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​cAMPing in the Immune System: A Real "Turn Off"​

Article from 2009-07-01

By Tom Brock, Ph.D.

To the pathologist peering through the microscope, "increased cellularity" translates as "inflammation". That is, if the tissue in question has
more leukocytes than normal, it's inflamed. Simply by being there, those leukocytes provide testimony of an earlier alarm raised by resident
cells calling for immune support. In addition, they lead the pathologist to look for additional clues of an ongoing struggle for control within
the tissue, as well as telltale indicators as to where the battle will lead.

Remarkably, providing much of the story behind this scenario is an old friend of the cell biologist: cAMP. In leukocytes, cAMP is the signal
to chill. The alarm that called in those leukocytes most likely did so by eliminating their intracellular cAMP. The future of the battle, the
progression or amelioration of the inflammation, may well be decided by the leukocyte cAMP level. This mini-review looks at some of the
key regulators in this system.

cAMPing for Beginners

Signaling through cAMP (Figure 1) is quite complex, to the point that one could focus on a single aspect for a lifetime of research. For
example, the principle enzyme, adenylate cyclase, which converts ATP to cAMP, has at least 10 isoforms (distinct gene products) in
humans. While the actions of adenylate cyclase isoforms are activated by Gs-coupled receptors and inhibited by Gi-coupled receptors, one
isoform is inhibited by calcium and two are activated by calmodulin. The inhibition of cAMP production by Gi is relevant for both reducing
basal cAMP production (which can be appreciable) in unstimulated cells and blocking the increased cAMP generation in cells stimulated
with ligands that activate Gs-coupled receptors.

An important pathway that is cAMP-dependent involves the membrane-associated Exchange Proteins Activated by cAMP (Epac), Epac-1,
and Epac-2 (Figure 1). These are two of several guanine nucleotide exchange factors (GEFs) that target the Ras GTPase homologues Rap1
and Rap2. The Rap proteins are activated when bound GDP is replaced with GTP by a GEF, like Epac. Hydrolysis of GTP to GDP in situ
inactivates Rap. The cAMP-Epac-Rap pathways are involved in regulating a variety of different cell-specific processes, ranging from cell
motility to gene expression.

The prototypical pathway activated by cAMP, involving protein kinase A (PKA), is likewise complex and wide ranging. In resting cells, PKA
exists as a tetramer of two regulatory subunits holding two catalytic subunits in an inactive state. The association of cAMP with the
regulatory subunits allows dissociation of the tetramer, allowing the free and active catalytic subunits of the kinase to phosphorylate target
proteins. Because the catalytic subunits are relatively small they can diffuse through nuclear pores to phosphorylate proteins within the
nucleus. In humans, there are three genes encoding PKA catalytic subunits and four encoding regulatory subunits, so the regulation of
expression and protein function can be complex. Moreover, there are at least three genes that encode inhibitors of PKA, proteins that bind
PKA catalytic subunits, promote their export from the nucleus and impair their kinase function. Finally there is a diverse family of A-kinase
anchoring proteins (AKAPs), which act as platforms for PKA action: AKAPs bind PKA, by the regulatory domains, as well as PKA substrates,
other kinases, and phosphatases. This conceivably allows better regulation of phosphorylation and dephosphorylation of target proteins.

Finally, cAMP is degraded to AMP by phosphodiesterases (PDEs). There are several PDE isoforms, and some are cAMP-specific, some
target cGMP only, and some break down both. Inhibitors of PDEs can prolong physiological processes mediated by cyclic nucleotides by
delaying their degradation. As the different PDEs have different roles, inhibitors of specific PDEs are clinically important. Inhibitors of
PDE4, the major cAMP-metabolizing isoform in immune cells, are used in the treatment of asthma, allergic diseases, and inflammation.

More cAMP, Less Inflammation

The appearance of COX-2 protein is a recognized marker of inflammation: expression of the gene for COX-2 is rapidly induced by pro-
inflammatory mediators, leading to the presence of COX-2 protein in cells within inflamed tissues. The COX-2 protein is unstable and
typically is degraded following the resolution of inflammation. By comparison, the protein COX-1 is stable and present in healthy tissues.
Interestingly, COX-1 and COX-2 give rise to different PGs in some cell types. For example, COX-1 couples with specific PGH2 isomerases
to produce primarily thromboxane, PGD2, and PGI2 in peritoneal macrophages, while COX-2 couples with specific synthases to generate
PGE2 and PGI2.1 The paired production of PGE2 and PGI2 is a common response to inflammatory mediators, being found in endothelial and
epithelial cells, fibroblasts, and leukocytes. As a result, the appearance of COX-2 shifts the profile of lipid mediators generated by these
cells during inflammation.

PGE2 is notorious as the agent of pain and fever. Aspirin and other NSAIDs reduce pain and fever by inhibiting the COX enzymes and
preventing PGE2 synthesis. PGE2 evokes its effects by activating the ‘E-prostanoid', or EP, receptors.2 The four EP receptors are G-protein
coupled and differ in their intracellular signaling and distribution on different cell types. The signaling through EP receptors on neurons in
the periphery and the hypothalamus, driving pain and fever, is complicated and involves multiple EP receptors.

In immune cells, PG signaling is simpler. In leukocytes, PGE2 activates primarily EP2 and EP4 and PGI2 binds to the Prostaglandin I Receptor
(IP receptor). These receptors are Gs-coupled, increasing cAMP levels. This suppresses leukocyte function, reducing (as applicable)
chemotaxis, cell adhesion and spreading, phagocytosis, reactive oxygen generation, bacterial killing, antigen presentation, LT synthesis, and
the production of TNF-α, IFN-γ, IL-2, IL-8, IL-12, MIP-1α, and MIP-1β.1-3 These effects can be mediated by Epac, PKA, or both. By acting in
unison to elevate cAMP, the COX-2 products PGE2 and PGI2 reduce inflammation. Other Gs-coupled receptors include those for adenosine
(A2a and A2b), calcitonin, dopamine (D1 and D5), FSH, glucagon, histamine H2, luteinizing hormone, melanocortin, and secretin.

No cAMP Means: Action!

Many of the effects that are suppressed in leukocytes by PGE2 or PGI2 are listed as classical effects induced by LTB4. In fact, LTB4 plays a
central role in the recruitment and activation of essentially all leukocytes from the blood into inflammatory settings. LTB4 evokes its effects
through two GPCRs, BLT1 and BLT2.2 Many LTB4 effects are blocked by pertussis toxin, indicating a role for Gi, which blocks adenylate
cyclase activity and allows cAMP levels to drop through PDE4 action.

A similar story holds for sphingosine-1-phosphate (S1P; see related story on page 40). S1P acts through five different GPCRs, with S1P1
and S1P3 signaling primarily through Gi. S1P1 is expressed on lymphocytes, dendritic cells, eosinophils, macrophages, and mast cells;
activation of S1P1 by S1P universally triggers chemotaxis and typically enhances cell function.4 Again, these effects are pertussis toxin
sensitive, underscoring the role of cAMP reduction in stimulating cell activity.

There are two interesting twists to the S1P story. First, the levels of S1P are high in circulating blood but low in tissues and lymph. The high
level in blood means that circulating leukocytes are continuously activated by S1P, which leads to temporary internalization of these
receptors, making these cells refractory to activation by S1P. As a result, S1P does not effectively recruit leukocytes from the blood. On the
other hand, it is important in recruiting cells from the lymph.

This leads to the second twist: chemical mimics of sphingosine and S1P can be used to down-regulate S1P receptors on T cells in the
lymph.5,6 Synthetic sphingosine mimetics, like KRP 203 and FTY720, are phosphorylated in vivo by sphingosine kinase to S1P-like
molecules. S1P analogs (e.g., SEW2871, KRP 203-phosphate, FTY720-phosphate) bind to, but don't activate, S1P receptors. Binding leads
to persistent down-regulation of S1P receptors, blocking the ability of lymphocytes to respond to S1P and migrate. This approach to
immunosuppression is being used to block allograft rejection and shows promise in other settings.

Implications
Some GPCRs can be regulated, upon ligand binding, by phosphorylation via GPCR kinases (GRKs). Phosphorylation induces receptor
binding to arrestin molecules, which serves to terminate G protein activation and promote receptor internalization or desensitization.
Because both S1P1 and S1P3 can be phosphorylated by GRKs and desensitized by arrestin, S1P mimics can be used to shut down signaling
through these receptors and suppress cell activation. Many other Gi coupled receptors also bind arrestin, including the adenosine A1,
Burkitt lymphoma, CB1, CB2, C5a, and P2Y12 receptors, suggesting that these might also be modulated pharmacologically through this
mechanism.

Alternatively, inadvertent internalization or desensitization of Gs-coupled receptors could impair leukocyte calming, leading to persistent
inflammation. For example, if PGE2 could not activate EP2 and EP4 on leukocytes, then an important anti-inflammatory signal would be
lost. Limited data suggest that these receptors can be bound by arrestin, leaving the door open that arrestin-mediated loss of EP2/EP4
responsiveness could contribute to certain pathologies. Similar patterns may also be found for other Gs-coupled receptors.

Figure

Figure 1. Signaling through the second messenger cAMP

References
1. Brock, T.G., McNish, R.W., and Peters-Golden, M. J. Biol. Chem. 274, 11660-11666 (1999).

2. Shimizu, T. Annu. Rev. Pharmacol. Toxicol. 49, 123-150 (2009).

3. Serezani, C.H., Ballinger, M.N., Aronoff, D.M., et al. Am. J. Respir. Cell Mol. Biol. 39, 127-132 (2008).
 4. Kee, T.H., Vit, P., and Melendez, A.J. Clin. Exp. Pharmacol. Physiol. 32, 153-161 (2005).

5. Drennan, M.B., Elewaut, D., and Hoqquist, K.A. Eur. J. Immunol. 39, 925-930 (2009).

6. Marsolais, D., and Rosen, H. Nat. Rev. Drug Discov. 8, 297-307 (2009).

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