The Role of Protein Kinase A Anchoring via the RII Regulatory Subunit in the Murine Immune System1
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免疫学杂志 2005年第11期
Abstract
Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RII regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RII regulatory subunit of PKA (–/–) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and –/– mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the –/– mice are similar to WT controls. In vitro, splenocytes from –/– mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8+ IFN- producing effector cells were stimulated upon infection of WT or –/– mice with Listeria monocytogenes. These data represent the first study of the role of RII in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RII –/– mice, suggesting either that RII is not required for normal immune function or that other proteins are able to compensate for RII function.
Introduction
T cell activation and proliferation are inhibited by the second messenger cyclic nucleotide, cAMP. Transcription factors NFAT and NF-B exhibit decreased activity in the presence of cAMP, resulting in reduced levels of IL-2 transcription (1, 2). The expression of several cell surface molecules such as CD-69, ICAM-1, and LFA is also diminished in the presence of cAMP, contributing to the ensuing absence of cell proliferation and inflammation (3, 4, 5, 6).
One mechanism by which cAMP may inhibit T cells is through activation of the serine/threonine protein kinase, protein kinase A (PKA). 3 PKA is a ubiquitously expressed, broad-specificity protein kinase. The catalytic activity of PKA is controlled via interactions between two catalytic subunits and a dimer of regulatory subunits. The catalytic subunits are inactive when bound to the regulatory subunits. Binding of cAMP by the regulatory subunits initiates the release and activation of the catalytic subunits. PKA has four regulatory subunit isoforms RI and and RII and . PKA containing RI isoforms is referred to as type I PKA, whereas PKA with RII isoforms is type II PKA (for review, see Ref. 7). Early reports suggested that RI and RII were the main regulatory subunit isoforms in human T cells (8); however, recent reports have demonstrated significant levels of expression of the RI and RII subunits in human T cells (9, 10). The expression of the regulatory subunits in murine T cells had never been characterized. As part of this study, we performed Western analyses and determined that murine T cells express all four regulatory subunits, RI, RI, RII, and small amounts of RII protein. RI, RII, and RII are expressed at lower levels in spleen and thymus than in brain, whereas RI is expressed more highly in spleen and thymus than in brain.
The spatial and temporal regulation of type II PKA activity is believed to occur through interactions with A-kinase anchoring proteins (AKAPs) (11). The regulatory subunit of PKA contains a conserved N-terminal domain that interacts with an amphipathic helix in the AKAP. The interaction between AKAPs and PKA functions to localize pools of PKA near activating elements, such as G proteins and transmembrane receptors, and in close proximity to substrates such as ion channels, mitochondria, cytoskeletal components, and cytoplasmic enzymes (12, 13, 14). PKA/AKAP binding can be disrupted using the anchoring inhibitor peptide, Ht31. Ht31 binds to the regulatory subunit of PKA at the conserved N-terminal domain preventing interaction with AKAPs (15). We previously identified several members of the AKAP family in human T cells, and Williams identified AKAPs in murine T cells (16). Using the Ht31 anchoring inhibitor peptide, Williams illustrated that anchored PKA is necessary for the cAMP-mediated inhibition of murine T cell activation in vitro. In addition, he demonstrated that anchored PKA contributes to maintaining the murine T cell in a resting state (17).
McKnight and colleagues (18) generated knockout mice for each regulatory subunit of PKA. RI knockout mice are embryonic lethal with severe developmental abnormalities. RI, RII, and RII knockout mice are all viable and fertile with varied cell type-specific phenotypes. Knockout of RI results in deficiencies in hippocampal long-term potentiation and long-term depression (19, 20). A targeted disruption of the RII gene, which is highly expressed in adipose tissue, displays marked alterations in both white and brown adipose tissue metabolism (21). Finally, although there is to date no reported phenotype for the RII –/– mice, the catalytic subunit is delocalized in mouse sperm lacking the RII regulatory subunit (22, 23).
To begin studying the role of PKA anchoring in cAMP-mediated inhibition of T cell activation in vivo, we studied the immune system of mice lacking the RII regulatory subunit of PKA. We found no gross morphological differences in the components of the immune system from the RII –/– mice. Spleen and thymus dissections from WT and knockout mice look the same. Single cell suspensions from these organs yield similar numbers of cells from WT and knockout mice. Flow cytometry analysis illustrates that the cell populations in the spleen and thymus of the knockout mice are also normal compared with WT controls, suggesting that T cell development is unaffected in the knockouts and that T cell homeostasis in the periphery is also typical. In vitro, splenocytes respond to CD3/CD28 and PMA/ionomycin stimulation and produce IL-2. Furthermore, CD4 purified T cells were able to differentiate into Th1 and Th2 cells as determined by cytokine production. Finally, the development of CD8+ effector cells is not altered in RII –/– mice, as similar frequencies of peptide-specific cells develop in knockout and WT mice following infection with the intracytoplasmic pathogen L. monocytogenes. To our knowledge, these are the first studies examining the role of RII in T lymphocytes in vivo. The results suggest either that the RII subunit is not important for T cell biology or that other proteins are compensating for RII function.
Materials and Methods
Sixteen to eighteen hours before use, 96-well nitrocellulose plates (Millipore) were coated with 100–500 ng/well anti-mouse IFN- capture Ab (BD Pharmingen) diluted in PBS and added in a volume of 100 μl. The plates were washed with sterile medium or sterile PBS 1 h before use and then blocked with cell culture medium (RPMI 1640 or DMEM) containing 5–10% FCS. The Ag-presenting population used for the ELISPOT assays is the RMAS (mouse lymphoid) cell line. RMAS cells, H2b haplotype, are defective for peptide transporters that mediate the translocation of peptides into the endoplasmic reticulum for subsequent association with MHC class I molecules. Because of this defect, the "peptide empty" class I molecules that traffic to the cell membrane are unstable and dissociate. However, at reduced temperatures, such as 25°C, peptide empty class I molecules will remain on the cell surface and present peptides that are added exogenously. The exogenous peptides will associate with the MHC class I molecules and stabilize the complex. If the cells are then placed at 37°C, the complex will remain on the cell surface and function as a target for peptide-specific CD8+ effector cells. For these experiments, RMAS cells were held at room temperature for 16–18 h, then resuspended at 1 x 106 cells/ml and pulsed with 1 x 10–7 M SIINFEKL peptide (an octameric peptide from OVA (OVA 257–264); SynPep). The peptide pulsed cells were incubated at room temperature for 2 h, washed twice with RPMI 1640, then added to the ELISPOT plates at 100,000 cells per well in a volume of 100 μl. Single cell suspensions of immune spleen cells from mice immunized 8 days previously with 800 CFU of OVA-expressing L. monocytogenes were prepared and added to the ELISPOT plates at 25,000–100,000 cells per well to the peptide pulsed RMAS cells. After 24 h incubation at 37°C the plates were washed four times with 0.05% Tween 20-PBS and a biotinylated anti-mouse IFN- detection Ab (BD Pharmingen) added at 500 ng/well in a volume of 100 μl. The plates were then incubated overnight at 4°C, washed four times with 0.05% Tween 20-PBS, and 100 μl of a 1:1,000 dilution of streptavidin-alkaline phosphatase (BD Pharmingen) then added. After 1 h at room temperature, the plates were washed four times with 0.05% Tween 20-PBS and then the detection substrate BCIP/NBT (KPL) was added to each well. After 5–20 min, the plates were washed with dH2O and allowed to dry. Spots are counted with a Zeiss microscopy unit equipped with KS Elispot software (Zeiss).
Results
PKA regulatory subunit isoform distribution
To facilitate our study of the role of PKA anchoring in T lymphocytes in the murine immune system, we performed a characterization of the distribution of the regulatory subunits in spleen and thymus. Western analyses for RI, RI, and RII, RII are presented in Fig. 1. Mouse tissue was prepared into lysate and 3 μg of brain, 0.3 μg of brain, 3 μg of spleen, and 3 μg of thymus extract were separated by SDS-PAGE and subjected to Western analysis using Abs from BD Pharmingen for RI, RI, and RII, and Abs from BIOMOL for RII. As illustrated, RI is expressed at higher levels in spleen and thymus than in brain (Fig. 1A). The difference in RI levels in spleen and thymus is due to slight alterations in protein levels visualized by Coomassie staining. The second panel of Fig. 1A, illustrates that RI is expressed at lower levels in spleen and thymus than in brain. This Ab cross reacts strongly with RI (24), thus care was taken to resolve the gel well and attribute staining based on size and in comparison with brain to RI. The third panel illustrates that RII is also expressed at much higher levels in brain than in spleen or thymus, because a 10-fold dilution of brain lysate (0.3 μg) is roughly equivalent to the protein detected in 3 μg of spleen or thymus lysate. Similarly, RII protein is expressed at much higher levels in brain than in spleen or thymus (Fig. 1A, bottom panel). Although the level of expression of each subunit can be compared between tissues, it is not possible to compare the level of expression of one isoform to another due to differences in Ab affinity. In summary, using the currently available reagents, the data suggest that all four regulatory subunit proteins RI, RI, RII, and RII are expressed in murine spleen and thymus.
Characterization of the immune systems of RII –/– mice
The RII regulatory subunit of PKA has been implicated as a mediator of cAMP signaling specificity via localization of the PKA catalytic subunit with AKAPs. To determine whether RII is required for cAMP-mediated inhibition of T cell activation in vivo, we characterized the immune systems of RII –/– mice. The generation of the RII –/– mice is described elsewhere (22). To characterize the immune systems of these mice, we began with dissection of spleen and thymus. The spleen (Fig. 2, bottom panel) and thymus (Fig. 2, top panel) from the knockout mice (Fig. 2, C–E) were the same size as those from WT mice (Fig. 2, A and B). RII –/– and WT thymus weighed 0.14 ± 0.01 g, RII –/– spleens weighed 0.099 ± 0.005 g, and WT spleens weighed 0.076 ± 0.003 g. Weights are averages of three organs from age-matched mice ± SEM.
Single cell suspensions from the spleen and thymus revealed similar cell numbers from WT and knockout mice (data not shown). Flow cytometric analysis of cells isolated from the spleen or thymus was conducted to look at T cell development (Fig. 3). For each comparison, two WT and four knockout mice were sacrificed. One representative of three experiments is shown. Data are presented as CD8 vs CD4 dot blots accompanied by CD3 histogram profiles for each quadrant (Fig. 3, A and B), and graphically, as percentage of events (Fig. 3C). Similar numbers of double positive CD4+CD8+ (top right), single positive CD4+CD8– (bottom right), and CD4–CD8+ (top left), and double negative CD4–CD8– (bottom right) cells were counted in the thymus of WT (Fig. 3, A and C) and RII –/– mice (Fig. 3, B and C). Likewise, similar numbers of single positive, CD4+CD8– and CD4–CD8+, cells were identified in the spleen (Fig. 3C). These results, in combination with histology from five knockout and two WT spleen and thymus that revealed no differences in organ development or cellular morphology (data not shown), suggest that thymic development and T cell homeostasis are normal in the RII –/– mice.
Response of splenocytes to anti-CD3/anti-CD28 or PMA/ionomycin stimulation and cAMP-mediated inhibition of T cell activation
cAMP is known to be a potent inhibitor of T cell activation. Williams demonstrated that the anchoring inhibitor peptide Ht31 stimulates T cell activation, suggesting that anchored PKA is necessary for maintaining the T cell in a resting state. In addition, Ht31 blocked camp-mediated inhibition of T cell activation (17). To determine whether PKA anchoring via RII is required for either T cell activation or camp-mediated inhibition of T cell activation, we isolated splenocytes from WT and RII –/– mice, stimulated them with either anti-CD3 and anti-CD28 or PMA and ionomycin, and measured IL-2 secretion. As illustrated in Fig. 4, stimulated splenocytes from RII –/– mice produce similar amounts of IL2 as WT by either anti-CD3/anti-CD28 (Fig. 4A) or PMA/ionomycin (Fig. 4B). Similarly, IL-2 production from stimulated knockout and WT splenocytes was equally inhibited by cAMP. Inhibition of IL-2, by cAMP production in cells stimulated with anti-CD3/anti-CD28 was always greater than inhibition in cells treated with PMA/ionomycin.
Th1/Th2 differentiation in RII –/– mice
Zhang and colleagues (25) reported apparently normal immune systems in cAMP response element-binding protein dominant-negative transgenic mice. However, upon further investigation they discovered that the mice had defective Th cell differentiation, which resulted in increased susceptibility to activation-induced cell death. To examine the role of RII in Th cell differentiation, we assayed the ability of splenocytes isolated from WT and RII –/– mice to differentiate in the presence of IL-4, anti-IL-12, and anti-IFN-, conditions that promote Th2 cell differentiation, or in the presence of IFN- and anti-IL-4, conditions that promote Th1 cell differentiation. Th1 and Th2 cell differentiation were then assayed by measuring IL-4 secretion (Fig. 5A) and IFN- secretion (Fig. 5B) using ELISA. The results presented in Fig. 5 illustrate that splenocytes isolated from both WT and RII –/– mice were able to produce similar levels of IL-4 and IFN-, indicating that differentiation into Th1 and Th2 cells is unaltered in the RII –/– splenocytes.
Response to infection with L. monocytogenes
The experiments described above have focused on immunologic parameters that can be studied ex vivo. To assess whether any defects in RII –/– mice can be detected in response to immunologic signals that are expected to occur in vivo, animals were infected with the intracytoplasmic pathogen L. monocytogenes (for review of the immune response to L. monocytogenes, see Ref. 26). The subsequent development of peptide-specific effector cells was then determined. WT C57BL/6 or RII –/– mice were immunized with a subclinical dose of L. monocytogenes, and the frequency of peptide-specific CD8+ effector cells was measured 8 days later, a time reflective of the peak of the cell-mediated immune response and a time in which the bacterium is completely cleared from the animals. For these studies we used a recombinant strain of L. monocytogenes that expresses OVA (27), thus allowing for frequency analysis of CD8+ T cells specific for the H2-Kb presented SIINFEKL peptide. Frequencies of IFN- secreting CD8+ effector cells present in immune spleen cell populations were measured by ELISPOT assays. Fig. 6 illustrates that robust SIINFEKL-specific responses develop in both the WT mice and the RII –/– mice following immunization with OVA-expressing L. monocytogenes, with frequencies of peptide-specific effector cells in the range of 200–250 per 100,000 immune cells. These data illustrate that development of peptide-specific CD8+ effector cells following infection with L. monocytogenes is not altered in RII –/– mice.
Western analysis of regulatory subunits in RII –/– mice
To examine the possibility that RI, RI, or RII is compensating for RII function via increased protein levels, we performed Western analysis on spleen and thymus from WT and RII –/– mice (Fig. 7). Densitometric quantitation of the RI bands (first panel) indicates no differences between WT and RII –/– mice. The second panel illustrates the Western analysis and quantitation for RI subunit. The apparent increase in protein expression in the third lane is the result of increased total protein load as seen by Coomassie staining and therefore cannot be attributed to a compensational increase in RI expression. As anticipated, and as illustrated for other tissues in the initial characterization of the RII –/– mice, RII is present in spleen, thymus, and brain of WT mice but absent from the spleen, thymus, and brain of the RII –/– mice. Coomassie stain is used to illustrate protein load in the WT and –/– brain samples (third panel and bottom figure). Finally, the fourth panel illustrates no significant differences in the expression of RII between WT and RII –/– mice. These data indicate that none of the regulatory subunits exhibit measurable increases in protein expression in the spleen or thymus when comparing WT and RII –/– mice.
Discussion
It has been known for years that T cell activation is inhibited by cAMP. One of the main effectors of cAMP is the cAMP-dependent protein kinase, PKA. PKA is a broad-specificity Ser/Thr protein kinase whose fidelity is maintained through interactions with AKAPs, proteins that anchor PKA in subcellular locations to facilitate substrate phosphorylation. Three articles have now been published on the presence of AKAPs in T lymphocytes (16, 17, 28). The most recent, by Williams (17), reports that when the anchoring inhibitor peptide, Ht31, is added to T cells in vitro, cAMP no longer inhibits T cell activation.
To begin studying the importance of PKA anchoring in T cell activation in vivo, we began by examining the immune system and immune response of RII –/– mice. We found no detectable differences in the gross morphology of the spleen or thymus of the RII –/– mice. In addition, we found the number of double positive, double negative, and single positive T cells in the thymus, and the number of single positive T cells in the spleen of RII –/– mice to be comparable with WT cell numbers. Finally, histology on the spleen and thymus of knockout mice look the same as those from WT mice. These results suggest that T cell development and selection in the thymus and maintenance in the periphery are unaffected by deletion of the RII regulatory subunit. The ability of the T cells to be activated was assayed by measuring IL-2 secretion in response to stimulation. T cells isolated from the RII –/– mice were stimulated to the same extent as WT mice by anti-CD3/anti-CD28 and by PMA/ionomycin stimulation and were inhibited to the same extent as WT mice by cAMP. No differences were seen in the ability of the T cells to differentiate into Th1 or Th2 cells and produce IFN- or IL-4, respectively. Finally, to examine the outcome of an immune response that develops solely in vivo, WT and RII –/– mice were infected with an OVA-expressing recombinant strain of the bacterial pathogen L. monocytogenes (see reference given above). Following immunization with a subclincal dose of bacteria (800 CFU), no differences could be detected in the frequencies of peptide-specific CD8+ effector T cells that subsequently developed. These results confirm the ex vivo data showing that the immune response potential of the RII –/– mouse functions are not altered in comparison with the WT C57BL/6 control mice.
The ability of Ht31 peptide to block the effects of cAMP in vitro suggests that anchored PKA is an important regulator of T cell activation (17). Our data illustrate that the RII subunit of PKA is not required for regulation of T cell activation. Although early reports suggested that RI and RII were the main regulatory subunit isoforms in human T cells (8), recent reports have demonstrated significant levels of expression of the RI and RII subunits in human T cells (9, 10). The expression of the regulatory subunits in murine T cells had never been characterized. As part of this study, we performed Western analysis and determined that murine T cells also express all four regulatory subunits in varying amounts in comparison with brain lysate. Although we can make statements about the expression of one subunit in different tissues, we cannot make a statement about the relative expression of one subunit in relation to another subunit due to differences in Ab affinities. For example we are able to conclude that RI is expressed at higher levels in spleen and thymus than in brain, and that RI, RII, and RII are all expressed at lower levels in spleen and thymus than in brain. However, we cannot draw any conclusions as to which subunit is the most highly expressed in brain, spleen, or thymus. We went on to examine by Western analysis whether any of the regulatory subunits were expressed at increased levels in the RII –/– mice, results that have been suggestive of compensation in other studies. For example, RI compensation has been shown in adult skeletal muscle and sperm of RII –/– mice, in the hippocampus and cerebral cortex of RI –/– mice, and in adipose tissue of RII –/– mice (18). In the RII –/– mice, RI compensation results in a PKA holoenzyme with a significantly increased basal activity, probably caused by a lower Ka of activation associated with the RI holoenzyme (21). The increased basal PKA activity leads to increased expression of the uncoupling protein 1 and altered lypolysis rates in white adipose tissue. In comparisons between WT and RII –/– spleen and thymus, we detected no differences in the levels of expression of the RI, RI, or RII subunits. In each of the above examples, increased quantities of RI are thought to be required for compensatory action, suggesting that our data may appear to argue against a role for compensation. However, we feel that proteins do not need to exhibit greater abundance to be compensating functionally. Compensation can also occur through a change in localization of the protein without increasing the quantity of the protein. Unfortunately, we are unable to determine the subcellular localization of the R subunits with the current available reagents, therefore this hypothesis will have to await the development of reagents before it can be tested. In other cell types, the RII –/– mice catalytic subunit of PKA remained colocalized with the L-type Ca2+ channel suggesting that RI is anchoring the catalytic subunit in the absence of RII (22). This interaction is known to be disrupted by the anchoring inhibitor peptide Ht31 (29). Surface plasmon resonance and equilibrium dialysis measurements confirmed that RI can associate with the amphipathic helix (Kd 2.1 μM), however, with a reduced affinity compared with RII (4 nM) (22). In addition, RI inhibited binding of RII to Ht31 protein in a solid phase overlay assay with an IC50 of 433 nM (22). Thus compensation by RI may very well be sufficient to regulate T cells in response to the stimuli used in these experiments.
In addition to compensation by RI binding to RII AKAPs, dual specificity AKAPs (D-AKAPs), which bind RI and RII, have also been identified (30, 31). D-AKAP1, also known as AKAP149, and D-AKAP2 have been identified in T cells (16, 30, 31). The importance of RI AKAPs and the RI-AKAP interaction will continue to grow as the evidence for the involvement of type 1 PKA in T cell activation and regulation keeps increasing. Type 1 PKA, PKA containing the RI regulatory subunit, has now been shown to be involved in regulating signaling through the TCR via phosphorylation of Csk (32, 33). Whether RI AKAPs are involved in this regulation and which RI AKAPs these may be are still unanswered questions. Unfortunately, RI –/– mice are embryonic lethal, preventing the performance of the above experiments in RI –/– mice or RI/RII double –/– mice. As mentioned above, the RI –/– mice and RII–/– mice are viable and fertile and exhibit cell type-specific defects. Examining the immune systems of these mice may lead to insights on the role of PKA anchoring in T cells in vivo. In addition, the generation of conditional, T cell-specific, RI –/– mice and conditional RI/RII double –/– mice will be valuable tools to further study the role of PKA anchoring in T cell activation in vivo.
Finally, Elliot et al. (34) have recently described new roles for RII and RI in the regulation of IL-2 production in human T lymphocytes. RII was shown to suppress CREB transcriptional activity and c-FOS production in T cells following TCR stimulation. In a follow-up study, overexpression of RII resulted in 90% reduction in IL-2 mRNA and IL-2 protein following T cell activation (24). Serine phosphorylation on S114 was shown to be required for both nuclear localization of RII and reduction of IL-2 production. In contrast, CD154 mRNA and cell surface expression were constitutively up-regulated by RII overexpression. These results suggest that RII regulatory subunit plays an important role in regulating T cell activation in human T lymphocytes. Unexpectedly, this work also illustrates that over expression of the RI subunit caused a 3- to 4-fold increase in IL-2 production following TCR stimulation and no change in CD154 expression. These authors present the argument that based on their data "... PKA can mediate variable and, in some cases, opposing signals in T cells." Examination of the immune systems of the RI, RII, or RII/RII double knockout mice will prove to be very interesting in future studies.
Footnotes
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 with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by Veterans Affairs Merit Review Funds (to D.W.C. and H.G.A.B.) and by National Institutes of Health Grant AI44376 (to H.G.A.B.).
2 Address correspondence and reprint requests to Dr. Daniel W. Carr, Veterans Affairs Medical Center, RD8, 3710 SW US Veterans Hospital Road, Portland, OR 97239. E-mail address: carrd{at}ohsu.edu
3 Abbreviations used in this paper: PKA, protein kinase A; AKAP, A-kinase anchoring protein; WT, wild type; -ME, 2-mercaptoethanol; D-AKAP, dual specificity AKAP; –/–, knockout; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP.
Received for publication February 10, 2004. Accepted for publication April 1, 2005.
References
Tsuruta, L., H. J. Lee, E. S. Masuda, N. Koyano-Nakagawa, N. Arai, K. Arai, T. Yokota. 1995. Cyclic AMP inhibits expression of the IL-2 gene through the nuclear factor of activated T cells (NF-AT) site, and transfection of NF-AT cDNAs abrogates the sensitivity of EL-4 cells to cyclic AMP. J. Immunol. 154: 5255-5264.
Ho, H. Y., H. H. Lee, M. Z. Lai. 1997. Overexpression of mitogen-activated protein kinase kinase kinase reversed cAMP inhibition of NF-B in T cells. Eur. J. Immunol. 27: 222-226.
Panettieri, R. A., Jr, A. L. Lazaar, E. Pure, S. M. Albelda. 1995. Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF--induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. J. Immunol. 154: 2358-2365.
Johnson, K. W., K. A. Smith. 1990. cAMP regulation of IL-2 receptor expression: selective modulation of the p75 subunit. J. Immunol. 145: 1144-1151.
Krause, D. S., C. Deutsch. 1991. Cyclic AMP directly inhibits IL-2 receptor expression in human T cells: expression of both p55 and p75 subunits is affected. J. Immunol. 146: 2285-2296.[
Kasyapa, C. S., C. L. Stentz, M. P. Davey, D. W. Carr. 1999. Regulation of IL-15-stimulated TNF- production by rolipram. J. Immunol. 163: 2836-2843.(Robynn V. Schillace*, Sar)
Intracellular cAMP may inhibit T cell activation and proliferation via activation of the cAMP-dependent protein kinase, PKA. PKA signaling is maintained through interactions of the regulatory subunit with A-kinase anchoring proteins (AKAPs). We demonstrated that T cells contain AKAPs and now ask whether PKA anchoring to AKAPs via the RII regulatory subunit is necessary for cAMP-mediated inhibition of T cell activation. We studied the immune systems of mice lacking the RII regulatory subunit of PKA (–/–) and the ability of cells isolated from these mice to respond to cAMP. Dissection of spleen and thymus from wild-type (WT) and –/– mice, single cell suspensions generated from these organs, and flow cytometry analysis illustrate that the gross morphology, cell numbers, and cell populations in the spleen and thymus of the –/– mice are similar to WT controls. In vitro, splenocytes from –/– mice respond to anti-CD3/anti-CD28 and PMA/ionomycin stimulation and produce IL-2 similar to WT. Cytokine analysis revealed no significant difference in Th1 or Th2 differentiation. Finally, equivalent frequencies of CD8+ IFN- producing effector cells were stimulated upon infection of WT or –/– mice with Listeria monocytogenes. These data represent the first study of the role of RII in the immune system in vivo and provide evidence that T cell development, homeostasis, and the generation of a cell-mediated immune response are not altered in the RII –/– mice, suggesting either that RII is not required for normal immune function or that other proteins are able to compensate for RII function.
Introduction
T cell activation and proliferation are inhibited by the second messenger cyclic nucleotide, cAMP. Transcription factors NFAT and NF-B exhibit decreased activity in the presence of cAMP, resulting in reduced levels of IL-2 transcription (1, 2). The expression of several cell surface molecules such as CD-69, ICAM-1, and LFA is also diminished in the presence of cAMP, contributing to the ensuing absence of cell proliferation and inflammation (3, 4, 5, 6).
One mechanism by which cAMP may inhibit T cells is through activation of the serine/threonine protein kinase, protein kinase A (PKA). 3 PKA is a ubiquitously expressed, broad-specificity protein kinase. The catalytic activity of PKA is controlled via interactions between two catalytic subunits and a dimer of regulatory subunits. The catalytic subunits are inactive when bound to the regulatory subunits. Binding of cAMP by the regulatory subunits initiates the release and activation of the catalytic subunits. PKA has four regulatory subunit isoforms RI and and RII and . PKA containing RI isoforms is referred to as type I PKA, whereas PKA with RII isoforms is type II PKA (for review, see Ref. 7). Early reports suggested that RI and RII were the main regulatory subunit isoforms in human T cells (8); however, recent reports have demonstrated significant levels of expression of the RI and RII subunits in human T cells (9, 10). The expression of the regulatory subunits in murine T cells had never been characterized. As part of this study, we performed Western analyses and determined that murine T cells express all four regulatory subunits, RI, RI, RII, and small amounts of RII protein. RI, RII, and RII are expressed at lower levels in spleen and thymus than in brain, whereas RI is expressed more highly in spleen and thymus than in brain.
The spatial and temporal regulation of type II PKA activity is believed to occur through interactions with A-kinase anchoring proteins (AKAPs) (11). The regulatory subunit of PKA contains a conserved N-terminal domain that interacts with an amphipathic helix in the AKAP. The interaction between AKAPs and PKA functions to localize pools of PKA near activating elements, such as G proteins and transmembrane receptors, and in close proximity to substrates such as ion channels, mitochondria, cytoskeletal components, and cytoplasmic enzymes (12, 13, 14). PKA/AKAP binding can be disrupted using the anchoring inhibitor peptide, Ht31. Ht31 binds to the regulatory subunit of PKA at the conserved N-terminal domain preventing interaction with AKAPs (15). We previously identified several members of the AKAP family in human T cells, and Williams identified AKAPs in murine T cells (16). Using the Ht31 anchoring inhibitor peptide, Williams illustrated that anchored PKA is necessary for the cAMP-mediated inhibition of murine T cell activation in vitro. In addition, he demonstrated that anchored PKA contributes to maintaining the murine T cell in a resting state (17).
McKnight and colleagues (18) generated knockout mice for each regulatory subunit of PKA. RI knockout mice are embryonic lethal with severe developmental abnormalities. RI, RII, and RII knockout mice are all viable and fertile with varied cell type-specific phenotypes. Knockout of RI results in deficiencies in hippocampal long-term potentiation and long-term depression (19, 20). A targeted disruption of the RII gene, which is highly expressed in adipose tissue, displays marked alterations in both white and brown adipose tissue metabolism (21). Finally, although there is to date no reported phenotype for the RII –/– mice, the catalytic subunit is delocalized in mouse sperm lacking the RII regulatory subunit (22, 23).
To begin studying the role of PKA anchoring in cAMP-mediated inhibition of T cell activation in vivo, we studied the immune system of mice lacking the RII regulatory subunit of PKA. We found no gross morphological differences in the components of the immune system from the RII –/– mice. Spleen and thymus dissections from WT and knockout mice look the same. Single cell suspensions from these organs yield similar numbers of cells from WT and knockout mice. Flow cytometry analysis illustrates that the cell populations in the spleen and thymus of the knockout mice are also normal compared with WT controls, suggesting that T cell development is unaffected in the knockouts and that T cell homeostasis in the periphery is also typical. In vitro, splenocytes respond to CD3/CD28 and PMA/ionomycin stimulation and produce IL-2. Furthermore, CD4 purified T cells were able to differentiate into Th1 and Th2 cells as determined by cytokine production. Finally, the development of CD8+ effector cells is not altered in RII –/– mice, as similar frequencies of peptide-specific cells develop in knockout and WT mice following infection with the intracytoplasmic pathogen L. monocytogenes. To our knowledge, these are the first studies examining the role of RII in T lymphocytes in vivo. The results suggest either that the RII subunit is not important for T cell biology or that other proteins are compensating for RII function.
Materials and Methods
Sixteen to eighteen hours before use, 96-well nitrocellulose plates (Millipore) were coated with 100–500 ng/well anti-mouse IFN- capture Ab (BD Pharmingen) diluted in PBS and added in a volume of 100 μl. The plates were washed with sterile medium or sterile PBS 1 h before use and then blocked with cell culture medium (RPMI 1640 or DMEM) containing 5–10% FCS. The Ag-presenting population used for the ELISPOT assays is the RMAS (mouse lymphoid) cell line. RMAS cells, H2b haplotype, are defective for peptide transporters that mediate the translocation of peptides into the endoplasmic reticulum for subsequent association with MHC class I molecules. Because of this defect, the "peptide empty" class I molecules that traffic to the cell membrane are unstable and dissociate. However, at reduced temperatures, such as 25°C, peptide empty class I molecules will remain on the cell surface and present peptides that are added exogenously. The exogenous peptides will associate with the MHC class I molecules and stabilize the complex. If the cells are then placed at 37°C, the complex will remain on the cell surface and function as a target for peptide-specific CD8+ effector cells. For these experiments, RMAS cells were held at room temperature for 16–18 h, then resuspended at 1 x 106 cells/ml and pulsed with 1 x 10–7 M SIINFEKL peptide (an octameric peptide from OVA (OVA 257–264); SynPep). The peptide pulsed cells were incubated at room temperature for 2 h, washed twice with RPMI 1640, then added to the ELISPOT plates at 100,000 cells per well in a volume of 100 μl. Single cell suspensions of immune spleen cells from mice immunized 8 days previously with 800 CFU of OVA-expressing L. monocytogenes were prepared and added to the ELISPOT plates at 25,000–100,000 cells per well to the peptide pulsed RMAS cells. After 24 h incubation at 37°C the plates were washed four times with 0.05% Tween 20-PBS and a biotinylated anti-mouse IFN- detection Ab (BD Pharmingen) added at 500 ng/well in a volume of 100 μl. The plates were then incubated overnight at 4°C, washed four times with 0.05% Tween 20-PBS, and 100 μl of a 1:1,000 dilution of streptavidin-alkaline phosphatase (BD Pharmingen) then added. After 1 h at room temperature, the plates were washed four times with 0.05% Tween 20-PBS and then the detection substrate BCIP/NBT (KPL) was added to each well. After 5–20 min, the plates were washed with dH2O and allowed to dry. Spots are counted with a Zeiss microscopy unit equipped with KS Elispot software (Zeiss).
Results
PKA regulatory subunit isoform distribution
To facilitate our study of the role of PKA anchoring in T lymphocytes in the murine immune system, we performed a characterization of the distribution of the regulatory subunits in spleen and thymus. Western analyses for RI, RI, and RII, RII are presented in Fig. 1. Mouse tissue was prepared into lysate and 3 μg of brain, 0.3 μg of brain, 3 μg of spleen, and 3 μg of thymus extract were separated by SDS-PAGE and subjected to Western analysis using Abs from BD Pharmingen for RI, RI, and RII, and Abs from BIOMOL for RII. As illustrated, RI is expressed at higher levels in spleen and thymus than in brain (Fig. 1A). The difference in RI levels in spleen and thymus is due to slight alterations in protein levels visualized by Coomassie staining. The second panel of Fig. 1A, illustrates that RI is expressed at lower levels in spleen and thymus than in brain. This Ab cross reacts strongly with RI (24), thus care was taken to resolve the gel well and attribute staining based on size and in comparison with brain to RI. The third panel illustrates that RII is also expressed at much higher levels in brain than in spleen or thymus, because a 10-fold dilution of brain lysate (0.3 μg) is roughly equivalent to the protein detected in 3 μg of spleen or thymus lysate. Similarly, RII protein is expressed at much higher levels in brain than in spleen or thymus (Fig. 1A, bottom panel). Although the level of expression of each subunit can be compared between tissues, it is not possible to compare the level of expression of one isoform to another due to differences in Ab affinity. In summary, using the currently available reagents, the data suggest that all four regulatory subunit proteins RI, RI, RII, and RII are expressed in murine spleen and thymus.
Characterization of the immune systems of RII –/– mice
The RII regulatory subunit of PKA has been implicated as a mediator of cAMP signaling specificity via localization of the PKA catalytic subunit with AKAPs. To determine whether RII is required for cAMP-mediated inhibition of T cell activation in vivo, we characterized the immune systems of RII –/– mice. The generation of the RII –/– mice is described elsewhere (22). To characterize the immune systems of these mice, we began with dissection of spleen and thymus. The spleen (Fig. 2, bottom panel) and thymus (Fig. 2, top panel) from the knockout mice (Fig. 2, C–E) were the same size as those from WT mice (Fig. 2, A and B). RII –/– and WT thymus weighed 0.14 ± 0.01 g, RII –/– spleens weighed 0.099 ± 0.005 g, and WT spleens weighed 0.076 ± 0.003 g. Weights are averages of three organs from age-matched mice ± SEM.
Single cell suspensions from the spleen and thymus revealed similar cell numbers from WT and knockout mice (data not shown). Flow cytometric analysis of cells isolated from the spleen or thymus was conducted to look at T cell development (Fig. 3). For each comparison, two WT and four knockout mice were sacrificed. One representative of three experiments is shown. Data are presented as CD8 vs CD4 dot blots accompanied by CD3 histogram profiles for each quadrant (Fig. 3, A and B), and graphically, as percentage of events (Fig. 3C). Similar numbers of double positive CD4+CD8+ (top right), single positive CD4+CD8– (bottom right), and CD4–CD8+ (top left), and double negative CD4–CD8– (bottom right) cells were counted in the thymus of WT (Fig. 3, A and C) and RII –/– mice (Fig. 3, B and C). Likewise, similar numbers of single positive, CD4+CD8– and CD4–CD8+, cells were identified in the spleen (Fig. 3C). These results, in combination with histology from five knockout and two WT spleen and thymus that revealed no differences in organ development or cellular morphology (data not shown), suggest that thymic development and T cell homeostasis are normal in the RII –/– mice.
Response of splenocytes to anti-CD3/anti-CD28 or PMA/ionomycin stimulation and cAMP-mediated inhibition of T cell activation
cAMP is known to be a potent inhibitor of T cell activation. Williams demonstrated that the anchoring inhibitor peptide Ht31 stimulates T cell activation, suggesting that anchored PKA is necessary for maintaining the T cell in a resting state. In addition, Ht31 blocked camp-mediated inhibition of T cell activation (17). To determine whether PKA anchoring via RII is required for either T cell activation or camp-mediated inhibition of T cell activation, we isolated splenocytes from WT and RII –/– mice, stimulated them with either anti-CD3 and anti-CD28 or PMA and ionomycin, and measured IL-2 secretion. As illustrated in Fig. 4, stimulated splenocytes from RII –/– mice produce similar amounts of IL2 as WT by either anti-CD3/anti-CD28 (Fig. 4A) or PMA/ionomycin (Fig. 4B). Similarly, IL-2 production from stimulated knockout and WT splenocytes was equally inhibited by cAMP. Inhibition of IL-2, by cAMP production in cells stimulated with anti-CD3/anti-CD28 was always greater than inhibition in cells treated with PMA/ionomycin.
Th1/Th2 differentiation in RII –/– mice
Zhang and colleagues (25) reported apparently normal immune systems in cAMP response element-binding protein dominant-negative transgenic mice. However, upon further investigation they discovered that the mice had defective Th cell differentiation, which resulted in increased susceptibility to activation-induced cell death. To examine the role of RII in Th cell differentiation, we assayed the ability of splenocytes isolated from WT and RII –/– mice to differentiate in the presence of IL-4, anti-IL-12, and anti-IFN-, conditions that promote Th2 cell differentiation, or in the presence of IFN- and anti-IL-4, conditions that promote Th1 cell differentiation. Th1 and Th2 cell differentiation were then assayed by measuring IL-4 secretion (Fig. 5A) and IFN- secretion (Fig. 5B) using ELISA. The results presented in Fig. 5 illustrate that splenocytes isolated from both WT and RII –/– mice were able to produce similar levels of IL-4 and IFN-, indicating that differentiation into Th1 and Th2 cells is unaltered in the RII –/– splenocytes.
Response to infection with L. monocytogenes
The experiments described above have focused on immunologic parameters that can be studied ex vivo. To assess whether any defects in RII –/– mice can be detected in response to immunologic signals that are expected to occur in vivo, animals were infected with the intracytoplasmic pathogen L. monocytogenes (for review of the immune response to L. monocytogenes, see Ref. 26). The subsequent development of peptide-specific effector cells was then determined. WT C57BL/6 or RII –/– mice were immunized with a subclinical dose of L. monocytogenes, and the frequency of peptide-specific CD8+ effector cells was measured 8 days later, a time reflective of the peak of the cell-mediated immune response and a time in which the bacterium is completely cleared from the animals. For these studies we used a recombinant strain of L. monocytogenes that expresses OVA (27), thus allowing for frequency analysis of CD8+ T cells specific for the H2-Kb presented SIINFEKL peptide. Frequencies of IFN- secreting CD8+ effector cells present in immune spleen cell populations were measured by ELISPOT assays. Fig. 6 illustrates that robust SIINFEKL-specific responses develop in both the WT mice and the RII –/– mice following immunization with OVA-expressing L. monocytogenes, with frequencies of peptide-specific effector cells in the range of 200–250 per 100,000 immune cells. These data illustrate that development of peptide-specific CD8+ effector cells following infection with L. monocytogenes is not altered in RII –/– mice.
Western analysis of regulatory subunits in RII –/– mice
To examine the possibility that RI, RI, or RII is compensating for RII function via increased protein levels, we performed Western analysis on spleen and thymus from WT and RII –/– mice (Fig. 7). Densitometric quantitation of the RI bands (first panel) indicates no differences between WT and RII –/– mice. The second panel illustrates the Western analysis and quantitation for RI subunit. The apparent increase in protein expression in the third lane is the result of increased total protein load as seen by Coomassie staining and therefore cannot be attributed to a compensational increase in RI expression. As anticipated, and as illustrated for other tissues in the initial characterization of the RII –/– mice, RII is present in spleen, thymus, and brain of WT mice but absent from the spleen, thymus, and brain of the RII –/– mice. Coomassie stain is used to illustrate protein load in the WT and –/– brain samples (third panel and bottom figure). Finally, the fourth panel illustrates no significant differences in the expression of RII between WT and RII –/– mice. These data indicate that none of the regulatory subunits exhibit measurable increases in protein expression in the spleen or thymus when comparing WT and RII –/– mice.
Discussion
It has been known for years that T cell activation is inhibited by cAMP. One of the main effectors of cAMP is the cAMP-dependent protein kinase, PKA. PKA is a broad-specificity Ser/Thr protein kinase whose fidelity is maintained through interactions with AKAPs, proteins that anchor PKA in subcellular locations to facilitate substrate phosphorylation. Three articles have now been published on the presence of AKAPs in T lymphocytes (16, 17, 28). The most recent, by Williams (17), reports that when the anchoring inhibitor peptide, Ht31, is added to T cells in vitro, cAMP no longer inhibits T cell activation.
To begin studying the importance of PKA anchoring in T cell activation in vivo, we began by examining the immune system and immune response of RII –/– mice. We found no detectable differences in the gross morphology of the spleen or thymus of the RII –/– mice. In addition, we found the number of double positive, double negative, and single positive T cells in the thymus, and the number of single positive T cells in the spleen of RII –/– mice to be comparable with WT cell numbers. Finally, histology on the spleen and thymus of knockout mice look the same as those from WT mice. These results suggest that T cell development and selection in the thymus and maintenance in the periphery are unaffected by deletion of the RII regulatory subunit. The ability of the T cells to be activated was assayed by measuring IL-2 secretion in response to stimulation. T cells isolated from the RII –/– mice were stimulated to the same extent as WT mice by anti-CD3/anti-CD28 and by PMA/ionomycin stimulation and were inhibited to the same extent as WT mice by cAMP. No differences were seen in the ability of the T cells to differentiate into Th1 or Th2 cells and produce IFN- or IL-4, respectively. Finally, to examine the outcome of an immune response that develops solely in vivo, WT and RII –/– mice were infected with an OVA-expressing recombinant strain of the bacterial pathogen L. monocytogenes (see reference given above). Following immunization with a subclincal dose of bacteria (800 CFU), no differences could be detected in the frequencies of peptide-specific CD8+ effector T cells that subsequently developed. These results confirm the ex vivo data showing that the immune response potential of the RII –/– mouse functions are not altered in comparison with the WT C57BL/6 control mice.
The ability of Ht31 peptide to block the effects of cAMP in vitro suggests that anchored PKA is an important regulator of T cell activation (17). Our data illustrate that the RII subunit of PKA is not required for regulation of T cell activation. Although early reports suggested that RI and RII were the main regulatory subunit isoforms in human T cells (8), recent reports have demonstrated significant levels of expression of the RI and RII subunits in human T cells (9, 10). The expression of the regulatory subunits in murine T cells had never been characterized. As part of this study, we performed Western analysis and determined that murine T cells also express all four regulatory subunits in varying amounts in comparison with brain lysate. Although we can make statements about the expression of one subunit in different tissues, we cannot make a statement about the relative expression of one subunit in relation to another subunit due to differences in Ab affinities. For example we are able to conclude that RI is expressed at higher levels in spleen and thymus than in brain, and that RI, RII, and RII are all expressed at lower levels in spleen and thymus than in brain. However, we cannot draw any conclusions as to which subunit is the most highly expressed in brain, spleen, or thymus. We went on to examine by Western analysis whether any of the regulatory subunits were expressed at increased levels in the RII –/– mice, results that have been suggestive of compensation in other studies. For example, RI compensation has been shown in adult skeletal muscle and sperm of RII –/– mice, in the hippocampus and cerebral cortex of RI –/– mice, and in adipose tissue of RII –/– mice (18). In the RII –/– mice, RI compensation results in a PKA holoenzyme with a significantly increased basal activity, probably caused by a lower Ka of activation associated with the RI holoenzyme (21). The increased basal PKA activity leads to increased expression of the uncoupling protein 1 and altered lypolysis rates in white adipose tissue. In comparisons between WT and RII –/– spleen and thymus, we detected no differences in the levels of expression of the RI, RI, or RII subunits. In each of the above examples, increased quantities of RI are thought to be required for compensatory action, suggesting that our data may appear to argue against a role for compensation. However, we feel that proteins do not need to exhibit greater abundance to be compensating functionally. Compensation can also occur through a change in localization of the protein without increasing the quantity of the protein. Unfortunately, we are unable to determine the subcellular localization of the R subunits with the current available reagents, therefore this hypothesis will have to await the development of reagents before it can be tested. In other cell types, the RII –/– mice catalytic subunit of PKA remained colocalized with the L-type Ca2+ channel suggesting that RI is anchoring the catalytic subunit in the absence of RII (22). This interaction is known to be disrupted by the anchoring inhibitor peptide Ht31 (29). Surface plasmon resonance and equilibrium dialysis measurements confirmed that RI can associate with the amphipathic helix (Kd 2.1 μM), however, with a reduced affinity compared with RII (4 nM) (22). In addition, RI inhibited binding of RII to Ht31 protein in a solid phase overlay assay with an IC50 of 433 nM (22). Thus compensation by RI may very well be sufficient to regulate T cells in response to the stimuli used in these experiments.
In addition to compensation by RI binding to RII AKAPs, dual specificity AKAPs (D-AKAPs), which bind RI and RII, have also been identified (30, 31). D-AKAP1, also known as AKAP149, and D-AKAP2 have been identified in T cells (16, 30, 31). The importance of RI AKAPs and the RI-AKAP interaction will continue to grow as the evidence for the involvement of type 1 PKA in T cell activation and regulation keeps increasing. Type 1 PKA, PKA containing the RI regulatory subunit, has now been shown to be involved in regulating signaling through the TCR via phosphorylation of Csk (32, 33). Whether RI AKAPs are involved in this regulation and which RI AKAPs these may be are still unanswered questions. Unfortunately, RI –/– mice are embryonic lethal, preventing the performance of the above experiments in RI –/– mice or RI/RII double –/– mice. As mentioned above, the RI –/– mice and RII–/– mice are viable and fertile and exhibit cell type-specific defects. Examining the immune systems of these mice may lead to insights on the role of PKA anchoring in T cells in vivo. In addition, the generation of conditional, T cell-specific, RI –/– mice and conditional RI/RII double –/– mice will be valuable tools to further study the role of PKA anchoring in T cell activation in vivo.
Finally, Elliot et al. (34) have recently described new roles for RII and RI in the regulation of IL-2 production in human T lymphocytes. RII was shown to suppress CREB transcriptional activity and c-FOS production in T cells following TCR stimulation. In a follow-up study, overexpression of RII resulted in 90% reduction in IL-2 mRNA and IL-2 protein following T cell activation (24). Serine phosphorylation on S114 was shown to be required for both nuclear localization of RII and reduction of IL-2 production. In contrast, CD154 mRNA and cell surface expression were constitutively up-regulated by RII overexpression. These results suggest that RII regulatory subunit plays an important role in regulating T cell activation in human T lymphocytes. Unexpectedly, this work also illustrates that over expression of the RI subunit caused a 3- to 4-fold increase in IL-2 production following TCR stimulation and no change in CD154 expression. These authors present the argument that based on their data "... PKA can mediate variable and, in some cases, opposing signals in T cells." Examination of the immune systems of the RI, RII, or RII/RII double knockout mice will prove to be very interesting in future studies.
Footnotes
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 with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This research was supported by Veterans Affairs Merit Review Funds (to D.W.C. and H.G.A.B.) and by National Institutes of Health Grant AI44376 (to H.G.A.B.).
2 Address correspondence and reprint requests to Dr. Daniel W. Carr, Veterans Affairs Medical Center, RD8, 3710 SW US Veterans Hospital Road, Portland, OR 97239. E-mail address: carrd{at}ohsu.edu
3 Abbreviations used in this paper: PKA, protein kinase A; AKAP, A-kinase anchoring protein; WT, wild type; -ME, 2-mercaptoethanol; D-AKAP, dual specificity AKAP; –/–, knockout; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP.
Received for publication February 10, 2004. Accepted for publication April 1, 2005.
References
Tsuruta, L., H. J. Lee, E. S. Masuda, N. Koyano-Nakagawa, N. Arai, K. Arai, T. Yokota. 1995. Cyclic AMP inhibits expression of the IL-2 gene through the nuclear factor of activated T cells (NF-AT) site, and transfection of NF-AT cDNAs abrogates the sensitivity of EL-4 cells to cyclic AMP. J. Immunol. 154: 5255-5264.
Ho, H. Y., H. H. Lee, M. Z. Lai. 1997. Overexpression of mitogen-activated protein kinase kinase kinase reversed cAMP inhibition of NF-B in T cells. Eur. J. Immunol. 27: 222-226.
Panettieri, R. A., Jr, A. L. Lazaar, E. Pure, S. M. Albelda. 1995. Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF--induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. J. Immunol. 154: 2358-2365.
Johnson, K. W., K. A. Smith. 1990. cAMP regulation of IL-2 receptor expression: selective modulation of the p75 subunit. J. Immunol. 145: 1144-1151.
Krause, D. S., C. Deutsch. 1991. Cyclic AMP directly inhibits IL-2 receptor expression in human T cells: expression of both p55 and p75 subunits is affected. J. Immunol. 146: 2285-2296.[
Kasyapa, C. S., C. L. Stentz, M. P. Davey, D. W. Carr. 1999. Regulation of IL-15-stimulated TNF- production by rolipram. J. Immunol. 163: 2836-2843.(Robynn V. Schillace*, Sar)