FACScan, Becton Dickinson) using appropriate mAb (PharMingen, San Diego, CA). Purified, naive lymphocytes contained 89–93% of CD3+ CD45 RA+. Contaminating cells were CD8+ (4–7%), CD45 RO+ (3–5%), and CD3- CD56+ (3–10%). The purity of naive lymphocytes was also tested by functional assay: Stimulation with an optimal dose of phytohemagglutinin (PHA; 2 µg/ml) did not induce a detectable mitogenic response. Conversely, an optimal response was observed upon addition to cultures of 10% of dendritic cells (DC) or anti-CD28 antibody (Becton Dickinson; data not shown).
Generation of monocyte-derived DC (Mo-DC)
Mo-DC were obtained according to protocol by Sallusto et al. [31
]. Briefly, monocytes were isolated from peripheral blood mononuclear cells by adherence on plastic dishes coated with 2% gelatin. Adherent cells were removed by incubation with phosphate-buffered saline–EDTA and were cultured for 5 days at a density of 3 x 106 cells/ml in RPMI 1640 containing 10% FCS supplemented with 50 ng/ml granulocyte macrophage-colony stimulating factor and 1000 U/ml IL-4 (Sigma Chemical Co.). The purity of isolated populations was tested by flow cytometry using the appropriate mAb.
Anergy assay
To test the capability of HIVenv to induce a prolonged hyporesponsivity state, namely anergy, naive lymphocytes were incubated with viral glycoprotein, rested in fresh medium, and stimulated with mitogens or allogenic Mo-DC. During the incubation step, 3–5 x 106 purified T cells were cultured in the presence or absence of 1–2 µg/ml rgp120 for 12–18 h. At the end of this incubation, lymphocytes were tested for viability, washed extensively, and rested in fresh medium for further 3–8 days (resting step). At the end of the resting step, Lymphoprep (Nyegaard and Co.) density gradient removed death cells, and living cells were tested for responsiveness to mitogens or allogenic Mo-DC (stimulation step). As tested in additional experiments, the purification step with Lymphoprep did not interfere with responsiveness and survival of lymphocytes. Proliferation assays were performed by culturing 105 lymphocytes/wells for 72 h in round-bottom 96 wells/plate with increasing doses of PHA (Sigma Chemical Co.) plus accessory cells (5% of plastic-adherent, allogenic cells). Mitogenic response to Mo-DC was tested by stimulation of T cells with increasing amounts of allogenic Mo-DC. 3H-Thymidine (Amersham International, Buckinghamshire, UK) was added to cultures during the last 12 h of incubation.
Immunoblotting and immunocomplex kinase assays
Purified CD4 CD45 RA cells (5x106) were preincubated for 30 min on ice in the presence or absence of rgp120 (2 µg/ml) and then incubated at 37°C for different periods of time. Lymphocytes were lysed in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 100 µg/ml phenylmethylsulfonyl fluoride, 100 µg/ml tosylphenylalanine chloromethyl ketone, 1 µg/ml leupeptin, 0.83 µg/ml chymostatin, 10 µg/ml soybean trypsin inhibitor, 1 mM sodium orthovanadate, and 0.5 mM dithiothreitol. The lysates were boiled for 5 min and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis was performed using mAb to Lck, phospho-ERK, ERK-2, cAMP response element-binding protein (CREB), and phospho-CREB (Santa Cruz Biotechnology, Santa Cruz, CA) and was developed using the enhanced chemiluminescence Western blotting kit (Amersham International).
PKA assays were performed in a final volume of 25 µl at 30°C for 10 min. The kinase reaction mixture contained 100 µM adenosine 5'-triphosphate (ATP), [32P] ATP 10 µCi/100 ml, 10 mM MgCl2, 20 mM Hepes (pH 7.4), and 100 µM Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide; Sigma Chemical Co.). Specificity of reaction was verified by adding 100 µM protein kinase inhibitor (PKI; P0300) synthetic rabbit PKA-specific inhibitor pseudo-substrate (Sigma Chemical Co.) to a duplicate of each experimental point. Kemptide phosphorylation was monitored by spotting 20 µl reaction mixture on phosphocellulode filters (Whatman, P81). After washing with phosphoric acid, 75 mM, radioactivity was determined by counting the filters in 4 ml scintillation liquid (Ecolite, ICN, Costa Mesa, CA). Free catalitic-PKA (C-PKA) activity was determined as PKI-inhibitable filter-bound radioactivity. Control assay indicated that 100 µM PKI used did not inhibit the binding of phosphorylated Kemptide to phosphocellulose filters. Data were expressed as picomoles of [32P] phosphate transferred to the peptide substrate during 10 min incubation.
Statistical analysis
Data were analyzed by Wilcoxon test for paired comparison by using the StatView SE+ Graphic program for Macintosh (Abacus Concepts, Cary, NC).
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Figure 1. Effects induced by transient exposure of naive T cells to purified HIVenv. Purified CD4+ CD45 RA lymphocytes were exposed for 12 h to 2 µg/ml rgp120, washed, and rested for 72 h in fresh medium. At different times, aliquots of T cells were stained with Annexin V and analyzed by flow cytometry. The left panel reports the percentage of Annexin V-positive cells upon 12 h of exposure to rgp120 at the end of the resting step and 72 h from addition of PHA 2 µg/ml (stimulation step). Responsiveness of T cells to PHA or allogenic DC is shown in the middle and right panels, respectively. The results of the proliferation assays are expressed as the percent of the maximal response, assuming 100% the response of HIVenv-untreated lymphocytes to an optimal dose of PHA (2 µg/ml; 75,000±12,000 cpm of 3H-thymidine incorporation) or to 50,000 allogenic DC/well (35,000±5400 cpm). Each data point represents the mean of three independent experiments ± SD. *, Statistical significant differences (P<0.001) were observed between the proliferation values measured in HIVenv-treated and untreated samples stimulated with 1 µg/ml PHA or 5000 allogenic DC/well, respectively (center and right panels).
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was also evaluated. After 12 h of incubation with HIVenv, 80% of CD4 and 40% CXCR4 molecules were removed from the cell surface, and no change in CD3- expression was observed (Fig. 2
). During the resting step, CD4 and CXCR4 surface levels progressively recovered until at later time points, no difference was found between HIVenv-treated and untreated lymphocytes (Fig. 2)
. These data indicate that the effects induced by HIVenv on CD4 expression were transient and required the continuous presence of the HIVenv in the culture medium. Conversely, a pulsed exposure to HIVenv was able and sufficient to induce a prolonged hyporesponsiveness in naive T lymphocytes to PHA and allogenic DC stimulation (Fig. 2
, inset).
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Figure 2. Kinetic analysis of the effects induced by HIVenv on naive T cells. CD4+ naive lymphocytes were exposed to HIVenv for 12 h, washed, and rested in fresh medium. The expression of CD4, CXCR4, and CD3- has been tested by flow cytometry at different time points. The results have been reported as the percentage of the maximum corresponding to the cell-surface expression levels measured in untreated lymphocytes. Inset reports the results of the anergy assay. Briefly, upon exposure to HIVenv, T cells were washed and rested for 72 h in fresh medium before being stimulated with T cell responsiveness to PHA (1 µg/ml) or allogenic DC (5000 cell/well). The results have been reported as the percentage of the maximum proliferation measured in HIVenv-unexposed lymphocytes stimulated with the optimal doses of PHA or allogenic DC. (3H-Thymidine incorporation was 41,000±7000 and 25,000±3500 cpm, respectively.) Each data point reports the mean of three independent experiments ± SD.
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Figure 3. Proximal signaling induced by HIVenv in CD4+ naive T cells. Naive CD4+ lymphocytes were exposed for different time to HIVenv (2 µg/ml) or stimulated with phorbol 12-myristate 13-acetate and ionomicin (P/I). (A) Upon 30 min from stimuli, postnuclear cell lysates were immunoprecipitated with an anti-Lck antibody. The authophosphotransferase activity was measured on immunocomplexes by an in vitro kinase assay (IVKA). Aliquots of lysates were immunoblotted for Lck (WB). (B) Immunoblots for the phosphorylated ERK (p-ERK) and ERK-2. (C) PKA phosphotransferase activity and immunoblots for phospho-CREB (p-CREB) and CREB. The results are representative of three independent experiments.
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Figure 4. Effects of anti-CD4 antibody and SDF-1 on HIVenv-induced anergy. Naive CD4+ lymphocytes were exposed to HIVenv (2 µg/ml) in the presence or absence of Leu3a (0.5 µg/ml) or SDF-1 (100 nM). (Left) Results of the anergy assay (see Materials and Methods). T cell responsiveness to allogenic (Allo) DC has been expressed as the percentage of the maximum proliferation measured in HIV-untreated T lymphocytes exposed to 50,000 allogenic DC/well corresponding to 35,000 ± 8000 cpm 3H-thymidine incorporation. Each data point reports the mean of three independent experiments ± SD. (Right) Ability of Leu3a and SDF-1 to prevent the binding of HIVenv to the cell surface. In this set of experiments, naive CD4+ lymphocytes were exposed for 2 h to biotinylated HIVenv (250 ng/ml) in the presence or absence of Leu3a (0.5 µg/ml) or SDF-1 (100 nM). Phycoerythrin (PE)-conjugated avidin was used to detect the binding of biotinylated HIVenv. The filled, gray profile refers to PE-avidin staining, measured on T cells preincubated with biotinylated HIVenv (maximal staining). Open, thick profiles refer to PE-avidin staining measured in cells preincubated with biotinylated HIVenv in the presence of Leu3a or SDF-1. Open, dotted profile refers to PE-avidin staining measured in lymphocytes not exposed to biotinylated HIVenv (negative control). The result is representative of three independent experiments.
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Figure 5. CD4 engagement by HIVenv or Leu3a is not required for the induction of T cell anergy in naive lymphocytes, which were cultured for 12 h with regular medium, Leu3a (0.5 µg/ml), SDF-1 (100 nM), HIVenv (2 µg/ml), or sCD4-gp120 complexes (sCD4-HIVenv; 2 µg/ml). To evaluate T cell responsiveness, lymphocytes were rested for 3 days in fresh medium before being stimulated with 10,000 cells/well allogenic DC (top). The results are expressed as the percentage of the maximal response measured in lymphocytes cultured in regular medium (38,000±2500 cpm). Aliquots of cells exposed for 12 h to the stimuli described previously were double-stained with CD4 and CXCR4 and analyzed by flow cytometry (middle). The results are expressed as the percentage of the maximum mean fluorescence intensity measured on lymphocytes cultured with regular medium alone. (Bottom) Phosphotransferase activity of the C-PKA measured in naive T cells exposed for 30 min to the reported stimuli (Leu3a, 0.5 µg/ml; SDF-1, 100 nM; HIVenv, 2 µg/ml; sCD4-HIVenv, 2 µg/ml). The results are representative of four independent experiments.
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The pharmacological block of PKA prevented the HIVenv-induced anergy
HIVenv and sCD4-HIVenv complexes share a comparable ability to induce anergy in naive T cells, a phenomenon associated with their capability to stimulate the PKA pathway. This association strongly suggested an involvement of PKA in the mechanism of anergy. Therefore, we pretreated lymphocytes with H89 and PP1, selective inhibitors of PKA and tyrosine kinases, before being exposed to this moiety. This set of experiments showed that the pharmacological block of PKA but not of the tyrosine kinase activity prevented the hyporesponsiveness induced by lymphocyte exposure to the viral glycoprotein. These data provided direct evidence about the involvement of the PKA pathway in the mechanism of anergy induction (Fig. 6
).
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Figure 6. Pharmacological block of PKA prevented HIVenv-induced anergy. Naive T lymphocytes were exposed for 12 h to rgp120 (2 µg/ml) in the presence or absence of PKA and tyrosine kinase inhibitors (H89 10 µM and PP1 10 µM, respectively). Lymphocytes were washed, rested for 96 h, and exposed for 72 h to an increasing number of allogenic DC. 3H-Thymidine was added in the last 12 h of culture. The results are expressed as the percentage of the maximal response measured in HIVenv-untreated lymphocytes stimulated with 50,000 allogenic DC (47,000+4000 cpm) and are calculated on three independent experiments. Statistical significant differences (P<0.001) were observed between the proliferation values measured in HIVenv-treated lymphocytes cultured with or without PP1 and untreated lymphocytes stimulated with 5000 allogenic DC/well cultured in regular medium or in the presence of HIVenv and H89.
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The ability of gp120 to modulate the biological response of lymphoid cells via a CD4-independent mechanism has been proposed by several authors. Fantuzzi and colleagues [38 ] have recently shown that the treatment of freshly isolated monocytes with rgp120-IIIB resulted in a dose-dependent enhancement of secretion of monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, and regulated on activation, normal T expressed and secreted. This phenomenon is independent by interaction of gp120 with CD4 but relates to the specific interaction of the viral glycoprotein with CCR5 and CXCR4 coreceptors. Other authors have found that in contrast to the CD4-dependent gp120 signaling via CCR5, the envelope-mediated CXCR4 signaling is CD4-independent and can induce chemotaxis of CD4 and CD8 T cells [39 ]. In our experimental model, treatment with Leu3a, a gp120 mimicking anti-CD4 antibody, induces a marked CD4 down-regulation but was unable to stimulate the PKA pathway as well as to induce anergy in naive T cells. Conversely, sCD4-HIVenv complexes, which are able to bind CXCR4 but not CD4, stimulate the PKA pathway and induce anergy in naive lymphocytes. These findings strongly support the hypothesis that CD4 molecules are required for the recruitment of the envelope glycoprotein to the T cell surface but are unable to mediate the negative signaling responsible for the induction of the prolonged hyporesponsiveness to mitogens. These data are in contrast with a number of reports showing that the engagement of CD4 by gp120 may induce pleiotropic effects and interfere with the activation process of T cells [13 14 15 16 , 20 21 22 ]. Other studies have also documented that gp120 can itself transduce intracellular signals via CD4 and CXCR4/CCR5 receptors, including the activation of the MAPK pathway [40 , 41 ]. The majority of these studies have been performed on transformed T cell lines or on preactivated T lymphocytes. When signaling has been investigated on normal, quiescent T cells, it has been observed that gp120 may induce differential effects on and activate different signal cascades in resting and preactivated lymphocytes [23 , 41 ]. One apparent distinction between primary, quiescent T lymphocytes compared with preactivated lymphocytes or the transformed Jurkat cell line is that in the former, a significant proportion of CD4 is outside the lipid rafts [42 , 43 ], whereas in the latter, it is located almost exclusively in raft structures [44 , 45 ]. This localization may be one of the factors accounting for the HIVenv signaling responses observed in the naive T lymphocytes and may explain the differential effects exerted by the envelope glycoprotein on the different T cell subsets.
The ability of HIV to stimulate PKA activity was originally shown a decade ago by Hofmann et al. [28
] by exposing normal peripheral blood lymphocytes to inactivated virions. Here, we clarify the molecular bases of this phenomenon by identifying HIVenv as a viral derivative responsible for PKA activation in naive T cells, showing for the first time that this biochemical change is not dependent on CD4-mediated signals. The coupling of the chemokine receptor CXCR4 to the PKA pathway has been documented recently in embryonic retinal ganglion cell neurons and in peripheral blood monocyte [46
, 47
]. Here, we confirm these findings, revealing for the first time the ability of SDF-1 to trigger the PKA pathway and to induce prolonged effects on the activation threshold in CD4+ naive T cells. Activation of the PKA pathway has a variety of transcriptional and post-transcriptional effects on different immune mediators. It has been reported that increased levels of cAMP have an important post-transcriptional effect on CD40 ligand expression, IL-10 secretion, and T helper cell type 2 (Th2) activation, and it inhibits T cell proliferation, IL-2 and IL-12 production, and Th1-mediated effector functions [48
49
50
51
52
53
]. In addition, it has been shown that TCR stimulation of naive CD4 T cells in the presence of cAMP-elevating agents leads to a population of CD4 CD45RA T cells with functional and phenotypic features of effector Th2 cells [54
]. Here, we found that HIVenv glycoprotein can induce anergy in naive T cells by stimulating the PKA pathway, a phenomenon whose relevance remains to be determined in the context of the mechanisms affecting homeostatic proliferation, clonal expansion, and terminal differentiation of this subset of lymphocytes in AIDS. Despite this, our findings offer an ensemble of the complex and differential effects that HIV derivatives can exert on the various hematopoietic cell types.
Received May 22, 2003; revised August 11, 2003; accepted August 12, 2003.
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