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Originally published online as doi:10.1189/jlb.0303103 on July 15, 2003

Published online before print July 15, 2003
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(Journal of Leukocyte Biology. 2003;74:531-541.)
© 2003 by Society for Leukocyte Biology

Divergence in NK cell and cyclic AMP regulation of T cell CD40L expression in asthmatic subjects

Denise Wingett*,{dagger},{ddagger},§,1 and Christopher P. Nielson*,{dagger},{ddagger}

* Research Service, Department of Veterans Affairs Medical Center, Boise, Idaho;
{dagger} Department of Medicine, Division of Gerontology and Geriatric Medicine, University of Washington, Seattle;
{ddagger} MSTI/MSMRI Research Institute of St. Luke’s Regional Medical Center, Boise, Idaho; and
§ Department of Biology, Boise State University, Boise, Idaho

1Correspondence: Boise VA Medical Center, Research Service 151, 500 W. Fort St., Boise, ID, 83702. E-mail: denise.wingett{at}med.va.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cells are central in the pathogenesis of asthma, and the associated ligand, CD40L, plays an important role by increasing production of immunoglobulin E and inflammatory mediators. ß-Adrenoceptor agonists are commonly used in asthma, although little is known regarding effects on CD40L expression and T cell activation. Here, we demonstrate that cyclic adenosine monophosphate (cAMP) and ß-adrenoceptor agonists differentially regulate CD40L in asthma. cAMP increased naïve T cell CD40L expression in asthmatics (9.8±8.5 increase in percent CD40L-positive cells), and expression in control subjects was inhibited (7.1±6.0 decrease in percent CD40L-positive cells; P< 0.05). Cell depletion and reconstitution experiments were used to determine that cAMP enhancement of CD40L required cell-to-cell contact with an asthma-associated natural killer (NK) cell subset. The NK cell subset expressed elevated levels of CD95, and in vitro-generated CD95+ NK2 cells also produced similar effects on CD40L expression. Our findings suggest that a subset of NK cells with elevated CD95 expression is associated with asthma and can reverse cAMP inhibitory effects on T cell CD40L with the potential to increase disease exacerbation.

Key Words: allergy • cAMP • cellular activation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asthma is now recognized as an inflammatory disease in which T cells play an important role in disease pathophysiology. Activated CD4+ T cells have been identified in bronchial biopsies [1 ], peripheral blood [2 , 3 ], and bronchoalveolar lavage [4 , 5 ] in association with allergen challenge or asthma exacerbations. T cells have also been implicated in animal models of antigen-induced airway hyper-responsiveness [6 , 7 ] with a central role for the T helper cell type 2 (Th2) subset in production of inflammatory mediators and cytokines including interleukin (IL)-4, IL-5, IL-10, and IL-13 involved in eosinophil activation and immunoglobulin E (IgE) generation [8 ].

Observations that T cells and IgE are important in asthma suggest that T cell CD154 (CD40L) may be relevant in disease pathogenesis. CD40L, a 33–39 kDa type II membrane protein expressed on activated T cells [9 , 10 ], interacts with CD40 on B cells, monocytes, and dendritic cells [9 , 11 ]. CD40L was initially recognized to be essential for B cell–T cell cognate interactions, resulting in B cell activation, differentiation, Ig class-switching, and IgE production [12 13 14 ]. More recent studies have demonstrated the importance of CD40L in antigen-presenting cell cytokine production and expression of costimulatory molecules, including CD80, CD86, and intercellular adhesion molecule-1 [15 16 17 ]. Potentially related to the critical role of CD40L in IgE production [12 , 13 ], CD40L is elevated in T cells from allergic patients [18 19 20 ]. Most directly relevant to asthma, CD40L is required for induction of IgE responses and development of bronchial hyper-responsiveness to inhaled allergen [21 ] and contributes to pulmonary inflammation and eosinophilia in murine models of allergic airway inflammation [22 ].

Consistent with the critical regulatory role of CD40L, expression of this protein is transient and tightly controlled. Optimal CD40L expression requires T cell receptor (TCR) and CD28 costimulation and reaches maximum levels ~6 h following T cell activation [23 ]. Cyclic adenosine monophosphate (cAMP) regulation of CD40L may be of particular importance, as cAMP is critical to many T cell signaling pathways, and actions of commonly used ß-agonist bronchodilators and inflammatory mediators including prostaglandin E2 are mediated by cAMP [24 ]. cAMP can increase or decrease CD40L expression, depending on the activation stimulus, costimuli, and T lymphocyte subset. For example, cAMP decreases CD40L expression in CD4+ T cells activated by TCR ligation but increases expression with calcium-dependent activation or appropriate costimulation [25 ]. T cell subtype is also relevant, as cAMP has minimal effects on naïve (CD45RA+) T cell CD40L but markedly inhibits memory (CD45RO+) T cell CD40L at equivalent concentrations [26 ]. These differences are likely related to distinct signal transduction in each cell subset, as naïve T cells have a greater activation threshold and have a greater calcium signal than memory cells [27 28 29 30 ]. Naïve T cell regulation is particularly relevant to asthma and atopy, as atopic subjects have greater numbers of naïve cells [31 , 32 ], naïve T cells are efficient inducers of IgE [32 , 33 ], and naïve cells from atopic subjects may remain CD45RA+ following antigen activation as a result of a defect in the CD45RO phenotype switch [32 ]. In the present study, we explore the effects of ß-adrenoceptor agonists and cAMP on the regulation of T cell CD40L in naïve CD4+ T cells. We find that cAMP can increase T cell CD40L expression in asthmatic subjects and inhibit expression in control subjects and that the regulatory effect is controlled by a natural killer (NK) cell subset sharing characteristics with the recently described NK2 cell subset.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
All asthmatic subjects were previously diagnosed with asthma, had a history of wheezing, dyspnea, or coughing compatible with the diagnosis, and met the American Thoracic Society criteria for asthma [34 , 35 ]. Asthmatic subjects had airflow obstruction [forced expiratory volume in 1 s (FEV1)<80% of predicted value] with significant ß-agonist reversal (>15%) or airway hyper-responsiveness following broncho-provocation using methacholine [provocative challenge 20 (PC20) of <8 mg/ml]. Patients using inhaled or systemic corticosteroids were excluded from study. ß-Agonists, leukotriene modifiers, and antihistamines were withheld for a minimum of 24 h before study. The study included allergic and nonallergic asthmatics, as defined by IgE levels or self-described symptoms and physician diagnosis.

Asymptomatic asthmatics met above criteria for asthma but experienced only seasonal symptoms. These subjects were symptom-free for a minimum of 2 weeks before study. Nonasthmatic control subjects had a life-long absence of allergy or asthma symptoms, and an absence of airway hyper-responsiveness was demonstrated for specific studies by pulmonary function and methacholine challenge testing (PC20 of >8 mg/ml).

All subjects were nonsmokers or ex-smokers that had discontinued smoking at least 1 year before study inclusion. All subjects withheld caffeine and fasted a minimum of 12 h before blood sampling and were excluded by upper respiratory traction infection within 14 days of blood sampling. Except as indicated (see Figs. 7 and 8 ), data presented in each figure represent a single culture from any given subject. Written, informed consent was obtained from all subjects, and the University of Washington Institutional Review Board (Seattle) approved the study.



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  		Figure 7. Asthmatic NK cells regulate T cell CD40L. CD4+CD45RA+ T cells were cocultured with NK cells isolated from the same PBMC preparation, and effects of cAMP on CD40L were evaluated. CD4+CD45RA+ (>95% CD4+, 93% CD45RA+) and NK cell populations (>94% CD56+CD3-) were obtained by negative selection. CD4+CD45RA+ cells (2x105) were added to wells containing immobilized CD3/CD28 Ab and cultured in the absence or presence of autologous NK cells (2x105). Cultures were treated with 100 µM dbcAMP or vehicle control for 6 h, and T cell CD40L expression was determined by flow cytometry. (A) A representative histogram (n=6) derived from a symptomatic asthmatic is presented, and inset numbers depict the percentage of positively staining cells and MFI, respectively. The lighter line depicts staining with isotype Ab, and CD40L staining is in black. (B) CD40L expression in symptomatic (n=6) and asymptomatic (n=6) asthmatics differs in response to cAMP and NK cell coculture. Cocultures were set-up as described above, and cAMP-induced change in CD40L+ T cells = [(%CD40L+ cellscAMP-treated cultures–%CD40L+control cultures)/%CD40L+ T cells control cultures] x 100. One asthmatic subject was classified into symptomatic and asymptomatic groups, depending on symptoms at the time of blood sampling.

 


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  		Figure 8. Asthmatic NK cells express CD95. Freshly isolated PBMC were stained with anti-CD3 FITC, anti-CD56 PC5, and anti-CD95 PE and were analyzed by flow cytometry within 2 h of drawing blood. Data are expressed as the percentage of CD95-positive cells among CD56+CD3--gated NK cells. Horizontal bars indicate mean values, statistical significance was determined using Welch-ANOVA, and the asthma group showed statistical difference (P<0.05) from the normal or asymptomatic subject groups. Asthmatic subjects demonstrated current asthma symptoms, normal control subjects had a life-long absence of asthma or allergy symptoms, and asymptomatic asthmatics had an absence of asthma symptoms for a minimum of 2 weeks. Two asthmatic subjects were classified into symptomatic and asymptomatic groups, depending on the presence or absence of symptoms.

 
Isolation of T cell subsets and NK cells
Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque (Histopaque-1077, Sigma Chemical Co., St. Louis, MO) gradient centrifugation using heparinized phlebotomy samples [36 ]. Cells were washed three times with Hank’s buffer (Sigma Chemical Co.) and incubated at 1 x 106 cells/ml in RPMI 1640 (Sigma Chemical Co.) containing 10% fetal bovine serum. CD45RA+ cells were obtained by positive immunomagnetic selection using anti-CD45RA monoclonal antibody (mAb) and the Miltenyi Variosort magnetic separation system according to the manufacturer’s protocol (Miltenyi Biotech, Auburn, CA). CD45RA+ cell preparations (>96% viability) were >95% CD45RA-positive and contained ~45% CD4-positive cells, and the remainder of cells were CD4-negative (i.e., the majority was CD8 T cells, B cells, and NK cells). CD45RA+CD4+ cells (see Fig. 5 ; purity, >95% CD4+, 93% RA+) were obtained by a CD4-negative selection using a cocktail of antibodies to deplete CD8+ T cells and non-T cells (CD8, CD19, CD14, CD16, CD56, CD8, glycophorin A) followed by CD45RA-positive selection using magnetically labeled CD45RA Ab (Miltenyi Biotech). CD45RA+CD16- cells (see Fig. 6 ; purity, >95% RA+, <1% CD16+) were obtained by CD16 immunomagnetic depletion using a StemCell Technologies (Vancouver, B.C., Canada) protocol and labeled Ab, followed by a subsequent CD45RA-positive selection, as described above. Negatively selected CD45RA+CD4+ cells (see Fig. 7 ; purity, >95% CD4+, 93% RA+) were obtained by incubating PBMC with magnetically labeled Ab directed against CD45RO, CD8, CD19, CD14, CD16, CD56, CD8, and glycophorin A (StemCell Technologies) with collection of unlabeled T cells (typically >96% CD4+, >95% CD3+, >95% CD45RA+). NK cell populations (purity, >94% CD3-, CD16+, CD56+) were obtained by negative immunomagnetic selection using Ab directed against CD3, CD4, CD14, CD19, CD66b, and glycophorin A (StemCell Technologies), according to the manufacturer’s protocol.



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  		Figure 5. Requirement of CD45RA+ CD4- cells for cAMP-induced CD40L. CD45RA+ cells or purified CD4+CD45RA+ cells were concurrently isolated from asthmatic donors. CD45RA positive cells (>94% purity) were isolated by positive selection and contained both CD4- and CD4+ cells. For this cell population, changes in naïve T cell CD40L expression were evaluated using flow cytometric gating of CD4+ events. The second cell population (CD45RA+ CD4+) was isolated by CD4-negative selection followed by positive selection for CD45RA+ cells and was typically >95% CD4+, 93% CD45RA+. Both T cell populations were activated with CD3/CD28 Ab and treated with vehicle control or 100 µM dbcAMP for 6 h, and CD40L expression was evaluated by flow cytometry. Data from three asthmatic donors are presented, and error bars depict SE.

 


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  		Figure 6. Depletion of CD16+ cells eliminates cAMP enhancement of CD40L. Two populations of cells were concurrently isolated from asthmatic subjects. CD45RA cells (>95% CD45RA+) were isolated by positive selection and contained an average of 32% CD56+, 14% CD16+ NK cells. The second population, CD45RA+CD16-, was obtained by CD16 depletion followed by CD45RA-positive selection (purity >95% CD45RA+, <4% CD56+, <1% CD16+). Equivalent numbers of cells (2x105 cells/well) were activated with CD3/CD28 Ab and treated with vehicle or 100 µM dbcAMP. After 6 h of culture, CD40L expression was assessed by flow cytometry by gating on CD4+ events. Staining with isotype Ab is shown in gray and CD40L staining in black. Inset numbers depict the percentage of CD40L positively staining cells and MFI, respectively. Data from a representative experiment are presented (n=6), and statistical significance of cAMP effects on each cell population was determined using a two-tailed, paired t-test.

 
Cell culture
Purified CD45RA+ or CD45RA+CD4+ cell populations were cultured in RPMI/10% fetal calf serum (FCS) at 1 x 106 cells/ml in 200 µl total volume in 96-well microtiter plates. Cells were activated using immobilized CD3 Ab [1.0 µg/well clone OKT3, American Type Culture Collection (ATCC), Manassas, VA] and CD28 Ab (0.25 µg/well clone CD28.2, PharMingen, San Diego, CA) and were concurrently treated with various concentrations of dibutyryl (db)cAMP (Sigma Chemical Co.), 3-isobutyl-1-methylxanthine (IBMX; Sigma Chemical Co.), salmeterol (Glaxo Group Research, Greenford, Middlesex, UK), or appropriate vehicle control. After 6 h of culture, CD40L expression was determined by flow cytometry. For coculture experiments, 2 x 105 NK cells were added to wells containing 2 x 105 CD4+CD45RA+ cells and cultured for 6 h in the presence or absence of dbcAMP. For transwell experiments described in Table 1 , CD4+CD45RA+ cells and autolougous NK cells were simultaneously isolated from asthmatic subjects. T cells (1.5x105) were cultured in lower chambers of 96-well plates, precoated with CD3/CD28 Ab, and 8 x 104 NK cells were cultured in upper transwell chambers (Nunc, Naperville, IL). Control wells evaluated T cells cultured in the absence of NK cells ± transwells.


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  		Table 1. cAMP Regulation in Transwell Cultures of T and NK Cells

 
In vitro generation of NK2 cells
NK2 cells were generated as described previously [37 ]. PBMC (2x106 cells) were cultured in 24-well plates in RPMI 1640 supplemented with 5% human serum. Mitomycin C-treated (50 µg/ml) RPMI 8866 cells (a kind gift from Giorgio Trinchieri, Wistar Institute, Philadelphia, PA; 4x105) were added to wells in the presence of 50 ng/ml IL-4 (PeproTech, Inc., Rocky Hill, NJ) and 10 µg/ml anti-human IL-12 (clone C8.6, R&D Systems, Minneapolis, MN). Cells were expanded with fresh media containing IL-4 and anti-IL-12 every 2–3 days. At days 7–8, NK cells were purified by negative immunomagnetic selection (StemCell Technologies) to >92% purity based on CD16 and CD56 expression and were immediately used in T cell coculture experiments.

Flow cytometry
Immunofluorescent staining and flow cytometry were performed as described previously [36 ]. Cells were stained with fluorescently labeled Ab for 30 min at 4°C, washed, fixed, and analyzed on a three-color Epics flow cytometer (Coulter, Miami, FL). In experiments using CD45RA+ positively selected cells, CD40L expression on naïve T cells was performed by gating on CD4-positive events (5000–10,000 events), and expression of percent CD40L positively staining cells and median fluorescence intensity (MFI) was determined by comparison with isotype control. In coculture experiments combining NK and CD45RA+CD4+ cells, CD40L expression on naïve T cells was evaluated by gating on CD4+ (bright) cells (5000–10,000 events). Using this gating scheme, NK cells were excluded from analysis, as they are absent or low in CD4 expression. Three-color flow cytometry was used to evaluate CD95 expression on NK cells present in freshly isolated PBMC. NK cells were defined as CD3-CD56+ using a minimum of 5000 gated events, and CD95 expression in this population was evaluated using a phycoerythrin (PE)-conjugated Ab. Fluorescently labeled Ab (CD40L, CD45RA, CD45RO, CD3, CD4, CD16, CD56, CD69, and CD95) were purchased from Coulter. The appropriate concentration of each Ab was determined by titration for optimal staining before experimental use.

Matrix metalloproteinase-9 (MMP-9) zymography
The ability of T cell CD40L expression levels to affect MMP-9 production by THP-1 cells (ATCC) was determined by coculture experiments followed by zymography on conditioned media. CD45RA+ naïve T cells (>96% purity, positively selected) were activated to induce variable levels of CD40L expression (0.25 µM ionomycin±100 µM dbcAMP, 6 h), fixed with 1% paraformaldehyde, and washed three times to remove residual fixative. T cells were analyzed by flow cytometry to determine CD40L expression levels or cocultured with THP-1 cells at a 4:1 ratio (T cells/THP-1 at 1x105/well) in RPMI supplemented with 4.5 g/L glucose, 1.0 mM HEPES, 1.0 mM sodium pyruvate, 5 x 10-5 M ß-mercaptoethanol, 2 mM L-glutamine, and 10% FCS. After 36 h, supernatants were analyzed by zymography using 10% acrylamide–0.1% gelatin gels, as described previously [38 , 39 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
cAMP differentially regulates CD40L in asthma
Previous work from our laboratory has demonstrated that cAMP regulation of T cell CD40L is complex and can inhibit or enhance CD40L, depending on the activation stimulus [26 ]. As ß-agonist bronchodilators affect cAMP and are commonly used in asthma, cAMP regulation of CD40L was compared between asthmatic and control T cells. CD45RA+ positively selected cells were activated with immobilized CD3/CD28 Ab, and effects of cAMP on T cell CD40L expression were evaluated using flow cytometry. In general, cAMP increased CD40L expression in asthmatic subjects yet decreased CD40L in T cells from control subjects (Fig. 1 ). The cAMP-induced increase in percentage of CD40L-positive cells in asthmatic subjects was 9.8 ± 8.5 SD (P<0.05), and the decrease in control subjects was -7.1 ± 6.0 (P<0.05). The character of the cAMP response remained consistent, and 10/12 asthmatics repeatedly demonstrated increased CD40L expression following dbcAMP treatment. Similar patterns of regulation were observed in MFI values, and cAMP increased CD40L MFI in asthmatics by 21.0 ± 9.7% and decreased staining in control subjects by 21.4 ± 3.7% (P<0.05, SE). No differences in baseline CD40L expression levels could be discerned between asthmatic and control subjects (values ranged between 1 and 3% CD40L+ in unstimulated cultures).



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  		Figure 1. cAMP increases CD40L on CD45RA+ cells from asthmatics and decreases expression in control subjects. Positively selected CD45RA+ cells (2x105 cells/well, >95% purity) were cultured in 96-well plates precoated with immobilized CD3/CD28 Ab. Cells were treated with vehicle (control) or 100 µM dbcAMP for 6 h, and CD40L expression on CD4+ T cells present in CD45RA+ cultures was determined by flow cytometry with gating of CD4+ events. (A) cAMP-induced changes in the percentage of CD40L positively staining cells (%CD40L+ staining in cAMP-treated cultures–%CD40L+ in control cultures) from 16 asthmatic subjects and 10 controls are presented. Horizontal bars indicate mean values, and statistical significance (P<0.05) between asthmatic and control responses was evaluated using a two-tailed, unpaired t-test. The average age of asthmatic subjects was 50 ± 17 years (three females, 12 males) and 36 ± 8 years (three females, seven males) for controls, and all control subjects tested negative for bronchial hyper-responsiveness. Histogram (B) depicts cAMP enhancement of CD40L in a representative asthmatic (37% CD40L+/20.6 MFI in control vs. 55%/17.6 in cAMP-treated). Histogram (C) depicts cAMP inhibition of CD40L in a representative control subject (44%/9.3 in control vs. 33%/6.9 in cAMP-treated). The thin gray line depicts staining with isotype-control Ab (iso); the heavy gray line depicts CD40L staining in control; and the black line depicts CD40L staining in cells treated with dbcAMP.

 
Effects of cAMP to decrease CD40L expression in control subjects yet increase expression in asthmatics were dose-dependent (Fig. 2 ). cAMP inhibited CD40L in control subjects at all concentrations tested (50–250 µM), and cAMP enhanced CD40L in asthmatics at concentrations ranging from 50 to 200 µM. At concentrations greater than 250 µM (i.e., 300–500 µM), cAMP also inhibited asthmatic expression (data not shown). Given that the greatest difference between asthmatic and control responses was observed at 50–100 µM dbcAMP, and similar concentrations were used in related studies of T cell activation [40 , 41 ], these concentrations were chosen for experiments described in this study.



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  		Figure 2. Dose-dependent regulation of CD40L by dbcAMP. CD45RA+ cells were obtained from asthmatic (n=3) and control subjects (n=8) by positive selection. Cells were activated with immobilized CD3/CD28 Ab and concurrently treated with vehicle control or varying concentrations of dbcAMP for 6 h, and CD40L expression on CD4+ T cells was determined by flow cytometry. Error bars depict SE.

 
Experiments evaluated whether a similar pattern of CD40L regulation was observed using other cAMP-inducing agents. The ß-agonist salmeterol and the cAMP phosphodiesterase (PDE) inhibitor IBMX dose-dependently increased CD40L expression in 6 h TCR-activated asthmatic CD45RA+ T cells (Fig. 3 ) but showed no appreciable effect on cells from control subjects. A similar CD40L increase was observed in cultures treated with salmeterol or IBMX for 24 h (data not shown).



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  		Figure 3. ß-Adrenergic agonist and PDE inhibitor increase asthmatic CD40L. CD45RA+ cells were isolated from asthmatic subjects by positive selection, activated with CD3/CD28 Ab, and concurrently treated with vehicle control, salmeterol, or IBMX for 6 h. CD40L expression on CD4+ T cells was determined using flow cytometry. (A) A representative histogram is shown where the %CD40L-positive staining and MFI in control cells (iso) are 66%/9.98, 200 nM salmeterol-treated cells (sal) is 77%/12.5, and 50 µM IBMX-treated cells is 86%/24.0. (B) The percentage of CD40L positively staining CD4+ T cells from two asthmatic donors following treatment with salmeterol, IBMX, or dbcAMP is presented.

 
Although it is impossible to exclude the possibility that study results may be affected by patient medication use, all subjects withheld ß-agonists and antihistamines for a minimum of 24 h to reduce confounding drug effects. The majority of asthmatic subjects (56% for this experiment and typically greater for subsequent experiments) withheld these medications for a minimum of 48 h, and the remaining population required the use of medications to control symptoms. No subjects had taken inhaled leukotriene modifiers or systemic corticosteroids for a period of at least 1 month. Based on the study sample size, no correlations between subjects using ß-agonists or antihistamines could be determined.

cAMP regulation of CD40L is distinct from other T cell activation markers
To determine whether increased CD40L in asthmatic subjects reflects a generalized effect of cAMP on T cell activation, an additional surface marker expressed early during T cell activation (CD69) was evaluated. CD45RA+ T cells from asthmatic subjects were activated in the absence or presence of dbcAMP, and CD69 expression was evaluated after 6 h of culture (Fig. 4 ). As anticipated, cAMP caused a statistically significant increase (P<0.05; 32.8±4.6% SE to 42.6±5.4%) in CD40L expression in asthmatic T cells and decreased CD69 expression (74.8±5.0% to 64.6±6.9%) in the same subjects. Although cAMP-inhibitory effects on CD69 did not reach statistical significance with the small sample size evaluated (n=4), effects of cAMP on CD69 and CD40L are of opposite polarity. Thus, actions of cAMP to increase CD40L in asthma may be relatively specific.



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  		Figure 4. cAMP regulation of CD69 expression. Positively selected CD45RA+ cells (>95% purity) from asthmatic subjects were cultured in 96-well plates and activated with immobilized CD3/CD28 Ab. Cells were concurrently treated with vehicle or 100 µM dbcAMP for 6 h. Flow cytometry with gating of CD4+ events was used to evaluate CD40L or CD69 expression on T cells. Data from four independent experiments are presented, and error bars show SE. Statistical significance of cAMP effects on CD40L was determined using a two-tailed, paired t-test.

 
A CD45RA+CD4- cell regulates CD40L expression
As the initial result demonstrating altered regulation of CD40L in asthma (Fig. 1) was obtained by gating on CD4+ T cells from positively selected CD45RA+ cultures, asthma-specific effects of cAMP may be associated with a subpopulation of CD4- CD45RA+ cells (i.e., NK cells, B cells, CD8+ T cells). To address this question, cAMP regulation was compared between CD45RA+ cell preparations (containing CD4- and CD4+ cells) and purified cultures of naïve CD45RA+ CD4+ cells concurrently isolated from the same asthmatic subject (Fig. 5 ). As expected, cAMP produced a statistically significant increase (P<0.05, two-tailed, paired t-test) in CD40L expression in CD45RA+ cultures containing CD4- cells (48±2% SE to 60±4%). However, in purified cultures of CD45RA+ CD4+ cells, cAMP failed to increase CD40L and actually resulted in decreased expression (76±6% to 71±2%). Thus, a CD4- CD45RA+ cell population appears to be required for cAMP to increase T cell CD40L expression.

To further characterize the CD4- cell population, CD40L expression was evaluated with cell preparations depleted of CD16-positive cells (a NK cell marker). CD45RA+CD16- cells were obtained by depletion of CD16+ cells followed by a subsequent CD45RA-positive selection and compared with CD45RA positively selected cells derived from the same PBMC preparation. As shown in a representative histogram (Fig. 6 ), effects of cAMP to increase T cell CD40L expression only occurred in asthmatic CD45RA+ cultures (51±4% SE to 63±2%) and not in cultures depleted of CD16+ cells (52±4% to 48±4%). The ability of cAMP to increase CD40L expression in CD45RA+ cultures was statistically significant (P<0.05) as were cAMP inhibitory actions on CD40L in cultures devoid of CD16+ cells.

Asthmatic NK cells mediate cAMP enhancement of CD40L
Additional experiments were performed to verify asthmatic NK cells alter the cAMP response of T cells. Negatively selected CD45RA+CD4+ naïve T cells were isolated from symptomatic asthmatics and were cocultured with autologous, negatively selected NK cells (CD16+, CD56+, CD3-). In the absence of NK cells, cAMP had negligible effects on CD40L expression (40–41%; Fig. 7A ). In contrast, cAMP induced a marked increase in CD40L expression when NK cells were cocultured with naïve T cells (41–77%). T cell viability did not appear affected by NK cell coculture, and regulatory effects of NK cells did not appear related to changes in cell density, as the addition of equivalent numbers of naive T cells had no effect (data not shown). Results also demonstrate that cAMP effects on CD40L are independent of CD45RA-positive selection.

As NK cells from asthmatic subjects had a marked effect on CD40L regulation, NK cells from symptomatic and asymptomatic asthmatic subjects were compared. Both subject groups were defined as asthmatic at study enrollment, according to criteria described in Materials and Methods. For this experiment, symptomatic asthmatics were further defined by self-described wheeze, cough, breathlessness, or sputum the day of blood sampling, and asymptomatic asthmatics reported an absence of symptoms for a minimum of 2 weeks. As expected, cAMP increased CD40L expression in cocultures of autologous NK cells and naïve T cells from symptomatic asthmatic subjects (Fig. 7B ; average cAMP-induced change in CD40L was -1.0±1.7% SE compared with 35.3±12.1% in T+NK cocultures; P<0.05; n=6). In contrast, cAMP failed to increase CD40L in T/NK cell cocultures from asymptomatic asthmatics (cAMP-induced change in CD40L was -2.2±3.1% compared with 1.5±1.4% in T+NK cocultures; P>0.05; n=6). Similarly, NK cells from nonasthmatic control subjects had no discernible effects on CD40L expression in the presence or absence of cAMP (data not shown). Similar IgE values were observed for both subject groups at study enrollment (200.3±60.0 SE IU/ml symptomatic compared with 169.6±80.4 asymptomatic). In addition, mean FEV1 values of 78.5% (n=4; range, 78–79%) or metacholine PC20 values of 1.9 mg/ml (n=2; range, 0.25–3.5) were observed in symptomatic subjects at study enrollment, and FEV1 of 68.7% (n=3; range, 60–78%) or PC20 of 3.9 mg/ml (n=3; range, 1.4–2.97) was observed in asymptomatic subjects.

Additional experiments evaluated the mechanism by which asthmatic NK cells regulate T cell CD40L expression. Autologous T and NK cells were concurrently isolated and cultured together or separated by a semipermeable membrane to evaluate whether cell-to-cell contact or soluble factors are involved. The 96-well Transwell culture system used (Table 1 ) required a lower NK:T cell ratio than conditions used for Figure 7 . Although the magnitude of response was expectedly lower than in Figure 7 , the effect of NK cells to increase CD40L in the presence of cAMP was still observed and statistically significant when cell-to-cell contact was allowed (P<0.05, two-tailed, paired t-test). A similar response was observed when empty transwells were placed above T + NK cocultures (data not shown). In contrast, when T cells and NK cells were physically separated, no such increase in CD40L expression was observed, indicating cell contact is required.

CD95 expression on NK cells is increased in asthma
The unique, regulatory property of NK cells from asthmatics with active symptoms suggests that a distinguishing NK cell phenotype might exist. As CD95 (Fas) expression may be associated with newly described NK1 and NK2 cell subsets [37 , 42 ], this marker was evaluated on freshly isolated PBMC from control and asthmatic subjects. As shown in Figure 8 , asthmatic NK cells (defined as CD56+CD3-) expressed significantly more (P<0.05) CD95 compared with cells from normal control subjects (13.2±2.6% SE vs. 3.0±0.3%) or asymptomatic asthmatics (5.0±1.0%). Study enrollment IgE values were 339.8 IU/ml ± 145.9 SE in symptomatic asthmatics, 271.0 ± 110.0 in asymptomatic subjects, and 20.8 ± 7.0 in control subjects. The mean study enrollment FEV1 or PC20 was 65.2 ± 4.4% SE (n=8) and 3.5 ± 1.4 mg/ml (n=5) for symptomatic asthmatics and 78.0% (n=1) and 3.2 ± 1.2 mg/ml (n=6) for asymptomatic asthmatics. Overall, results indicate that an increased frequency of CD95+ NK cells is associated with active symptoms.

Regulatory function of in vitro-primed NK cells
As altered CD95 expression on NK cells has been described as a potential marker for the NK2 subset [42 ], and these cells are capable of regulating T cell function and Th2 cytokine production [37 , 42 ], this subset may be relevant to asthma and CD40L regulation. NK2-like cells were generated in vitro by culture with IL-4 and anti-IL-12 blocking Ab, as described previously [42 ], and were cocultured with autologous, naïve T cells in the presence or absence of dbcAMP. The data presented in Figure 9A indicate that the overall T cell response to cAMP in the absence of NK2-like cells was slight and caused a statistically insignificant reduction in CD40L expression (–2.3% change in CD40L; P>0.05). In contrast, when identical T cells were cultured in the presence of in vitro-generated NK2 cells, cAMP induced a modest but significant increase in CD40L expression (13.5% change in CD40L; P<0.05). These results indicate that NK cells primed in vitro with IL-4 and IL-12 blocking Ab modulate T cell cAMP responses with the ability to increase CD40L similar to NK cells from asthmatic subjects.



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  		Figure 9. In vitro-generated NK2 cells regulate CD40L. (A) Freshly isolated CD4+CD45RA+ T cells (2x105) were obtained from control (nonasthmatic/nonallergic) donors, activated with CD3/CD28 Ab, and cocultured in the absence or presence of autologous NK2 cells (2x105). Triplicate cultures were treated with 50 µM dbcAMP or vehicle control for 6 h and were stained for flow cytometry. Expression of CD40L was performed by gating on CD4+ events. cAMP-induced change in CD40L+ T cells = [(%CD40L+ cellscAMP-treated cultures–%CD40L+control cultures)/%CD40L+ T cellscontrol cultures] x 100. Data from five independent experiments are presented, and each symbol represents the change in CD40L expression in the presence or absence of NK2 cells. (B) In vitro-generated NK2 cells express CD95. CD56+CD3- NK cells were gated and analyzed for CD95 expression. A representative histogram is presented, and the inset number indicates the percentage of CD95 positively staining cells/MFI. Staining with isotype Ab is shown in gray and CD95 staining in black.

 
As shown in Figure 9B , CD95 expression on in vitro-generated NK2-like cells was elevated (43.0±13.6%; n=5). This finding is consistent with the increase frequency of CD95-positive NK cells in asthma, suggesting that a phenotypically related NK subset may be associated with asthma and may confer cAMP-enhancing effects.

Modest changes in CD40L produce biological effect
Previous studies have documented that modest increments in CD40L expression can produce significant biological effects with respect to Ab production, B cell proliferation, and monocyte responses in human and murine systems [38 , 43 , 44 ]. Although the magnitude of CD40L changes are remarkably similar to cAMP-induced increases described in this study, additional experiments were performed to further validate that abundance of CD40L is a critical factor that can limit biological response. The production of MMP-9 by monocytes was evaluated, as CD40L signaling via monocyte CD40 receptor induces MMP-9 [38 , 39 ], and a number of animal and human studies implicate MMP-9 in asthma pathogenesis and exacerbation [45 46 47 48 ]. Naive T cells were activated in the presence or absence of dbcAMP, cocultured with the monocytic THP-1 cell line, and MMP-9 production was evaluated by zymography. As shown in Figure 10 , a 1.8 ± 0.3-fold increase in MMP-9 production was paralleled by an increase (7.0±1.2% to 22.3±2.9%) in membrane-bound CD40L expression. Notably, the cAMP-induced change in percentage of CD40L-positive cells was of similar magnitude to increases observed in asthmatic T cells (Fig. 1 ; ~15% vs. ~10%). A T cell activation scheme involving calcium ionophore was used to simplify experiments, as cAMP-induced CD40L increases in ionophore-activated T cells have been described [25 ]. Overall, results are consistent with published studies demonstrating that relatively modest changes in CD40L expression can have marked effects on immune responses relevant to asthma pathogenesis or exacerbation.



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  		Figure 10. Changes in T cell CD40L expression correlate with MMP-9 production. CD45RA+ cells (>96% purity, positively selected) were activated with 0.25 µM ionomycin (Ion) ± 100 µM dbcAMP for 6 h. Cells were fixed with 1% paraformaldehyde and washed 3x, and aliquots were stained for CD40L expression by flow cytometry (A) or were cocultured with THP-1 cells at a 4:1 ratio (T:THP) for 36 h. MMP-9 in cell-free supernatants was analyzed by zymography and scanning laser densitometry (B). The inset gel depicts a representative zymogram; lane 1, THP cells; lane 2, THP cells treated with 5 µg/ml lipopolysaccharide; lane 3, THP and ionomycin-activated T cells; lane 4, THP and ionomycin/dbcAMP-treated T cells; lane 5, T cells alone. Error bars depict SE (n=3). OD, Optical density.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These data demonstrate that cAMP selectively increases T cell CD40L in asthmatic subjects and that a CD95+ NK cell subset is required for increased expression. These results are of importance not only because cAMP-induced increases in CD40L may be relevant to ß-agonist bronchodilator use but also because the presence of a characteristic NK cell population and changes in T cell CD40L regulation may be important in asthma pathogenesis. As even modest changes in CD40L expression have significant biological effects [38 , 43 , 44 ] (Fig. 10) , and CD40L is a critical control point in early immune response and IgE production [12 ], actions of an asthma-associated NK cell subset to facilitate ß-agonist and cAMP-induced increases in CD40L may have important clinical consequence. The importance of clinically apparent, adverse effects from ß-agonists still remains controversial [49 , 50 ]. However, the identification of actions that increase immune response may have major ramifications with respect to long-term exacerbation of the disease.

The difference in cAMP regulation from decreasing CD40L in control subjects to increasing CD40L in asthma is remarkable in view of the important regulatory role of CD40L. In fact, several studies have demonstrated that the level of CD40L expression on activated T cells is a critical factor limiting the immune response and that even modest increases produce substantial biological effects. Transgenic mice engineered with the CD40L gene under the control of the human IL-2 promoter have been used to demonstrate that a modest increase in surface CD40L (mean 29% CD40L-positive cells in transgenic mice vs. mean 18% positive cells in littermate controls) produces a marked increase (four- to fivefold) in total and high-affinity Ab production [43 ]. Importantly, this study evaluated the consequence of increasing CD40L expression levels while preserving physiological patterns of regulation by appropriate study design and promoter selection. Another study using nontransformed human T cell clones derived from the same donor determined that moderate changes in CD40L expression (mean 18% CD40L-positive compared with <=5% positive) are associated with a five- to 23-fold increase in B cell proliferation, 19-fold increase in IgG, and ninefold increase in IgM Ab production, which was neutralized by mAb specific for CD40L or CD40 [44 ]. Another study examining biological effects of recombinant CD40L determined that relatively minor increases in protein (25% increase, 0.6–0.8 µg/ml) produce a striking increase in MMP-3 production [38 ]. Notably, a 25% increase in protein concentration is of the same magnitude as changes in CD40L MFI staining observed in asthma (i.e., 21.0% increase in MFI). Lastly, data presented in Figure 10 are consistent with the ability of small changes in CD40L expression (7–22% CD40L-positive) to produce reproducible increases (1.8-fold) in monocyte MMP-9 expression, and this proteinase has been implicated in asthmatic pathogenesis, airway remodeling, and exacerbation [45 46 47 48 ]. Thus, deceptively small changes in CD40L expression, such as cAMP-induced increases observed in asthma, appear capable of affecting a number of immunological responses ranging from B cell proliferation, Ab production, and monocyte activation, which may be relevant to inflammatory processes leading to asthma pathogenesis or exacerbation.

The mechanisms underlying altered CD40L regulation in asthma were evaluated, and NK cell depletion and reconstitution experiments clearly implicate a regulatory role for NK cells in this process. Until recently, the main focus on NK cells has centered on their potent cytotoxic activity and production of IFN-{gamma}. However, these cells are becoming increasingly recognized as important regulators of the adaptive-immune response [42 , 51 52 53 ]. Recent evidence suggests NK cells regulate acquired T cell response in autoimmunity by affecting T cell proliferation, differentiation, and promotion of Th2 responses instead of Th1 responses [42 , 52 ]. Although the involvement of NK cells in asthma has not been directly investigated, a possible role has been suggested by studies indicating a correlation between NK cell activity and IgE levels in control subjects and increased NK cell activity and NK cell numbers in asthmatic peripheral blood [53 54 55 56 ]. Other supporting studies indicate peripheral blood NK cell activity is increased following bronchial challenge with specific antigen [57 ]. In addition, depletion of NK cells before immunization inhibited eosinophil and T cell lung infiltration and reduced expression of bronchoalveolar lavage IL-4 and IL-5 in murine models of allergen-induced airway inflammation [58 , 59 ]. These data are consistent with our observations indicating that asthmatic NK cells possess unique regulatory attributes affecting CD40L expression.

The mechanism by which asthmatic NK cells affect cAMP-regulated CD40L expression is of interest and pursued in this study. The release of cytokines by NK cells, particularly the newly recognized ability to release Th2 cytokines [37 , 42 ], was explored using a variety of experimental approaches including transwell cultures (Table 1) . The ability of cAMP to increase CD40L required direct NK–T cell contact and was not controlled by NK-derived, soluble mediators. Likewise, the addition of conditioned NK cell medium from asthmatic donors or the addition of exogenous cytokines (IL-4, IL-5, IL-10, and IL-18) failed to confer cAMP-enhancing effects, although IL-4 increased baseline CD40L expression (data not shown), as described previously [25 ].

These observations indicate asthmatic NK cells affect CD40L regulation by mechanisms involving alterations in NK cell-surface receptors with subsequent effects on T cell signal transduction. The consideration that NK cells include a number of cell subsets with similar morphological appearances but different function and phenotype is relatively new [37 , 42 , 60 61 62 ]. In support of this hypothesis, recent data demonstrate alterations in CD54 and L-selectin receptors on peripheral blood NK cells from asthmatic children with acute exacerbation [63 ]. Although disruption of CD54 signaling with blocking Ab failed to affect NK cell regulation of CD40L (data not shown), we pursued this line of investigation to determine whether additional NK phenotypic differences are associated with asthma.

To our knowledge, the observation that asthmatic NK cells express elevated levels of CD95 has not been previously recognized (Fig. 8) . We observed that NK cells from asthmatic subjects express significantly more CD95 compared with control subjects. It is of interest that the CD95 molecule has been described as a potential marker of the NK2 or NK1 subset depending on the authors. In a study by Peritt et al. [37 ], in vitro-generated NK2 cells expressed lower levels of CD95 relative to NK1 cells. The NK2 subset also expressed a pattern of cytokines normally ascribed to Th2 cells (i.e., IL-4, IL-5, IL10, IL-13, with low IFN-{gamma} production). The strength of this study resides in defined culture conditions used to polarize NK subsets combined with concomitant measures of Th1 and Th2 cytokine profiles. However, in vitro conditions and extended culture periods may contribute to responses not generally observed in vivo or associated with any particular disease state. A contrasting study by Takahashi et al. [42 ] demonstrated increased CD95 expression with concomitant Th2 cytokine production in NK cells isolated from patients in remission of multiple sclerosis. These attributes were described as a potential NK2 cell characteristic, as these properties were lost when disease relapse occurred. The strength of this study relies on the fact that elevated NK cell expression of CD95 was observed only in disease remission, was associated with concomitant NK cell production of IL-5, and was lost when relapse and presumably Th1 polarizing conditions predominated in vivo. Thus, it is presently unclear as to whether elevated NK cell expression of CD95 can be ascribed to either subset of NK cells or whether NK2 cells represent a truly distinct subset or an immature stage of NK cell differentiation [37 , 61 , 62 , 64 ]. Regardless, an important regulatory role of NK2 cells to bias T cell cytokine production toward a Th2 response [42 ] has been described and may be relevant to asthma pathogenesis.

The biological significance of increased CD95 expression on asthmatic NK cells remains speculative. However, it is of interest that only NK cells from asthmatics with active symptoms expressed elevated CD95 compared with control subjects or asymptomatic asthmatics (Fig. 8) . This observation suggests that NK cell CD95 expression may be a relatively sensitive marker of disease activity and may be clinically useful to identify NK subsets possessing low cytotoxic activity and enhanced cytokine capacity [60 ] or novel regulatory effects on T cell responses. It is also possible that elevated NK cell CD95 expression may be related to the regulation of survival and apoptosis of these cells. Elevated CD95 expression may serve as a means to eliminate this subset via activation of apoptotic pathways after the biological function of the cell has been fulfilled. It is also conceivable that soluble CD95 generated from this subset may play a protective role against CD95-mediated death of immune cells in asthma. Future studies investigating the biological role of elevated NK cell CD95 expression are required to address this issue.

As cytokine profiles high in IL-4 and low in IL-12 are associated with asthma [65 ] and required for in vitro generation of NK2 cells [37 , 42 ], experiments were performed to evaluate effects of NK2 cells on CD40L expression. NK2 cells were generated under polarizing culture conditions containing IL-4 and anti-IL-12-blocking Ab, as described previously [37 , 42 ]. These cells expressed CD95, as previously reported [37 ], and display similar regulatory properties to NK cells obtained from asthmatic subjects (Fig. 9) . These results indicate that the CD95+ NK cells observed in asthma (Fig. 8) share characteristics with the NK2 subset, although further studies including cytokine profiles are required before a final classification can be made.

Although further study is required to identify the exact pathway involved in cAMP/NK cell induction of CD40L, it is tempting to speculate that asthmatic NK cells may provide an accessory signal that increases T cell calcium mobilization. It is interesting that increases in T cell calcium flux provided by a variety of signals (i.e., CD2, CD99, endothelial cell costimulation, calcium ionophore) can reverse cAMP inhibition and result in increased CD40L expression [25 ] through a calcium-dependent calmodulin-dependent kinase IV pathway [26 ]. It is also of interest to note that elevated CD95 expression and the ability of asthmatic NK cells to regulate CD40L expression appear to require ongoing, inflammatory processes accompanying active asthma, as NK cells obtained from subjects previously diagnosed with seasonal or intermittent asthma but without current symptoms expressed minimal levels of CD95 and could not affect CD40L expression. These observations may explain why alterations in NK cell phenotype and function were not readily associated with IgE levels, pulmonary function, or asthma severity in this study, as pertinent information was obtained at study enrollment and not for all subsequent blood samplings.

As a NKT cell subset that generates IL-4 has been associated with allergic disease [66 ], some consideration was given to whether a NKT cell contributed to our observations [67 ]. NKT cells represent a subset of lymphocytes that are operationally defined by expression of T cell (i.e., CD3 and limited TCR-{alpha} chain) and NK cell markers (i.e., NK 1.1). NKT cells were unlikely to have contributed to enhancing effects, as these cells should have been present in CD16 depletion experiments (Fig. 6) yet eliminated in NK reconstitution experiments (Fig. 7) [66 ]. Additionally, no differences in the ratio of peripheral blood NKT cells/T cells were observed between asthmatics and controls (data not shown).

In conclusion, the results of this study suggest that NK cells may have a novel role in cAMP regulation of CD40L expression. A distinct NK cell subset that shares characteristics with the NK2 subset may be relevant to the pathogenesis and clinical presentation of asthma. Further evaluation of CD40L regulation and NK cell subsets in asthma may be of importance with respect to diagnostic evaluation and therapeutic intervention.


   ACKNOWLEDGEMENTS
 
The Office of Research and Development and Medical Research Service, Department of Veterans Affairs, the American Heart Association, and Mountain States Medical Research Institute/Mountain States Tumor Institute supported this work. We thank Margo Riggs for her invaluable assistance with pulmonary function testing and Kristen Forcier, Sarah Perusich, Eva Katahira, and Estelle Miller for their expertise with cell culture and flow cytometry.

Received March 12, 2003; revised April 30, 2003; accepted May 21, 2003.


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 DISCUSSION
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