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Increased cAMP levels and protein kinase (PKA) type I activation in CD4⁺ T cells and B cells contribute to retrovirus-induced immunodeficiency of mice (MAIDS): a useful in vivo model for drug testing¹

Department of Pathology, University of Liège, Liège, Belgium; and
^* Institute of Medical Biochemistry, University of Oslo, Oslo, Norway

²Correspondence: CHU Sart-Tilman, 4000 Liège, Belgium. E-mail: mmoutschen{at}chu.ulg.ac.be

SPECIFIC AIMS

We addressed the hypothesis that aberrant activation of cyclic AMP (cAMP) -dependent protein kinase A type I is involved in the pathogenesis of the murine AIDS (MAIDS) model and in the unusual CD90^- phenotype displayed by a large fraction of CD4⁺ T cells in retrovirally infected mice. Selective PKA type I antagonists were evaluated for their potential to restore T cell responses of infected mice in an ex vivo model.

PRINCIPAL FINDINGS

Increased levels of cAMP in CD4⁺ T cells and B cells of mice infected with the MAIDS virus
Mice were killed at different times after viral inoculation and cAMP levels were measured in three different lymphocyte subsets (CD8⁺ T cells, CD4⁺ T cells, and B cells) (Fig. 1 ). There was a major increase in cAMP levels in the CD4⁺ T cells of the infected mice. This phenomenon peaked at wk 4 with an 30-fold increase above cAMP levels of control mice. B cells displayed a similar increase in cAMP levels but the plateau was reached at wk 7, later than in CD4⁺ T cells. In contrast with CD4⁺ T cells and B cells, CD8⁺ T cells from infected mice were characterized by a less pronounced rise in cAMP levels, which were never higher than eightfold control levels.

View larger version (17K): [in this window] [in a new window]   Figure 1. Evolution of cAMP levels in the course of MAIDS in CD8+ T cells (A), CD4+ T cells (B), and B cells (C). CD8+ T cells, CD4+ T cells and B cells were negatively sorted from the lymph nodes of infected mice by FACS. After sonication, cAMP levels were measured by radioimmunoassay. At each time point, three mice were individually analyzed (means ±SD are shown).

Higher concentrations of cAMP in CD4⁺ CD90^- vs. CD4⁺ CD90⁺ T cells of the infected mice
CD4⁺ CD90^- T cells are strongly expanded in MAIDS and play an important role in the pathogenesis of the syndrome. In infected mice, highly purified CD4⁺ CD90^- T cells were characterized by a sixfold higher cAMP concentration than the CD4⁺ CD90⁺ T cells. The rare CD4⁺ CD90^- T cells, which exist in the germinal centers of normal mice, were also characterized by a higher cAMP level than the CD4⁺ CD90⁺ T cells of the same animals. Since cAMP has been shown to induce the down-regulation of CD90 in thymocytes at the mRNA level, we compared by semiquantitative RT-PCR the level of CD90 mRNA in sorted CD4⁺ CD90⁺ vs. CD90^- cells of the infected mice. In comparison with CD4⁺ CD90⁺, CD4⁺ CD90^- T cells had three- to fourfold lower levels of CD90 mRNA.

Increased sensitivity to inhibition by the cAMP analog 8-CPT-cAMP in murine AIDS
The cAMP analog 8-CPT-cAMP inhibits in vitro proliferative responses of T cells. The effect of this agonist on T cell receptor (TCR)/CD3-induced proliferation was investigated in five infected mice and four controls. T cells from infected mice revealed an increased sensitivity to inhibition by exogenously added 8-CPT-cAMP. In addition to a left shift, the slopes of the inhibition curves were significantly different, with Hill coefficients of 0.58 (0.54–1.52) for T cells from MAIDS mice vs. 2.24 (1.93–2.47) for normal T cells (P<0.05). These results demonstrate a contribution from elevated endogenous cAMP in priming cAMP binding site B of PKA type I with subsequent increase in the affinity of the A site for the exogenously added cAMP analog.

Whereas total PKA type I protein levels remain unchanged in LN lymphocytes from RadLV-Rs-infected mice, cytoplasmic PKA activity goes down
PKA type I subunits were compared in infected mice vs. controls. Immunoblotting of lymphocyte detergent-solubilized extracts revealed similar levels of RI in controls and infected mice after viral inoculation. Similarly, the level of C subunit was identical in infected animals and controls. Examination of PKA phosphotransferase activity in the postnuclear, detergent-solubilized extracts revealed that total levels of cAMP-dependent kinase activity were decreased in MAIDS lymph node cells whereas minor changes in the activity were observed in the absence of cAMP. This is consistent with a chronic activation and dissociation of PKA leading to partial degradation or inactivation of the C subunit and/or to translocation of C to other cellular compartments. Assessment of cAMP binding revealed no changes in total levels of PKA-R subunits.

The catalytic subunit of PKA is translocated to the nucleus in the lymphocytes of infected mice
Using immunocytochemistry on cytospins prepared from lymph node cell suspensions, we compared the subcellular localization of PKA-C in infected vs. control animals (Fig. 2 ). In control mice, the brown coloration indicating PKA-C specific immunostaining was mainly restricted to the cytoplasm, with a scarce speckled staining in some nuclei. In contrast, the nuclei appeared with a stronger and more homogenous staining in the infected mice. The demonstration of increased nuclear C subunit is consistent with the observation that total C subunit levels are unchanged whereas cytoplasmic PKA activity is reduced.

View larger version (69K): [in this window] [in a new window]   Figure 2. Subcellular localization of PKA-C subunit in LN lymphocytes from control (A) and infected (B) mice. Cytospins were immunostained with anti-PKA-C and counterstained with hematoxylin-eosin. Brown indicates the PKA-C-specific staining and blue represents the hematoxylin staining of nucleus.

PKA type I antagonist restores T cell proliferation from RadLV-Rs-infected mice
A sulfur-substituted cAMP analog (Rp-8-Br-cAMPS) working as a full antagonist for PKA type I was used to see whether it could restore lymphocyte proliferative responses in vitro. In T cells from infected mice, TCR/CD3-stimulated proliferation was less than 10% of controls. Incubation with the PKA type I antagonist allowed a concentration-dependent increase in TCR/CD3-induced proliferation that was more than fourfold at higher concentrations, whereas no enhancement was observed by treatment of control T cells. The stimulatory effect of the PKA I antagonist was not saturated even at the highest concentrations used. This indicates that the solubility of the compound, affinity, or availability to cells may be a limiting factor for the effect observed.

CONCLUSION AND SIGNIFICANCE

Cyclic AMP inhibits various aspects of immune responses in vitro. Moreover, the involvement of the cAMP-PKA type I pathway has recently been proposed to be involved in T cell dysfunction associated with HIV infection and common variable immunodeficiency, opening the way for new therapeutic options in these diseases. In this study, which focuses on murine AIDS, we demonstrate a dramatic up-regulation of cAMP in CD4⁺ T cells, B cells, and to a lesser extent CD8⁺ T cells. This cAMP increase induces hyperactivation of PKA type I and nuclear translocation of its C subunit in the lymphocytes of the infected animals. Furthermore, partial restoration of lymphocyte proliferative responses is obtained by treating T cells with PKA type I selective antagonists ex vivo.

An involvement of cAMP and PKA type I has never been described in MAIDS, although the participation of soluble factors in the induction of anergy was hypothesized by several groups years ago. Our findings may help explain several aspects of the murine syndrome. Lymphocytes from infected mice fail to respond to TCR stimulation even in the presence of a high concentration of IL-2. This evokes the existence of a block in the IL-2 receptor signaling pathway. cAMP was recently shown to induce a strong JAK3 down-regulation in T cells downstream of IL-2. Although cAMP also acts at other proximal and distal sites via PKA-dependent and PKA-independent mechanisms, down-regulation of JAK3 is a plausible mechanism by which cAMP could be responsible for the profound lack of IL-2 response, which characterizes MAIDS. MAIDS is also associated with a preferential involvement of CD4⁺ T cells whereas the alteration of CD8⁺ T cells is at least partially due to the lack of adequate help from CD4⁺ T cells. Our observation of a strikingly more pronounced increase of cAMP in CD4⁺ T cells and B cells than in CD8⁺ T cells is therefore compatible with this finding. If a soluble factor is indeed responsible for cAMP induction in MAIDS, what could explain the subset selectivity of its effect since normal CD8⁺ T cells are fully susceptible to the cAMP-inducing effects of different agents? Inflammatory states such as rheumatoid arthritis are characterized by a down-regulation of G receptor kinases (GRK) and therefore by an increased lymphocyte sensitivity to cAMP-inducing agents such as catecholamines. In MAIDS, where MHC class II-dependent processes are involved, CD4⁺ T cells and not CD8⁺ T cells could selectively down-regulate GRK.

The involvement of the cAMP-PKA type I pathway in the generation of the CD4⁺ CD90^- T cells is also an important finding because these cells play a major role in the pathogenesis of MAIDS. A large fraction of CD4⁺ T cells in normal germinal centers also lack CD90. Our results therefore suggest that cAMP-inducing mediators such as prostaglandin E₂ could be involved in the physiology of germinal center reaction. In human lymphocytes, CD7 is considered a functional equivalent of CD90. Indeed, both genes share similar promoter regions and exonic organization. Although most human peripheral CD4⁺ T cells are CD7⁺, several immunodeficient states, including HIV infection, allogeneic bone marrow transplantation, and immunosenescence, are associated with the expansion of CD4⁺ CD7^- T cells reminiscent of the increase of CD4⁺ CD90^- in MAIDS. Further investigation is required to determine whether cAMP is indeed involved in the human CD4⁺ CD7^- phenotype and whether PKA type I antagonists can be used as therapeutic agents in these pathological states. This could be of paramount importance in HIV infection where restoring strong helper T cell responses is a promising way to control HIV replication with less intensive antiretroviral regimens.FIGURE 3

View larger version (43K): [in this window] [in a new window]   Figure 3. No caption available.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0813fje ; to cite this article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0813fje

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