cAMP Up-Regulates Cell Surface Expression of CXCR4: Implications for and HIV-1 Infection

This information is current as Steve W. Cole, Beth D. Jamieson and Jerome A. Zack of September 27, 2021. J Immunol 1999; 162:1392-1400; ; http://www.jimmunol.org/content/162/3/1392 Downloaded from References This article cites 67 articles, 38 of which you can access for free at: http://www.jimmunol.org/content/162/3/1392.full#ref-list-1

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. cAMP Up-Regulates Cell Surface Expression of Lymphocyte CXCR4: Implications for Chemotaxis and HIV-1 Infection1

Steve W. Cole,2* Beth D. Jamieson,* and Jerome A. Zack*†

The receptor CXCR4 mediates lymphocyte chemotaxis in response to stromal cell-derived factor-1 (SDF-1) and func- tions as a coreceptor for -tropic strains of HIV-1. We examined the role of the cAMP-protein kinase A (PKA) signaling pathway in regulating expression of CXCR4. In response to exogenous dibutyryl cAMP or cAMP-inducing ligands, cell surface expression of CXCR4 was increased by up to 10-fold on CD3/CD28-stimulated PBMC and by up to sixfold on unstimulated PBMC. cAMP did not alter receptor mRNA levels or affect the size of the total CXCR4 pool. However, cAMP did significantly reduce CXCR4 internalization rates and thereby increased the fraction of the total CXCR4 pool expressed on the cell surface. cAMP-induced increases in CXCR4 expression counteracted SDF-1-induced receptor internalization and enhanced both chemo- tactic response to SDF-1 and cellular vulnerability to HIV-1 infection. Thus, altered expression may provide Downloaded from one mechanism by which cAMP-inducing ligands influence lymphocyte localization and HIV pathogenesis. The Journal of Im- munology, 1999, 162: 1392–1400.

ffective immune response requires the recruitment of Chemokine receptor expression has received a great deal of at- functionally distinct leukocyte subsets to appropriate tis- tention following the discovery that certain chemokine receptors

E sue sites (e.g., direction of hemopoietic progenitors and function in conjunction with CD4 to mediate HIV infection of http://www.jimmunol.org/ naive to lymphoid organs and mature lymphocytes to human cells (9). CXCR4 was initially identified as a coreceptor for peripheral sites of inflammation). This recruitment process is be- T cell-tropic strains of HIV-1 (10), and CCR5 and several other lieved to be mediated in part by , soluble messenger CC chemokine receptors were subsequently identified as corecep- molecules secreted by target tissue cells to signal their status as tors for -tropic strains of HIV-1 (11–13). CXCR4 can lymphoid organs or inflamed tissue (1–3). Differential localization also mediate CD4-independent infection of human cells by HIV-2 of leukocyte subsets is mediated in part by their differential ex- (14, 15). Varying levels of chemokine receptor expression can in- pression of chemokine receptors (4). For example, activated/mem- fluence cellular susceptibility to HIV infection (16, 17), and che- ory T lymphocytes display a higher density of the CC chemokine mokines and anti-receptor Abs can block HIV infection under cer- by guest on September 27, 2021 receptor CCR5 than do resting/naive T lymphocytes and, as a re- tain circumstances (18–23). sult, show greater chemotaxis in response to CC chemokines such Because chemokine receptors play a critical role in both normal as macrophage inflammatory protein-1 (MIP-1)3 (5). Conversely, immune system function and HIV pathogenesis, understanding the naive T lymphocytes express greater levels of the CXC chemokine factors that regulate their expression may provide options for ther- receptor CXCR4 than do memory T lymphocytes and, as a result, apeutic intervention in a variety of pathophysiologic settings (1– show greater chemotaxis in response to CXC chemokines such as 4). In addition to differential expression across distinct leukocyte SDF-1 (5). Thus, differential chemokine receptor expression may subpopulations, chemokine receptor density is also altered in the play a critical role in the differential trafficking patterns of naive vs mature T lymphocytes. Similar dichotomies in chemokine receptor presence of its natural ligand (24), over the course of mitosis (5, expression appear to distinguish CD4ϩ T lymphocytes supporting 24, 25), and during cellular maturation (26). can alter cellular immune responses from those that foster humoral re- the expression of some CC chemokine receptors (27, 28) although sponses (6–8). their effects on lymphocyte CXC chemokine receptors remain poorly defined. Chemokine receptors are members of a diverse family of cell surface receptors characterized by a seven-trans- membrane serpentine structure and signal transduction via hetero- *UCLA AIDS Institute, Department of Medicine, and †Department of Microbiology trimeric guanine nucleotide binding proteins (4, 29, 30). Cell sur- and Molecular Genetics, University of California at Los Angeles School of Medicine, face expression of G-protein-linked receptors is regulated both by Los Angeles, CA 90095 expression and by continual recirculation of receptors be- Received for publication July 13, 1998. Accepted for publication October 26, 1998. tween the cell surface and endosomal compartments (24, 31). Dis- 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 tinct signaling pathways govern these two modes of receptor reg- with 18 U.S.C. Section 1734 solely to indicate this fact. ulation. For example, whereas certain mitogenic stimuli suppress 1 This work was supported by National Institute of Allergy and Infectious Diseases CXCR4 expression by increasing receptor internalization via the Grant AI 33259. protein kinase C (PKC) signaling cascade, this pathway does not 2 Address correspondence and reprint requests to Dr. Steve W. Cole, Division of appear to mediate ligand-induced suppression of CXCR4 (24). Hematology-Oncology, Department of Medicine, Factor 11-934, UCLA School of Medicine, Los Angeles, CA 90095-1678. E-mail address: coles@nicco. With the exception of mitogenic stimuli and ligand-induced sscnet.ucla.edu down-regulation, little is known about the role of extracellular fac- 3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; tors in regulating lymphocyte expression of CXC chemokine re- SDF-1, stromal cell-derived factor-1; PKA, protein kinase A; dbcAMP, N6,2Ј-O- dibutyryl adenosine-3Ј,5Ј-cyclic monophosphate; ACTH, adrenocorticotropic hor- ceptors. However, the pronounced effects of cytokines, growth fac- mone; HSA, heat-stable Ag; NE, norepinephrine. tors, and hormones on lymphocyte localization (3, 32–34) suggest

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 1393 that extracellular factors may well play an important role in che- FACScan instrument (Becton Dickinson Immunocytometry). Data were mokine receptor expression. One major mode of extracellular in- analyzed using CELLQuest software (Becton Dickinson Immunocytom- fluence on lymphocyte function comes from a class of secreted etry), with gating to exclude dead cells and debris on the basis of forward- vs side-scatter profiles. Linear regression was used to assess the statistical molecules that activate the cellular cAMP-dependent protein ki- significance of dose-response relationships between dbcAMP concentra- nase A (PKA) (35). The cAMP-PKA signaling pathway represents tions and CXCR4 mean fluorescence intensity. a common second messenger system for a variety of distinct re- ceptors that bind a diverse array of hormones, neurotransmitters, CXCR4 gene transcription and peptide-signaling molecules (e.g., E series PGs, histamine, cat- CXCR4 mRNA was quantified by RT-PCR. Total cellular RNA from 107 echolamines, neurohypophyseal hormones (such as corticotropin- PBMC was extracted (RNeasy; Qiagen, Chatsworth, CA), subject to releasing factor and vasopressin), and pro-opiomelanocortin-de- DNase-1 treatment (Promega, Madison, WI), and reverse transcribed using rived peptides (such as adrenocorticotropic hormone)) (30). As a murine leukemia virus reverse transcriptase (Perkin-Elmer Cetus, Norwalk, CT) and a 20-thymidine oligonucleotide primer. One-tenth of the resultant result of cAMP signaling, PKA phosphorylates multiple intracel- cDNA was amplified by PCR using the CXCR4-specific primers 5Ј-TCA lular substrates, including elements of other signaling pathways TCT ACA CAg TCA ACC TCT ACA-3Ј and 5Ј-gAA CAC AAC CAC Ј ␤ (e.g., phospholipase C␥1) and regulators of gene transcription (e.g., CCA CAA gTC ATT-3 (47). To verify equivalent RNA loading, -actin cAMP response element binding protein (CREB)). The cAMP- cDNA was amplified in parallel using commercial primers (R&D Sys- tems). Primers were radio end-labeled with 32P, and samples were ampli- PKA signaling pathway plays a critical “switching” role in a va- fied using Taq DNA polymerase (Perkin-Elmer Cetus) with 34 cycles of riety of physiologic settings ranging from glycolysis to ontogenic denaturing at 94°C for 1 min, annealing at 54°C for 1 min, and extension differentiation. In the immune system, cAMP signaling modulates at 72°C for 1 min. Standards consisting of serial 10-fold dilutions of cDNA 7 cellular activation (35, 36) and alters production profiles from total cellular RNA extracted from 10 PBMC were amplified in par- Downloaded from (37–41). cAMP-inducing stimuli can also alter leukocyte traffic allel. Radiolabeled amplified products were resolved on a 6% polyacryl- amide gel and quantified by radioanalytic image analysis in comparison and localization (33, 34, 42), although the molecular basis for such with standard curves. All amplifications were RNA specific as demon- effects is not fully understood. strated by no-reverse-transcriptase negative controls. In the present study, we explore the role of the cAMP-PKA signaling pathway in modulating lymphocyte expression of CXCR4 internalization

CXCR4, the receptor for the chemokine SDF-1 (19, 23). In con- Receptor internalization was measured by retention of anti-CXCR4 Ab http://www.jimmunol.org/ trast to most other chemokine systems, which recruit cells to sites binding following elimination of cell surface-bound Ab via acid wash (24). of inflammation, the SDF-1/CXCR4 system is believed to localize Total cell-associated Ab binding reflects both receptors expressed at the cells to lymphoid organs (43–45). Consistent with this function, cell surface and those subsequently internalized into endosomal compart- ments. When surface-bound Ab is removed by acid wash, residual Ab CXCR4 is richly expressed on hemopoietic progenitor cells and binding reflects internalized receptors only (24). Removal of surface-bound immature thymocytes, on fully differentiated but antigenically na- Ab was confirmed by establishing that acid washing abrogated both anti- ive B and T lymphocytes, and on and cultured dendritic CD4 Ab binding at 37°C (CD4 does not internalize under normal circum- cells (5, 26, 46). Lymphocyte CXCR4 expression is down-regu- stances; Ref 48) and anti-CXCR4 Ab binding on cells maintained at 4°C to inhibit internalization (see Results). Receptor internalization was quantified lated during cellular activation (24), and CXCR4 is virtually absent

as the fraction of total anti-CXCR4 fluorescence intensity (pH 7 wash) by guest on September 27, 2021 from NK cells, , , and freshly isolated Lang- retained following acid wash (pH 3). Fluorescence intensity was measured erhans cells (5, 21, 47). Here, we examine the role of cAMP in by FACS analysis gated to exclude dead cells and debris on the basis of regulating CXCR4 expression on both activated and unstimulated forward- vs side-scatter profiles. We used t tests to assess the statistical PBMC. CD4ϩ cells represent a special focus of attention due to significance of differences in receptor internalization. their potential vulnerability to HIV infection. In addition, we con- CXCR4 compartmentalization sider the functional implications of cAMP regulation of CXCR4 for ligand-induced internalization, SDF-1-induced chemotaxis, The distribution of CXCR4 in intracellular vs cell surface compartments was assessed by flow cytometric quantitation of anti-CXCR4 Ab binding and vulnerability to infection with CXCR4-tropic HIV-1. following permeabilization of fixed cells (measuring both intracellular and extracellular binding) vs fixation in the absence of permeabilization (mea- Materials and Methods suring extracellular binding only). PBMC (106) were surface stained with Cell culture and activation anti-CD4 Ab according to the manufacturer’s protocol, fixed by suspension in 1 ml 4% paraformaldehyde, pelleted, and resuspended in 1 ml of saponin Healthy donor PBMC were isolated by Ficoll density gradient and cultured buffer (0.1% w/v saponin plus 0.05% azide in HBSS) for permeabilization. at 3 ϫ 105/ml in RPMI 1640 supplemented with 10% (v) human AB serum, Unpermeabilized cells were suspended in 1 ml HBSS. All cells were then 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM glutamine, at pelleted, 800 ␮l of suspension solution was aspirated, and 10 ␮l of anti-

37°C in an atmosphere of 5% CO2. PKA-activating ligands were added CXCR4 Ab was added. Following 30 min of incubation at room temper- once, at the beginning of culture, and included the membrane-permeable ature, cells were washed once in 1.5 ml of either saponin buffer (perme- cAMP analogue, N6,2Ј-O-dibutyryl adenosine-3Ј,5Ј-cyclic monophosphate abilized cells) or HBSS (unpermeabilized cells), pelleted, and resuspended (dbcAMP), the adenylyl cyclase activator Forskolin, and the physiologic in PBS for flow cytometry. Fluorescence intensity was measured by FACS cAMP inducers PGE2, histamine (H), adrenocorticotropic hormone analysis gated to exclude dead cells and debris on the basis of forward- vs (ACTH), epinephrine (E), and norepinephrine (NE) (all from Sigma, St. side-scatter profiles. Intracellular CXCR4 compartmentalization was quan- Louis, MO). Activated cells were costimulated with Abs to CD3 (0.1 tified by subtracting the mean fluorescence intensity of anti-CXCR4 Ab ␮g/ml adhered to flask by goat anti-mouse Ab; Southern Biotechnology, binding on unpermeabilized cells (extracellular CXCR4) from that of per- Birmingham, AL) and CD28 (0.1 ␮g/ml soluble; Biodesign, Kennebunk- meabilized cells (intracellular plus extracellular CXCR4). Statistical sig- port, ME) at the beginning of culture. CD4ϩ T cell cultures were estab- nificance of differences in intracellular compartmentalization was assessed lished by isolating cells on negative selection columns (R&D Systems, by t test. Nonspecific Ab binding was assessed by parallel staining with an Minneapolis, MN) and culturing them under conditions described above. isotype-matched phycoerythrin-conjugated anti-keyhole limpet hemocya- nin (KLH) Ab and did not differ across cAMP-treated and -untreated cells. CXCR4 cell surface expression Analyses subtracting nonspecific fluorescence intensity from anti-CXCR4 fluorescence intensity produced similar results. Cell surface expression of CXCR4 was quantified by flow cytometry using the phycoerythrin-conjugated 12G5 mAb (PharMingen, San Diego, CA). Ligand-induced down-regulation CD4ϩ, CD8ϩ, CD14ϩ, and CD19ϩ subsets were identified by FITC- and PerCP-conjugated mAb binding (Becton Dickinson Immunocytometry, CXCR4 down-regulation in response to exogenous ligand was assessed by Mountain View, CA). Ab staining was conducted according to the manu- flow cytometric quantification of cell surface CXCR4 expression on co- facturer’s protocol, and all flow cytometric data were acquired using a stimulated PBMC (as described above) at 5 and 30 min after addition of 1394 cAMP UP-REGULATES CELL SURFACE CXCR4

100 ng/ml SDF-1␣ (R&D Systems). Effects of ligand-induced internaliza- sion spontaneously increased over the first 12 h of culture and tion were quantified by expressing CXCR4 mean fluorescence intensity on subsequently remained stable for up to 3 days (Fig. 1). Equivalent ␣ SDF-1 -treated cells as a fraction of CXCR4 mean fluorescence intensity culture in the presence of 100–300 ␮M dbcAMP significantly in- on untreated cells. Recirculation of CXCR4 to the cell surface following SDF-1␣-induced creased CXCR4 cell surface expression by 12 h, with CXCR4 internalization was assessed by flow cytometric quantification of CXCR4 levels declining gradually thereafter but remaining significantly expression on costimulated PBMC at 0, 20 and 60 min after washing SDF- elevated above those of untreated cells (Fig. 1). dbcAMP treatment 1␣-treated cells twice in PBS and resuspending them in fresh culture me- did not significantly affect CXCR4 expression levels during the dium. Surface expression recovery rates were estimated by linear regres- sion of CXCR4 mean fluorescence intensity vs time since SDF-1 washout, first6hofculture as receptor expression spontaneously increased and recovery rates on costimulated cells and cells costimulated in the pres- on both untreated and dbcAMP-treated cells (data not shown). Pro- ence of dbcAMP were compared by t test. nounced effects of dbcAMP on CXCR4 expression were observed Chemotaxis only once CXCR4 levels reached a steady state (between 6 and 12 h of culture), suggesting that cAMP regulates the constitutive ␣ Chemotactic response to SDF-1 was measured by a membrane transmi- receptor set point rather than the receptor generation process itself. gration assay in which 5 ϫ 105 PBMC in 100 ␮l medium (RPMI ϩ 0.25% BSA) were loaded into the upper chamber of a Transwell insert with a cAMP up-regulation of CXCR4 was dose dependent (Fig. 1, Table 5-␮m pore size polycarbonate membrane separating cells from a lower I), and cAMP-inducing ligands produced similar effects, with 100 chamber containing graded concentrations of SDF-1␣. The number of cells ␮M Forskolin increasing CXCR4 cell surface expression by up to transmigrating during2hofincubation was quantified by FACS (gating sixfold at 24 h (Table I). CXCR4 expression was also increased by out dead cells and debris on the basis of forward- and side-scatter profiles), a variety of physiologic cAMP-inducing ligands, including PGE , and SDF-1␣-induced chemotaxis was calculated by subtracting back- 2 Downloaded from ground transmigration (0 ng/ml SDF-1␣) and standardizing relative to a histamine, ACTH, and the catecholamines epinephrine and NE ϩ ϩ 500-␮l sample containing 10% of the input cell number. SDF-1␣-induced (Table I). Similar dynamics were observed in CD4 , CD8 , and chemokinesis (ambidirectional motility) was assessed by transmigration CD19ϩ subsets (Fig. 1) although with differing kinetics in each ␣ ϩ rates in the presence of 300 ng/ml SDF-1 in both upper and lower cham- subset. On CD4 cells, cAMP-induced CXCR4 expression peaked bers (45). Statistical significance of differential chemotaxis rates was at 12 h and declined gradually thereafter, whereas peak expression assessed by t test and comparison of dose-response curves by linear ϩ ϩ regression. on CD8 and CD19 cells occurred at 24 h, followed by a rapid

decline toward baseline levels (Fig. 1). At all time points observed, http://www.jimmunol.org/ HIV-1 infection and expression CD19ϩ cells showed significantly greater levels of CXCR4 ex- ϩ ϩ Healthy donor PBMC were infected with CXCR4-tropic HIV-1NL4–3 (49) pression than did CD4 or CD8 cells (all p Ͻ 0.0001). In con- ␮ (0.05 infectious units per cell) for 30 min in the presence of 10 g/ml trast to cAMP effects on lymphocyte CXCR4, cAMP significantly polybrene. Following infection, cells were washed twice and costimulated ϩ as described above. To prevent viral spread, postinfection medium in- suppressed CXCR4 expression on CD14 cells (Fig. 1). cAMP did cluded the HIV protease inhibitor, Indinavir (100 nM; Merck, Rahway, not significantly alter cell surface CD4 expression at any time NJ). To determine whether effects of cAMP were mediated specifically by point examined (data not shown). altered CXCR4 expression, cells were preincubated for1hinthepresence Following CD3/CD28 costimulation, PBMC cell surface ex- of 1 mM recombinant SDF-1␣ to block interactions between HIV-1 and pression of CXCR4 was suppressed by approximately 70% rela-

CXCR4 (18). Equivalent preincubation in the presence of 1 mM recom- by guest on September 27, 2021 binant MIP-1␣ (R&D Systems) served as a nonblocking control. tive to unstimulated PBMC (Fig. 1) ( p Ͻ 0.001 at each time point). The number of infected cells was quantified by PCR analysis of proviral Equivalent costimulation in the presence of dbcAMP significantly DNA at 14 h post infection (AA55 and M667 primer pair specific for the up-regulated CXCR4, with peak expression levels significantly ex- R/U5 region of the viral long terminal repeat, quantified relative to input ␤ ceeding those of both costimulated PBMC and unstimulated cell number as indicated by -globin primers LA1 and LA2) (50). Briefly, Ͻ one primer of each pair was radio end labeled, and amplification was con- PBMC in the absence of cAMP ( p 0.0001) (Fig. 1). Elevated ducted for 25 cycles in parallel with control standards consisting of lin- CXCR4 cell surface expression was observed by 12 h of costimu- earized cloned HIV-1 DNA and known quantities of cellular DNA. Ra- lation, peaked at 24 h, and declined gradually back toward un- diolabeled amplified products were resolved on a 6% polyacrylamide gel stimulated levels at 48 and 72 h (Fig. 1). As with unstimulated and quantified by radioanalytic image analysis in comparison with standard curves. To ensure that viral spread did not occur, proviral DNA was also cells, cAMP-inducing ligands also up-regulated CXCR4 expres- quantified at 48 h postinfection. In no case did the fraction of provirus- sion on costimulated PBMC (Table I), with 100 ␮M Forskolin bearing cells at 48 h exceed that at 14 h postinfection. increasing CXCR4 levels by 10-fold. Similar CXCR4 expression HIV-1 gene expression was quantified by flow cytometric detection of a kinetics were observed for CD4ϩ, CD8ϩ, and CD19ϩ cells, with reporter gene product from a genetically altered HIV-1 . ϩ NL4–3 CXCR4 levels on CD19 cells significantly exceeding those on HIV-1 was created by cloning the murine heat-stable Ag (HSA) NL-r-HSAS CD4ϩ and CD8ϩ cells (Fig. 1). In the absence of cAMP, CXCR4 gene into a vacancy created by deleting nucleotides 5625 through 5742 in ϩ the vpr gene of HIV-1NL4–3 (51). Upon transcription and translation of expression on CD19 cells from costimulated cultures was signif- ϩ HIV-1NL-r-HSAS, murine HSA is expressed on the cell surface and can be icantly suppressed relative to CD19 cells from unstimulated cul- quantified by flow cytometric assessment of anti-murine CD24 Ab binding tures, implying some degree of cross regulation by T cell activa- (FITC-conjugated M1/69; PharMingen). This virus is pathogenic in vivo in ϩ SCIDhu mice, and HSA reporter gene expression correlates closely with tion (since CD19 cells should not be directly activated by CD3/ other means of verifying HIV infection, including PCR assessment of pro- CD28 costimulation). As with unstimulated cells, cAMP ϩ viral DNA and flow cytometric quantitation of intracellular p24 (51). significantly suppressed CXCR4 expression on CD14 cells (Fig. 1) and did not significantly alter CD4 expression at any time point Results examined (data not shown). cAMP up-regulates CXCR4 expression on both resting and activated lymphocytes cAMP does not alter CXCR4 gene expression Activation of the lymphocyte cAMP-PKA signaling pathway al- To determine whether cAMP-induced up-regulation of cell surface ters lymphocyte traffic and localization (33, 34, 42) and increases CXCR4 was mediated by increased gene expression, receptor HIV replication (52–54), although the mechanisms underlying mRNA levels were quantified by RT-PCR following 20 h of cul- such effects remain incompletely understood. To determine ture (preceding the period of peak difference in receptor expres- whether altered expression of chemokine receptors might play a sion). dbcAMP failed to significantly increase CXCR4 mRNA lev- role, we examined the effect of cAMP in regulating cell surface els in either resting or costimulated PBMC (Fig. 2). Across five expression of CXCR4. On unstimulated PBMC, CXCR4 expres- experiments, the average CXCR4 mRNA level in cAMP-treated The Journal of Immunology 1395

Table I. Effects of cAMP and cAMP-inducing agents on CXCR4 expression on PBMCa

CD3/CD28 Agent Unstimulated Costimulated

db-cAMP dose-response 100 ␮M 191% (1%) 487% (3%) 200 ␮M 302% (1%) 729% (13%) 300 ␮M 347% (1%) 826% (1%) cAMP-inducing agents Forskolin (10Ϫ4 M) 607% (3%) 1001% (10%) Ϫ4 Prostaglandin E2 (10 M) 198% (2%) 235% (2%) Norepinephrine (10Ϫ5 M) 151% (1%) 196% (5%) Epinephrine (10Ϫ5 M) 145% (1%) 159% (3%) Histamine (10Ϫ5 M) 184% (2%) 152% (4%) ACTH (10Ϫ4 M) NDb 155% (2%)

a Data represent mean (SE) percentage increase in CXCR4 fluorescence intensity relative to no-cAMP controls following 24 h of culture (three experiments). All values differ significantly from control value of 100% (p Ͻ .0001). b ND, not done. Downloaded from surface expression is not mediated by increased CXCR4 gene expression.

cAMP reduces CXCR4 internalization To determine whether altered receptor trafficking might play a role

in cAMP up-regulation of CXCR4 cell surface expression, recep- http://www.jimmunol.org/ tor internalization rates were quantified by flow cytometric mea- surement of internalized anti-CXCR4 Ab following removal of surface-bound Ab by acid washing. Residual Ab binding reflects internalized receptors not exposed to extracellular low pH (24). Low pH washing efficiently removed cell surface-bound Ab, as demonstrated by the abrogation of anti-CD4 Ab binding (Fig. 3A) and the abrogation of anti-CXCR4 Ab binding in cells incubated at 4°C to prevent internalization (Fig. 3B). Under normal incubation conditions (37°C), acid-resistant anti-CXCR4 Ab binding aver- by guest on September 27, 2021 aged 20% of total CXCR4 fluorescence intensity at 60 min (Fig. 3, C and D). Parallel incubation in the presence of dbcAMP de- creased acid-resistant anti-CXCR4 Ab binding by 39%, to an av- erage of 12% of total fluorescence intensity at 60 min ( p Ͻ 0.001). In contrast, dbcAMP did not significantly alter acid-resistant anti- CD4 binding (Fig. 3E), indicating a specific effect of cAMP on CXCR4 internalization. Similar effects were observed in isolated CD4ϩ T cell cultures, with dbcAMP decreasing acid stable anti- CXCR4 binding by an average of 44% (data not shown). Thus cAMP appears to up-regulate cell surface expression of CXCR4 in part by reducing receptor internalization rates. Consistent with reduced internalization, flow cytometric analy- sis of CXCR4 compartmentalization indicated that cAMP de- FIGURE 1. Effect of cAMP on CXCR4 expression. Unstimulated creased the fraction of CXCR4 in intracellular compartments. In- PBMC (left column) or CD3/CD28-costimulated PBMC (right column) tracellular CXCR4 constituted an average of 38% of total CXCR4 were cultured for 3 days in the presence of 0 (open squares), 100 (closed fluorescence intensity on costimulated PBMC, with 100 ␮M circles), or 300 ␮M dbcAMP (closed squares). Data represent the mean dbcAMP reducing this value by more than 50%, to an average of fluorescence intensity of anti-CXCR4 Ab binding as measured by flow 18% across four experiments ( p ϭ 0.004). dbcAMP did not sig- cytometry on total PBMC and CD4ϩ, CD8ϩ, CD19ϩ, and CD14ϩ subsets. Ϯ nificantly affect total CXCR4 fluorescence intensity (intracellular Each data point represents the mean ( SE) of three separate experiments ϭ Ϯ Ϯ (SE falls within the plotting symbol for data points lacking error bars). plus extracellular; mean 736 330 vs 822 216 fluorescence Dose-response relationships were assessed by linear regression with sta- units for costimulated PBMC vs PBMC costimulated in the pres- p Ͻ 0.01; ence of 100 ␮M dbcAMP; p ϭ ns), suggesting that cAMP alters ,ءء ;p Ͻ 0.05 ,ء .tistical significance at each time point indicated p Ͻ 0.0001. CXCR4 cell surface expression primarily by altering receptor ,ءءءء ;p Ͻ 0.001 ,ءءء compartmentalization rather than by increasing the size of the total CXCR4 receptor pool. Consistent with this mechanism, cAMP- induced down-regulation of CXCR4 on CD14ϩ cells was accom- cells differed from that of untreated cells by less than 5%. More- panied by a significant increase in the fraction of CXCR4 in in- over, dbcAMP-induced up-regulation of CXCR4 was not abro- tracellular compartments (mean ϭ 31% in costimulated cultures vs gated by the protein synthesis inhibitor cyclohexamide (data not 80% in cultures costimulated in the presence of dbcAMP, p ϭ shown), confirming that cAMP up-regulation of CXCR4 cell 0.049) without any change in total CXCR4 fluorescence intensity. 1396 cAMP UP-REGULATES CELL SURFACE CXCR4

Ligand-induced CXCR4 suppression is rapidly reversed upon removal of SDF-1, suggesting that such effects are mediated by internalization into endosomal compartments and subsequent re- circulation to the cell surface (31). To determine whether cAMP influences CXCR4 reexternalization rates, CXCR4 cell surface ex- pression was monitored on cells treated with 100 ng/ml SDF-1␣ for 1 h and then washed twice and resuspended in fresh medium. Following suppression to 57% of pre-SDF-1 levels, CXCR4 ex- pression levels on costimulated PBMC recovered by an average of 0.34% per minute during the first 60 min following SDF-1 re- moval. In cells costimulated in the presence of dbcAMP, CXCR4 recovery rates increased to 0.67% per minute ( p ϭ 0.029). Thus FIGURE 2. Effect of cAMP on CXCR4 mRNA expression. RT-PCR cAMP up-regulation of cell surface CXCR4 can influence both was used to quantify CXCR4 mRNA expression in unstimulated and CD3/ ligand-induced receptor internalization and reexternalization fol- CD28 costimulated PBMC cultured for 20 h in the presence of 0 or 100 lowing ligand removal. ␮M dbcAMP. Parallel determination of ␤-actin mRNA verified equivalent mRNA loading. Samples were quantified by radioanalytic image analysis cAMP-induced up-regulation of CXCR4 increases in comparison with dilution-titrated PBMC mRNA standards. Across five SDF-1␣-induced chemotaxis

experiments, CXCR4 mRNA levels in dbcAMP-treated cultures differed Downloaded from by less than 5% from those of untreated cultures. To explore the functional consequences of cAMP-induced up-reg- ulation of CXCR4, lymphocyte chemotactic response to SDF-1␣ was assessed in membrane transmigration assays. In PBMC co- stimulated for 24 h, SDF-1␣ produced dose-dependent increases in cAMP-induced CXCR4 up-regulation counteracts ligand-induced lymphocyte transmigration, with the fraction of transmigrating down-regulation cells increasing by 5.8% with each 100 ng/ml increase in SDF-1 CXCR4 undergoes rapid internalization after binding its natural concentration (Fig. 4). Costimulation in the presence of 100 ␮M http://www.jimmunol.org/ ligand, SDF-1 (18, 31). To determine whether cAMP-induced up- dbcAMP increased transmigration rates by approximately three- regulation of CXCR4 cell surface expression can offset ligand- fold (to 15.8% per 100 ng/ml, p Ͻ 0.0001) (Fig. 4). More pro- induced down-regulation, 100 ng/ml SDF-1␣ was added to PBMC nounced effects were observed using the adenylyl cyclase activator costimulated for 24 h in either the presence or absence of dbcAMP. Forskolin (100 ␮M), which increased transmigration rates by five- As shown in Table II, 100 ng/ml SDF-1␣ suppressed CXCR4 cell fold (28.6% per 100 ng/ml, p Ͻ 0.0001; data not shown). Physi- surface expression within 5 min, and this suppression persisted for ologic cAMP-inducing ligands also increased SDF-1␣-induced ϭ ␮ ␮ at least 30 min ( p 0.007). dbcAMP (10 M) (Table II) offset the chemotaxis, with 10 MNEorPGE2 each approximately dou- ϭ effects of SDF-1 and restored cell surface CXCR4 expression to bling transmigration rates ( p 0.004). by guest on September 27, 2021 levels that did not differ significantly from those observed on cells To distinguish cAMP’s effects on migration along a chemokine costimulated in the absence of SDF-1 ( p ϭ 0.277). dbcAMP (100 gradient (chemotaxis) from chemokine-induced alterations in ran- ␮M) (Table II) counteracted ligand-induced internalization effects dom motility (chemokinesis), transmigration rates were assessed in and elevated CXCR4 expression to levels significantly greater than the presence of 300 ng/ml SDF-1␣ on both sides of the membrane. those observed on costimulated PBMC (untreated with either PBMC showed minimal chemokinetic response to SDF-1␣ (trans- SDF-1 or dbcAMP; p ϭ 0.020). Thus cAMP up-regulation of migration rates differed from those of no-SDF controls by less than CXCR4 counteracts ligand-induced CXCR4 suppression. 5%), and dbcAMP did not significantly alter these effects (data not

FIGURE 3. Effect of cAMP on CXCR4 internalization. CXCR4 internalization was quantified by flow cytometric assessment of anti-CXCR4 Ab binding following acid stripping of cell surface-bound Ab. To assure that acid washing effectively stripped surface-bound Abs, anti-CD4 Ab binding was measured following wash at pH 7 (A, top)orpH3(A,bottom) (CD4 internalization is minimal on normal PBMC). To confirm the effect of acid washing on anti-CXCR4 surface binding, cells were maintained at 4°C to inhibit receptor internalization for 60 min before wash at pH 7 (B, top)orpH3(B,bottom). To quantify CXCR4 internalization during 37°C incubation, acid-resistant Ab binding (internalized) was expressed as a fraction of total Ab binding (internal plus external) (C) over 1–60 min of incubation. dbcAMP (100 ␮M) suppressed CXCR4 60-min internalization rates by an average of 39% across four experiments (p ϭ 0.001) (D) (SE falls within the plotting symbol for data points lacking error bars). In contrast, CD4 internalization rates were not significantly affected (E). Data represent the mean (SE) across four experiments, with statistical significance of comparisons at specific time points indicated. .p Ͻ 0.0001 ,ءءءء ;p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء The Journal of Immunology 1397

Table II. Effect of cAMP on SDF-1-induced CXCR4 down-regulationa

Time SDF-1 db-cAMP (ng/ml) (␮M) 5 min 30 min

0 0 145 (100%) 156 (100%) 100 0 104 (71%) 88 (56%) 100 10 140 (97%) 134 (86%) 100 100 445 (307%) 314 (201%)

a Data represent mean fluorescence intensity of anti-CXCR4 Ab binding to CD3/ CD28-costimulated PBMC at indicated times following resuspension of cells in fresh medium containing indicated concentrations of SDF-1␣. Parenthesized values repre- sent CXCR4 fluorescence relative to costimulated cells incubated in the absence of SDF-1␣.

␣ shown). Thus cAMP up-regulation of SDF-1 -induced chemo- FIGURE 4. Effect of cAMP on SDF-1␣-induced chemotaxis. Chemo- taxis was not mediated by increased random mobility. taxis was measured by 2-h transmigration across a 5-␮m transwell mem- brane separating PBMC from media including the indicated concentrations cAMP-induced up-regulation of CXCR4 increases vulnerability ␣ ϩ of recombinant SDF-1 . Transmigrating cells were quantified by flow cy- Downloaded from of CD4 cells to HIV-1 infection tometry. Regression analysis of dose-response curves indicated that 100 Ϫ1 PCR was used to quantify reverse-transcribed proviral DNA at ␮M dbcAMP increased transmigration rates from 0.058% (ng SDF-1␣) ␣ Ϫ1 Ͻ 14 h following infection by CXCR4-tropic HIV-1 . When to 0.158% (ng SDF-1 ) across five experiments (p 0.0001). Statistical Ͻ ء NL4–3 PBMC were infected following 24 h of costimulation, an average significance of comparisons at specific time points is indicated. , p 0.05; .p Ͻ 0.0001 ,ءءءء ;p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء of 4% bore proviral DNA 14 h later (Fig. 5A). Costimulation in the presence of dbcAMP increased proviral penetrance by approxi- http://www.jimmunol.org/ mately eightfold (Fig. 5A). These cAMP-induced increases in HIV-1 infectivity were mediated specifically by altered CXCR4 that cAMP-PKA signaling can increase cell surface expression of expression, as demonstrated by the fact that cAMP effects on pro- the CXC chemokine receptor CXCR4 by 6- to 10-fold on unstimu- viral penetrance could be blocked by preincubating cells in the lated and CD3/CD28-costimulated lymphocytes. These effects are presence of the CXCR4-specific ligand SDF-1␣ (1 mM), but not mediated primarily by cAMP-induced alterations in the balance of by parallel preincubation in the presence of the CCR5 ligand CXCR4 expressed in intracellular vs extracellular compartments. MIP-1␣ (1 mM; see Fig. 5A). Thus cAMP-induced up-regulation We also show that cAMP up-regulation of CXCR4 can offset the of CXCR4 expression increases cellular vulnerability to infection effects of ligand-induced internalization and accelerate recovery of by CXCR4-tropic HIV-1. by guest on September 27, 2021 cell surface CXCR4 expression following release from ligand-in- Studies using the HIV-1 reporter virus (murine HSA NL-r-HSAS duced suppression. Functional results of cAMP-induced up-regu- gene cloned into a vpr-deleted HIV-1 ; Ref 51) showed that NL4–3 lation of CXCR4 include increased chemotaxis in response to cAMP-induced increases in proviral penetrance were accompanied SDF-1␣ and increased vulnerability to infection by CXCR4-tropic by an increase in the fraction of cells expressing viral at 48 h ϩ HIV-1. cAMP up-regulates CXCR4 expression on both CD4 and postinfection (Fig. 5B). Similar effects were observed using phys- ϩ ϩ CD8 T lymphocytes as well as on CD19 B cells. In contrast to iologic inducers of cAMP (e.g., 10 ␮M NE increased HIV-1 gene these effects on lymphocytes, cAMP suppresses CXCR4 expres- expression to 230% of costimulated control levels, p ϭ 0.022). To ϩ sion on CD14 cells by promoting CXCR4 internalization. These ensure that cAMP-induced increases in HIV-1 gene expression did data imply that the diverse family of extracellular factors that ac- not reflect enhanced viral spread, cells were cultured in the pres- tivate the cAMP-PKA signaling pathway may function as natural ence of an HIV-1 protease inhibitor (100 nM Indinavir). PCR anal- modulators of leukocyte traffic and localization and incidentally ysis of proviral load at 48 h postinfection confirmed that antiret- ϩ render CD4 T cells more vulnerable to infection with CXCR4- roviral treatment prevented spread of provirus beyond cells tropic HIV-1. Because SDF-1 also regulates aspects of lympho- infected by 14 h (data not shown). Thus cAMP-induced up-regu- poiesis and tissue development (55, 56), cAMP regulation of lation of cell surface CXCR4 expression renders T lymphocytes CXCR4 expression may have additional implications for cellular more vulnerable to productive infection by CXCR-4-tropic HIV-1. maturation and morphogenesis. Kinetic analysis of HSA expression as a function of CXCR4 The present results suggest that cAMP may modulate lympho- density indicated that HSA initially appeared on cells expressing cyte circulation patterns by altering expression of chemokine re- high levels of CXCR4 (mean fluorescence intensity Ͼ 1000; data ceptors. Such results are consistent with evidence that cAMP-in- not shown). As HSA intensity increased over time, CXCR4 levels ducing ligands can selectively mobilize specific lymphocyte declined, with average expression dropping below 100 fluores- subsets into circulation while retaining other subsets in lymphoid cence intensity units on cells showing maximal HSA expression organs (33, 34, 42). cAMP-inducing ligands are distributed both (Fig. 5B). Such results may arise from several possible mecha- systematically via the endocrine system and locally via paracrine nisms, including down-regulation of CXCR4 expression by HIV secretion from cells of the nervous and immune systems. Potential gene expression and differential survival of cells expressing high paracrine activators of the lymphocyte cAMP pathway in vivo in- vs low levels of CXCR4. clude PGs and histamine secreted by myeloid cells at sites of in- flammation (57, 58), catecholamines secreted by sympathetic ner- Discussion vous system neurons terminating in lymphoid organ parenchymal Activation of the cAMP-PKA signaling pathway alters lympho- tissues (59), and ACTH secreted by activated lymphocytes (60). cyte traffic and localization (33, 34, 42), but the mechanisms un- Signaling via the cAMP-PKA pathway may interact with other derlying such effects remain poorly understood. Here, we show CXCR4-modulators to modify cellular localization in response to 1398 cAMP UP-REGULATES CELL SURFACE CXCR4

FIGURE 5. Effect of cAMP on HIV-1 infection and gene expression. PBMC were infected with HIV-

1NL4–3 (0.05 multiplicity of infection) following 24 h of CD3/CD28 costimulation in the presence of 0 or Downloaded from 100 ␮M dbcAMP. HIV-1 proviral DNA was assessed by PCR at 14 h following infection and quantified by radioanalytic image analysis in comparison to ␤ glo- bin DNA (A). To demonstrate the role of CXCR4 ex- pression in mediating cAMP effects on infectivity, 1 h preincubation in 1 mM SDF-1␣ was used to block

interactions between HIV-1 and CXCR4. Similar pre- http://www.jimmunol.org/ incubation in the presence of 1 mM MIP-1␣ served as a nonblocking control. HIV-1 gene expression was measured by flow cytometry for the murine HSA re- porter gene cloned into a vpr-deleted HIV-1NL4–3 (HIV-1NL-r-HSAS)(B). Reporter gene expression was assessed by anti-HSA (CD24) Ab binding following two days of CD3/CD28 costimulation in the presence of 100 nM Indinavir. by guest on September 27, 2021

antigenic signals. For example, CXCR4 expression on T lympho- Such effects may have significant clinical implications since the cytes typically declines following cellular activation (Refs. 24 and emergence of CXCR4-tropic HIV strains is associated with pro- 31, and Fig. 1), and this effect is particularly pronounced for gression from chronic infection to the development of life-threat- CD45RAϩ (naive) cells. This regulatory pathway may facilitate ening illness (61, 62). Several cAMP-inducing mediators are in- migration of activated cells out of lymphoid organs and into pe- creased during HIV infection (e.g., PGE; Ref. 63), and exogenous ripheral tissue sites (5). However, cAMP-inducing ligands may HIV-1 proteins can increase intracellular cAMP levels (64, 65). counteract this effect and thus promote retention of activated lym- Such effects could conceivably promote disease progression by phocytes in areas of high SDF-1 concentration. increasing T lymphocyte vulnerability to infection and thereby fa- CXCR4 plays an important role in HIV pathogenesis by func- cilitating replication of CXCR4-tropic viral strains. Such a dy- tioning in conjunction with CD4 as a coreceptor for virulent syn- namic may undermine the use of cAMP-inducing agents to sup- cytium-inducing viral strains (9). Activation of the lymphocyte press chemokine receptors on myeloid cells (as demonstrated cAMP-PKA signaling pathway can accelerate HIV-1 replication above) as an antiviral strategy (66). (52–54). The present data suggest that cAMP effects on HIV rep- The present data indicate that cAMP up-regulates lymphocyte lication may be mediated in part by increased cellular vulnerability CXCR4 expression primarily by altering receptor compartmental- to HIV infection as a function of up-regulated CXCR4 expression. ization. The negligible effects of cAMP on total CXCR4 pool size The Journal of Immunology 1399 and receptor gene expression are consistent with the absence of 19. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, and any known cAMP response element in the CXCR4 promoter (67, T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for Lestr/fusin and blocks HIV-1 entry. Nature 382:829. 68). Although cAMP up-regulation of cell surface CXCR4 is me- 20. Cocci, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, and diated by altered receptor trafficking, the molecular mechanism of P. Lusso. 1995. Identification of RANTES, MIP-1␣, and MIP-1␤ as the major HIV suppressive factors produced by CD8ϩ T cells. Science 270:1811. these effects remains to be clarified. Activated PKA may directly 21. Hori, T., H. Sakaida, A. Sato, G. Nakajima, H. Shida, O. Yoshie, and phosphorylate the receptor system itself (as in other receptor fam- T. Uchiyama. 1998. Detection and delineation of CXCR-4 (Fusin) as an entry and ilies; Ref. 30), or it may interact with other signaling pathways that fusion cofactor for T cell-tropic HIV-1 by three different monoclonal antibodies. J. Immunol. 160:180. influence CXCR4 compartmentalization (69). Another outstanding 22. McKnight, A., D. Wilkinson, G. Simmons, S. Talbot, L. Picard, M. Ahuja, question regards the teleologic rationale for receptor regulation by M. Marsh, J. A. Hoxie, and P. R. Clapham. 1997. Inhibition of human immu- cAMP-inducing ligands. Many cAMP-inducing factors exert sig- nodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent. J. Virol. 71:1692. nificant effects over lymphocyte function (e.g., PGs, cat- 23. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, echolamines, ACTH), but their physiologic roles remain poorly F. Arenzana-Seisdedos, O. Schwartz, J. M. Heard, I. Clark-Lewis, D. F. Legler, M. Loetscher, M. Baggiolini, and B. Moser. 1996. The CXC chemokine SDF-1 understood. A more comprehensive understanding of both the is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted sources and function of CXCR4 ligands may be helpful in eluci- HIV-1. Nature 382:833. dating the role of cAMP in the immune response more generally (1). 24. Signoret, N., J. Oldridge, A. Pelchen-Matthews, P. J. Klasse, T. Tran, L. F. Brass, M. M. Rosenkilde, T. W. Schwartz, W. Holmes, W. Dallas, M. A. Luther, T. N. C. Wells, J. A. 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