b-Agonist enhances type 2 T-cellsurvival and accumulation

Matthew J. Loza, PhD, Stephen P. Peters, MD, PhD, Susan Foster, PhD,

Islam U. Khan, PhD, and Raymond B. Penn, PhD Winston-Salem, NC

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Background: Neurohumoral modulation of immune system

function is poorly understood. b-Adrenergic receptor ligands

(b-agonists) subserve numerous physiologic processes but also

function as pathogenic or therapeutic agents in numerous

diseases with inflammatory components.

Objectives: We sought to establish the effects of b-agonists and

prostaglandin E2 (PGE2) on antigen-dependent and antigen-

independent accumulation of IL-131 (type 2) and IFN-g1 (type

1) T cells. We also sought to clarify the mechanisms mediating

the effects of these G protein–coupled receptor agonists.

Methods: Effects of b-agonists or PGE2 on T-cell subtype

accumulation were assessed in peripheral blood lymphocytes

cultured with aCD3/CD28 or IL-2 by using flow cytometry.

The role of cyclic AMP-dependent protein kinase (PKA) in

mediating agonist effects was assessed by means of

characterization of (1) phosphorylation of an intracellular

PKA substrate and (2) T cells from patients with lupus

possessing a natural defect in PKA activation.

Results: b-Agonists, in contrast to PGE2, increased IL-2–

induced accumulation of human type 2 T cells, an effect

attributable to differential activation of PKA affecting

regulation of cell proliferation and apoptosis. In T cells from

patients with lupus exhibiting defective PKA activation, both

b-agonists and PGE2 promoted an increase in type 2 T-cell

accumulation.

Conclusion: Gs-coupled receptors have the capacity to elicit

prosurvival signaling in type 2 T cells, which, in most instances,

is obscured by concomitant and antimitogenic PKA activation.

Clinical implications: b-Agonists and other Gs-coupled receptor

agonists have the potential to regulate T-cell development to

affect disease pathogenesis or the efficacy of therapies, and

variability of effect relates to the ability to stimulate PKA

activity. (J Allergy Clin Immunol 2007;119:235-44.)

Key words: Human, T cells, apoptosis, cytokines, signal transduc-tion, b-adrenergic receptor, prostaglandin E2, EP receptor, lupus,

asthma, heart failure, inflammation, cyclic AMP protein-dependent

kinase

From the Department of Internal Medicine, Center for Human Genomics,

Wake Forest University School of Medicine.

Supported in part by grants from the National Institutes of Health (HL58506 to

R.B.P. and AR39501 to I.U.K.) and the General Clinical Research Center of

the Wake Forest University School of Medicine (M01-RR07122 to I.U.K.).

M.J.L. is the recipient of American Heart Association Beginning Grant-in-

Aid 0665390U.

Disclosure of potential conflict of interest: The authors have declared that they

have no conflict of interest.

Received for publication June 2, 2006; revised September 15, 2006; accepted

for publication September 15, 2006.

Available online October 27, 2006.

Reprint requests: Raymond B. Penn, PhD, Center for Human Genomics,

Wake Forest University School of Medicine, Medical Center Blvd,

Winston-Salem, NC 27157. E-mail: rpenn@wfubmc.edu.

� 2007 American Academy of Allergy, Asthma & Immunology

doi:10.1016/j.jaci.2006.09.019

b-Adrenergic receptors (b-ARs) activated by endoge-nous or exogenous b-agonists regulate numerous cell,tissue, and organ system functions. In the cardiovascularsystem norepinephrine and epinephrine increase cardiaccontractility and relax vascular smooth muscle throughactions on b1- and b2-ARs, respectively. In the lunginhaled b-agonists are the principal therapy for reliefof acute bronchoconstriction in asthmatic subjects, actingon b2-ARs in airway smooth muscle to promote bronchor-elaxation. b-AR–mediated regulation of the immunesystem, however, is poorly understood. Although certaininflammatory cell functions are known to be attenuatedby b-agonists, the overall effects of b-agonists on inflam-matory processes are unclear. In multiple types of heartfailure, in which circulating catecholamine levels areincreased, inflammation plays an important pathogenicrole.1-3 Airway inflammation associated with asthma hasbeen reported to be either unaffected by or slightly in-creased by inhaled b-agonist therapy,4-6 and concerns re-garding disease exacerbations with continuous b-agonistmonotherapy have prompted the recommendation ofconcomitant steroid treatment.7 Few studies to date haveexamined the effects of b-agonists on human T-cellfunctions,8 particularly with respect to their effects onT-cell subtype accumulation.

Herein we report that b-agonists regulate T-cell sub-type accumulation in cultures of human peripheral bloodlymphocytes (PBLs) in a manner that contrasts dramati-cally to that caused by prostaglandin E2 (PGE2), despiteboth agonists acting on G protein–coupled receptors(GPCRs) that stimulate the heterotrimeric G protein Gs

and the downstream effector cAMP-dependent protein

Abbreviations usedAR: Adrenergic receptor

BAD: Bcl-2/bcl-XL antagonist, causing cell death

cAMP: Cyclic AMP

CFSE: Carboxyfluorescein diacetate succimidyl ester

CRTH2: Chemoattractant receptor–homologous molecule

expressed on TH2 cells

EP: Epiprostanoid

Epac: Exchange protein activated by cyclic AMP

GPCR: G protein–coupled receptors

ISO: Isoproterenol

PBL: Peripheral blood lymphocyte

PGE2: Prostaglandin E2

PKA: cAMP-dependent protein kinase

SLE: Systemic lupus erythematosus

VASP: Vasodilator-stimulated phosphoprotein

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kinase (PKA).8 Surprisingly, b-agonists promoted a sig-nificant increase in IL-2–stimulated accumulation oftype 2 T cells. Analyses of cell proliferation andAnnexin V staining indicate that the effects of b-agonistson type 2 cell accumulation are mediated primarily by re-ducing the rate of cell apoptosis. In T cells with defectivePKA activation (isolated from patients with systemic lu-pus erythematosus [SLE]9), both b-agonists and PGE2

promoted an increase in IL-2–stimulated type 2 T-cell ac-cumulation. These findings suggest that Gs-coupled recep-tors have the capacity to elicit prosurvival signaling in Tcells, which, in most instances, is obscured by concomi-tant and antimitogenic PKA activation.

METHODS

Culture and analysis of cytokine production

Venous blood was obtained from healthy human adult subjects

and, when indicated, from adult subjects with SLE (characterized

in Table E1 in the Online Repository at www.jacionline.org)

after informed consent was provided in accordance with a

Wake Forest University School of Medicine Institutional Review

Board–approved protocol and the Helsinki Declaration. None of

the patients with SLE had received cytotoxic drugs for at least 3

months, and steroids and nonsteroidal anti-inflammatory drugs

were stopped for 48 hours before blood drawing. PBLs, isolated by

using standard procedures,10 were cultured in RPMI 1640 media

supplemented with 5% pooled human plasma and either 50 U/mL

IL-2 or CD3 mAb (plate-bound, clone OKT3), CD28 (soluble, clone

9.3) mAb, and 50 U/mL IL-2. IL-12–neutralizing mAb was included

in all conditions to eliminate the confounding effects of variable

levels of IL-12 produced by the few, if any, contaminating mono-

cytes. When indicated, 1 mmol/L (6) isoproterenol (ISO; Sigma, St

Louis, Mo) and 1 mmol/L PGE2 (Cayman Chemicals, Ann Arbor,

Mich) were added at the start of culture only. At the indicated times,

cells were collected, viable cells were counted with an automated cell

counter (ViCell, BeckmanCoulter, Fullerton, Calif), and the remain-

ing cells were washed and then stimulated for cytokine production

(5 hours with 2 nmol/L phorbol 12-myristate 13-acetate plus 0.2

mg/mL calcimycin plus 5 mmol/L monensin). Intracellular IL-13

and IFN-g accumulation by T cells was detected by using immunoflu-

orescence flow cytometry, as previously described.8,11

Average division numbers and minimum progenitor numbers were

calculated for experiments of carboxyfluorescein diacetate succi-

midyl ester (CFSE)–labeled PBLs, as described previously.12-14

Briefly, the average number of divisions that the progenitors of the

population of interest undergo is calculated as follows:

D5 S CFSEn cell% � n =100�Þð½ , where n is defined as the division

number based on discrete CFSE peaks, and CFSEn cell % is defined

as the percentage of cells in the population of interest that are in the

CFSE peak corresponding to ‘‘n’’ divisions. Minimum progenitor

number is calculated by using the following formula:

P5SðCFSEn cell #=2n, where n is defined as the division number

based on discrete CFSE peaks, and CFSEn cell # is defined as the num-

ber of cells in the population of interest that are in the CFSE peak cor-

responding to ‘‘n’’ divisions. Minimum progenitor number (P)

represents the minimal number of cells necessary to produce the total

number of CFSE-labeled cells. Progenitor numbers lower than day 0

numbers indicate loss of progenitors.

Early apoptosis detection

Annexin V–positive cells in the standard viable lymphocyte gate,

determined by means of light-scatter characteristics on the flow

cytometer, represent early apoptotic cells (data not shown). This type

of analysis eliminates the confounding effects of apoptotic bodies

and differential rate of clearance of late apoptotic/necrotic cells. The

proportions of early apoptotic (Annexin V staining) chemoattractant

receptor–homologous molecule expressed on TH2 cells (CRTH2)–

positive (type 2) and CD561 (type 1) T cells were determined by

means of 4-color immunofluorescence flow cytometry on 4 consecu-

tive days of culture. Average daily percentage of Annexin V–positive

cells was calculated for each subject and reported as the mean 6 SD,

and the mean of these averaged values for each T-cell population was

reported as the mean 6 95% CI. A thorough discussion regarding the

use of CRTH2 and CD56 as surrogate markers for type 2 and type 1 T

cells is provided in Loza et al.8 Use of surrogate surface markers that

are expressed independently of stimulation for cytokine production

are required because detection of apoptosis is not compatible with

the method for cytokine detection as a result of the requirement for

fixation and permeabilization that renders detection of apoptosis by

Annexin V impossible (live nonapoptotic cells must be alive at the

time of analysis to prevent loss of membrane integrity).

Analysis of vasodilator-stimulatedphosphoprotein phosphorylation

Phosphorylation of vasodilator-stimulated phosphoprotein

(VASP)–Ser157 in T cells from freshly isolated PBLs was used

as a measure of PKA activity and was detected by means of flow

cytometry, simultaneously with CD5 expression, after 30 minutes of

stimulation, as previously described.8

Statistical analyses

Because of interindividual variability in proportions of cytokine-

positive T cells for the control conditions, data were normalized to

percentage change relative to the control condition to perform statistical

analyses. Error bars are for SDs of the mean, unless otherwise noted.

Significant differences in median from 100% of control value were

tested by using Wilcoxon signed-rank tests. Significance of differences

between means between vehicle, ISO, and PGE2 conditions were tested

by using ANOVA with Tukey-Kramer multiple comparison tests and

GraphPad Prism 4.03 software, as were linear regression correlation

coefficients, significance of slopes, and 2-tailed t tests, where indicated.

A P value of less than .05 was considered significant.

RESULTS

Effects of b-agonists and PGE2 onaccumulation of T-cell subsets

Freshly isolated adult human PBLs contain distinctsubsets of T cells that produce IL-13 and IFN-g, asassessed by means of flow cytometric analysis afterstimulation with phorbol 12-myristate 13-acetate andcalcimycin (Fig 1, A). Total T-cell numbers, includingthe IL-131 and IFN-g1 subpopulations, increase withCD3 plus CD28-mediated stimulation (Fig 1, B, and seeFig E1 in the Online Repository at www.jacionline.org).However, no significant increase in the proportion of IL-131 T cells occurs because the accumulation of IL-132

cells is similarly increased (Fig 1, C). The numbers ofIFN-g1 T cells increased in cultures with CD3 plusCD28-mediated stimulation, but the relative proportionof IFN-g1 T cells decreased.

Relative to the major IL-132 and IFN-g1 T-cell pop-ulations, IL-131 T cells preferentially proliferate inresponse to IL-2,12,13 increasing in both absolute number

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FIG 1. Accumulation of IL-131 and IFN-g1 T cells. Before (day 0) or after culture for 5 days with IL-2 and CD3

plus CD28 mAb or 6 days with IL-2, PBLs were stimulated for cytokine production. A, Representative experi-

ment of day 0 expression of IL-13 and IFN-g. Numbers (B) and proportions (C) of total (CD31), IL-131, and

IFN-g1 T cells for cultured cells were normalized to the respective day 0 values (5 100%) and plotted for

each subject tested (bars 5 median). *P < .05 for median versus day 0. PE, Phycoerythrin; FITC, fluorescein

isothiocyanate.

Bas

and proportion (P � .00003; Fig 1, B and C). Numbers ofIFN-g1 T cells decreased in response to IL-2 (P < .006)and increased only in cultures including IL-12 (data notshown).12,13

Both human type 2 and type 1 cells express epiprosta-noid (EP) prostanoid receptors and b2-ARs,8 2 GPCRsthat couple to the Gs family of heterotrimeric G proteinsand are capable of regulating proliferation and survival

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FIG 2. Effects of b-agonist and PGE2 on accumulation of T-cell subsets. A and B, PBLs were cultured for 5 days

with IL-2 and CD3 plus CD28 mAb or 6 days with IL-2 plus vehicle, ISO, or PGE2 and then stimulated for cyto-

kine production. Percentage change in numbers (Fig 2, A) and proportions (Fig 2, B) of total (CD31), IL-131, and

IFN-g1 T cells are plotted for each subject tested. P < .05: *from control condition (reflecting 0 on y-axes);

�between ISO- versus PGE2-treated conditions. C, Representative analysis of VASP phosphorylation in T cells

after indicated stimulation.

of numerous cell types through actions of the downstreameffector PKA. Effects of PGE2 and the b-agonist ISO onboth CD3 plus CD28– and IL-2–mediated accumulationof total, IL-131, and IFN-g1 T cells were assessed and de-picted in Fig 2. PGE2, previously shown to robustly acti-vate PKA in total and IL-131 human T cells (Fig 2, C),8

significantly inhibited total T-cell and IL-131 T-cell accu-mulation (in proportion and number) in PBLs stimulatedwith CD3 plus CD28 (Fig 2, A). IL-2–stimulated accumu-lation of IL-131 T cells (in number and proportion) wasalso significantly inhibited by PGE2 (Fig 2, B). PGE2 sig-nificantly reduced IL-2– but not CD3 plus CD28–stimu-lated accumulation of IFN-g1 T cells.

In contrast with the effects of PGE2, treatment withISO significantly inhibited CD3 plus CD28–stimulatedaccumulation of total T cells (P < .05) but had no signif-icant effect on CD3-stimulated accumulation of IL-131

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T cells. Interestingly, with the IL-2 stimulatory condition,despite some variability in response, ISO significantly in-creased IL-131 proportion (32%; 95% CI, 15% to 50%;P < .001) and cell number (22%; 95% CI, 5% to 38%;P < .01), with no effect on either total or IFN-g1 T-cellaccumulation. This was rather surprising in that despiteISO (relative to PGE2) being a weak activator of PKA inhuman IL-131 T cells (Fig 2, C),8 this characteristic mighthave been predicted to confer a diminished inhibitory ef-fect but not an augmenting effect. In separate analyses,when cultures were supplemented with 1 mmol/L ISO or1 mmol/L PGE2 each day of culture after the initial treat-ment, neither the augmenting effect of ISO nor the inhib-itory effect of PGE2 was significantly affected, suggestingthat the effects of ISO and PGE2 depended primarily onmodulation of early signaling events (data not shown).This was further suggested by observations that addition

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of the b2-AR–selective antagonist ICI 118,551 inhibitedthe effect of ISO if added before the addition of ISO butnot when added after 18 hours (data not shown). A rolefor agonist-mediated modulation of endogenous IFN-glevels was excluded because similar results were obtainedwhen IFN-g–neutralizing mAb was added to the cultures(data not shown).

Mechanisms mediating the effects ofb-agonists and PGE2 on accumulation

The accumulation of cytokine-producing T-cell subsetscan be modulated by changes in the proliferation or thesurvival of the cytokine-positive cells (or their progeni-tors). Modulation of proliferation can be distinguishedfrom modulation of cell survival by analyzing the numbersof divisions cytokine-positive cells undergo during culturewith the CFSE method.12-14 Coanalysis of dilution ofCFSE (labeling intensity decreases by one half with eachcell division) and cytokine production after culture allowscalculation of an average division number and a minimumprogenitor number. The latter is the minimum number ofcells from which the cytokine-positive population musthave derived.12-14 If the calculated progenitor number islower than the actual number of cytokine-positive cellsin the starting population, then this indicates a loss ofprogenitors, which can occur as a result of cell death orloss of ability to produce the cytokine. If the calculatedprogenitor number is higher than the starting number,then cells previously incapable of cytokine productionmust have differentiated to become capable, a situationwe did not observe for IL-131 T cells in any condition(not shown).

In cultures with IL-2– or CD3 plus CD28–mediatedstimulation, the minimum progenitor number for IL-131

and IFN-g1 T cells was always lower than the starting(day 0) number, indicating that there is always some de-gree of loss of progenitors during culture (Fig 3, A).12,13

Accumulation of IL-131 T cells is associated with and de-pendent on proliferation of the preexisting pool of IL-131

T cells.12,14 Given the established antiproliferative effectof PKA activity, a likely mechanism for the inhibition ofaccumulation of IL-131 T cells by PGE2 is inhibition ofproliferation. CFSE analyses (Fig 3, B, top) indicated apositive correlation between the inhibition of IL-2–stimu-lated accumulation of IL-131 T cells by PGE2 and inhi-bition of proliferation relative to the control conditionof IL-2 only (r2 5 0.65, P 5 .015). Inhibition of IL-131

T-cell accumulation by PGE2 also positively correlatedwith a relative loss of progenitors (r2 5 0.83, P 5 .002),indicating that both inhibition of proliferation and lossof progenitors contribute to the effects of PGE2 (Fig 3,C, top).

The mechanisms underlying the effects of ISO on IL-131 T-cell accumulation are less clear. In 5 cultures inwhich ISO increased the accumulation of IL-131 T cells,either no effect or a slight decrease in proliferation ofIL-131 T cells was observed. However, ISO-mediated in-creases in accumulation of IL-131 T cells were associatedwith a relative increase (ie, reduced loss) of IL-131 T-cell

progenitors, indicating that there is less loss of IL-131 T-cell progenitors (and their progeny) relative to the loss incontrol cultures with IL-2 alone. In the minority of cases(n 5 2) in which ISO had an inhibitory effect on IL-131

T-cell accumulation, the relative change in progenitornumber could not compensate for the decrease in averagedivision number. Therefore the effects of ISO on IL-131

T-cell accumulation are influenced to a greater extent bysurvival-maintenance of IL-131 cells than by modulationof proliferation.

Unlike for accumulation of IL-131 T cells, the PGE2-and ISO-mediated effects on accumulation of IFN-g1 Tcells in cultures with IL-2 did not correlate with changesin rate of proliferation (Fig 3, B, bottom). Rather, changesin IFN-g1 T-cell numbers strongly correlated with relativechanges in minimum progenitor number (r2 5 0.98 andP < .002 for both PGE2 and ISO; Fig 3, C, bottom).Similarly, the effects of PGE2 and ISO on CD3 plusCD28–stimulated increases in IL-131 T cells correlatedwith changes in minimum progenitor numbers (r2 5

0.85 and 0.98, P 5 .001 and .002 for PGE2 and ISO,respectively) but not average division number (Fig 3, Dand E, top). In the cases in which ISO had a positive ornegative effect on accumulation, there was a relative in-crease or decrease in the progenitor number, respectively.PGE2 and ISO had variable effects on IFN-g1 T cellsin cultures with CD3 plus CD28–mediated stimulation(Fig 2, B), and these effects were associated with changesin minimum progenitor number (r2 5 0.96 and 0.75, P 5

.0001 and .006 for PGE2 and ISO, respectively) but notaverage division number (Fig 3, D and E, bottom).Therefore the effects of ISO and PGE2 on CD3 plusCD28–stimulated accumulation of IFN-g1 T cellsoccurred through regulation of the numbers of IFN-g1

T-cell progenitors (or their progeny).Early apoptosis was detected in CRTH21 (type 2) and

CD561 (type 1) T cells by means of daily analysis ofAnnexin V binding to determine whether modulation ofapoptosis is associated with the change in the loss of pro-genitors. The use of CRTH2 and CD56 as markers oftype 2 and type 1 T cells, respectively, was required be-cause detection of apoptosis is not compatible with themethod of cytokine detection (see the Methods section).In the control culture condition of IL-2 only, the averageproportion of apoptosis detected each day in culture waslow in the CRTH21 and the predominant CRTH22

CD562 (type 0) T-cell populations and significantlyhigher in the CD561 T-cell population (Fig 4, A and B).Apoptosis in the CRTH21 population was significantly re-duced in cultures with ISO when compared with that incontrol (vehicle-treated) cultures (20% decrease; 95%CI, 9% to 31%; P < .001), but ISO did not significantlyaffect apoptosis in the CRTH22/CD562 or CD561 T-cellpopulations (Fig 4, C). The decrease in early apoptoticCRTH21 T cells in cultures with ISO was observedthroughout the 4 days of the assays (Fig 4, D). Theseresults indicate that reduced apoptosis contributes to the in-creased accumulation of IL-131 T cells and increasedmaintenance of their progenitors in cultures with b-agonist.

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FIG 3. Mechanisms for modulation of T-cell subset accumulation. CFSE-labeled PBLs were cultured for 6 days

with IL-2 (A-C) or 5 days with IL-2 and CD3 plus CD28 mAb (A, D, and E) plus vehicle, ISO, or PGE2 and then

stimulated for cytokine production. For the vehicle conditions, minimum progenitor numbers versus the re-

spective number of IL-131 (top) and IFN-g1 (bottom) T cells in the original (day 0) population for each subject

tested are shown (Fig 3, A). Percentage changes relative to the vehicle-treated conditions for average division

numbers and progenitor numbers versus percentage change in numbers of cytokine-positive T cells, with

symbols plotted for each subject tested, are shown (Fig 3, B-E).

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FIG 4. b-Agonist–mediated reduction in apoptosis in type 2 T cells. Apoptosis was analyzed in T-cell subsets

during culture with IL-2 plus vehicle or ISO. A, Representative analysis for vehicle condition on day 3. B, Av-

erage percentage of Annexin V–positive T cells for each subject (left) and mean for each T-cell population

(right). C, Percentage change for ISO relative to vehicle condition for each subject (left) and mean for each

T-cell population (right). D, Mean percentage decrease in proportion of Annexin V–positive cells in the

CRTH21 T-cell population for ISO relative to vehicle conditions for each day of culture. P < .05: *between

indicated populations; �from vehicle condition.

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FIG 6. Proposed regulation of T-cell numbers. Even though IL-131 T cells proliferate slightly less in the b-ag-

onist condition, IL-131 cells accumulate to greater numbers than in the IL-2–only condition because of reduced

apoptosis. In the PGE2 condition decreases in both proliferation and IL-131 cell progenitor number contribute

to a reduction in the final IL-131 cell number.

FIG 5. PKA dependence of modulation of IL-131 T-cell accumulation. A, Freshly isolated PBLs from patients

with SLE were cultured for 6 days with IL-2 plus vehicle, PGE2, or ISO and then stimulated for cytokine produc-

tion. The percentage change in IL-131 T cells is reported for each subject tested. B, VASP phosphorylation in

T cells from patients with SLE is indicated. Numbers in histograms are the percentage change in proportion

of IL-131 T cells for IL-2–stimulated cultures.

Association with cAMP and PKA

Experiments with various E-prostanoid analogues in-dicated that the EP2 receptor, which couples to Gs to acti-

vate PKA, is sufficient to mediate the observed inhibitory

effects of PGE2 on IL-131 T-cell accumulation, whereas

the effects of N6,O29-dibutyryl cyclic AMP suggest

PKA activation is sufficient to produce these effects (see

Fig E2 in the Online Repository at www.jacionline.org).Because a subset of patients with SLE exhibits a T cell–

specific defect in PKA,9,15,16 T cells from these patients

represent a useful natural model for exploring the role of

PKA in the regulation of T-cell accumulation. We there-

fore examined the effects of ISO and PGE2 on IL-2–

stimulated accumulation of T cells isolated from patientswith SLE (Fig 5, A). Although the effect of ISO in aug-menting IL-2–stimulated IL-131 T-cell accumulationwas only slightly greater than that observed for cellsfrom healthy subjects (Fig 2, B), the mean inhibitory effectof PGE2 was lost because of a strong augmenting effectobserved in approximately one half of the experimentsperformed. In an attempt to explain this effect and the var-iability in response among patients with SLE, concomitantanalysis of PKA-mediated intracellular VASP phospho-rylation8 was determined in 8 of the subjects. As shownin Fig 5, B, a failure of PGE2 to induce significantVASP phosphorylation coincided with augmentation ofIL-2–stimulated IL-131 cell accumulation, whereas

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more typical robust VASP phosphorylation was associ-ated with inhibition of accumulation similar to that ob-served with cells from healthy subjects (Fig 2, B). Ofinterest is the observation that defects in PKA activationappear to be restricted to T cells (ie, agonist-stimulatedVASP phosphorylation in natural killer cells is evident;see Fig E3 in the Online Repository at www.jacionline.org).

DISCUSSION

We have demonstrated disparate effects of 2 differentGs-coupled receptors on the antigen- and IL-2–stimulatedaccumulation of IL-131 T cells and have revealed a noveleffect of b-agonists in the promotion of type 2 T-cell sur-vival (Fig 6). These disparate effects of PGE2 andb-agonists are explained in part by the differential capac-ity of PGE2 and b-agonists to activate PKA. PGE2, thestronger PKA activator, inhibits accumulation throughPKA-dependent inhibition of proliferation. b-Agonistsonly minimally inhibit proliferation, which is consistentwith a lesser induction of PKA activity in type 2 T cells.8

Because the effect of b-agonists on proliferation is mini-mal, a concomitant prosurvival effect can result in a netincrease in IL-131 T-cell accumulation. That failure tosufficiently induce PKA activation contributes to this aug-mentation of IL-131 T-cell accumulation by Gs-coupledreceptors is revealed in analyses of (PKA-defective) Tcells from patients with lupus, in which PGE2 convertsfrom a strong inhibitor to an enhancer of IL-131 T-cellaccumulation.

The finding that b-agonists have an antiapoptotic effecton type 2 T cells is somewhat surprising. A possiblemechanism is PKA-mediated regulation of bcl-2/bcl-XL

antagonist, causing cell death (BAD). In a variety of celllines (eg, HEK293, COS1, CHO-1, B-cell lymphoma,and cardiac myocytes), PKA is reported to phosphorylateBAD to inhibit apoptosis, mediated by loss of a capacity ofphosphorylated BAD to interact with bcl-2 and bcl-XL.17-20

However, in human T cells bcl-2 and bcl-XL expressionlevels are either unaffected or slightly decreased by ISO(not shown), and our collective observations suggest acomplex relationship between PKA activation and apo-ptosis in type 2 T cells. Additional possible mechanismslinking b2-ARs to prosurvival signaling include cAMP-dependent, PKA-independent activation of exchangeprotein activated by cAMP (Epac), leading to activationof phosphatidylinositol-3 kinase and Akt. However,real-time PCR analysis suggests Epac mRNA levels inhuman T cells are either extremely low or absent, andthe Epac-activating cAMP analogue (8-pCPT-29-O-Me-cAMP) did not affect IL-2–stimulated accumulation (notshown). b2-AR–mediated activation of arrestin-dependentsignaling, possibly influencing nuclear factor kB–depen-dent signaling,21,22 is also a possible mechanism affectingT-cell survival. However, clarification of the role of ar-restins in this process requires molecular transfection/infection strategies to which primary human T cells are

(currently) resistant. Several additional cAMP/PKA-inde-pendent signaling pathways promoted by b2-AR havebeen proposed, including heterologous oligerization withother GPCRs and direct coupling of signaling moleculesto the b2-AR (reviewed by Giembycz and Newton23).

The effect of b-agonists in promoting an increase in IL-131 cell accumulation is only observed for IL-2 and notCD3 stimulation conditions, although the disparate effectsof ISO and PGE2 on CD3-stimulated type 2 T-cell accu-mulation are similarly explained by differential activationof PKA. IL-2– or IL-15–stimulated bystander increases intype 2 T cells have only recently been described.12,13 Ourpreliminary data indicate that IL-2–stimulated IL-131 T-cell accumulation is greater in atopic asthmatic subjectscompared with that in nonatopic nonasthmatic subjects,24

suggesting the biologic relevance of antigen-independentbystander accumulation.25 Bystander accumulation mightalso be important in chronic inflammation and autoimmu-nity.26 An implication of these and our current results isthat frequent use of inhaled b-agonists by asthmatic sub-jects might promote type 2/type 1 cell skewing by enhanc-ing the preferential accumulation of IL-131 T cells inresponse to T-cell IL-2 production stimulated by bacte-rial-viral infections or allergens (or IL-15 produced bymonocytic cells), without affecting accumulation ofIFN-g1 cells. In contrast, b-agonists would be expectedto temporarily inhibit both antigen-stimulated IL-131

and IFN-g1 production8 but not subset accumulation.Clearly, however, establishing whether our findings re-garding b-agonists’ effects in vitro can be extrapolatedto the effects of inhaled b-agonists on T-cell subtype accu-mulation in the lung will ultimately require analysisinvolving an in vivo, and ideally human, model.

Predominant type 2 T-cell responses are characteristicin asthma27-29 and also SLE.30-32 Our data suggest thatpatients with SLE with a defect in T-cell PKA activityare subject to augmentation of bystander type 2 cell accu-mulation promoted not only by b-agonists but also byother Gs-coupled receptor agonists as well. PGE2 couldbe a particularly relevant mediator of this effect. SLEexacerbations are marked by excessive inflammatory re-sponses, and PGE2 is a common inflammatory mediatorproduced by both immune (monocytes/macrophages)and nonimmune (epithelia, airway smooth muscle) cells.33

In patients with SLE with a T cell–specific PKA defect,PGE2 in concert with IL-2 from stimulated T cells or IL-15 from monocytic cells (IL-15 is upregulated in patientswith SLE34) could promote exaggerated accumulation ofIL-13–producing type 2 T cells.

Other conditions under which b-agonists might pro-mote an increase in bystander accumulation of IL-131

cells include various forms of heart failure, particularlythose involving viral cardiomyopathy. Viral infectionshave been implicated in acceleration of heart disease, pre-sumably through induction of local inflammation. The roleof T cells in this process is not clear, and it is unknownwhether type 1– or type 2–mediated inflammationpredominates. Levels of circulating catecholamines areincreased in patients with heart failure, which can lead

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244 Loza et al

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to enhanced accumulation of type 2 T cells, as describedabove for asthma and SLE. Chronic psychological stress,believed to promote atopic disease through ill-definedneurohumoral mechanisms, could also provide the contextfor the regulation of T-cell development by endogenouscatecholamines.35-37

Finally, our findings demonstrating the relationshipbetween agonist-induced PKA activation and type 2 T-cellaccumulation provide a potential explanation for thevariability in response observed in control subjects (Fig 2).It is well established that the multiple elements of GPCRsignal transduction (including receptor, G protein, andadenylyl cyclase) exhibit considerable variance in theirexpression and function caused by genetic, epigenetic, orbiologic influences. Such variance affecting agonist-induced PKA activation could readily explain the variationin the effects of b-agonists or PGE2 on type 2 cell accumu-lation, thus suggesting a prevalent role of GPCR-mediatedregulation of T-cell development.

In summary, we have demonstrated an unexpectedactivity of b-agonists on T-cell function. b-Agonist–induced accumulation of IL-13–producing T cells throughenhancement of cell survival mechanisms has importantimplications in understanding adrenergic regulation of theimmune system and its effect on the pathogenesis andmanagement of multiple disease states.

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