Johnson & Johnson Pharmaceutical Research & Development Scientists Uncover Previously Unknown Role for Class of Molecules

SAN DIEGO, Aug. 4, 2011 /PRNewswire/ -- Johnson & Johnson Pharmaceutical Research & Development, L.L.C. (J&JPRD) announced today the publication of research that uncovered a previously unknown role for a class of molecules that could lead to future advances in immunologic medicine.  The findings show a definitive link between EBI2, a G-protein-coupled receptor that controls B-cell migration, and some unexpected immunological effects of certain oxysterols.  Co-authored by the company's neuroscience and immunology discovery teams, the results of the initiative were published in a Letter in the July 28, 2011 issue of NATURE, "Oxysterols direct B-cell migration through EBI2."

EBI2 is highly expressed in the spleen and upregulated upon Epstein-Barr-virus infection(1).  Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production (2-6).  Oxysterols are oxygenated derivatives of cholesterol that elicit profound effects on immune and inflammatory responses, as well as on cholesterol metabolism(7-9).  Prior to the publication of these data, the biological effects of oxysterols have been credited largely to the activation of nuclear hormone receptors(10,11).  The results reported in this research communication demonstrated for the first time that an oxysterol – more specifically, 7,25-dihydroxycholesterol isolated from pig spleen extracts – is the endogenous ligand for EBI2, and as such, is capable of also potently activating a G-protein coupled receptor.  

"This finding reveals a new way in which the body may modulate the immune system in response to pathogenic attack and will help us understand better how B cells and other immune cells coordinate the humoral immune response," said Tim Lovenberg, senior research fellow, Neuroscience Discovery, J&JPRD, La Jolla, CA.  

The complete text of the Letter, "Oxysterols direct B-cell migration through EBI2," is available at: http://www.nature.com/nature/journal/v475/n7357/full/nature10226.html

About G-Protein-Coupled Receptors (GPCRs)

With the completion of the human genome, many genes have been uncovered with unknown functions. The 'orphan' G-protein-coupled receptors (GPCRs) are examples of genes without known functions. These are genes that exhibit the seven helical conformation hallmark of the GPCRs but that are called 'orphans' because they are activated by none of the primary messengers known to activate GPCRs in vivo. They are the targets of undiscovered transmitters and this lack of knowledge precludes understanding their function. Because they belong to the supergene family that has the widest regulatory role in the organism, the orphan GPCRs have generated much excitement in academia and industry.

About the Research

The Letter, co-authored by a team of J&JPRD neuroscientists, immunologists, and analytical chemists, describes work the team completed as part of an ongoing orphan GPCR program regarding the orphan G-protein-coupled receptor known as EBI2, and also called GPR183.  The J&JPRD orphan GPCR program has previously de-orphanized the histamine H3 receptor, histamine H4 receptor, the Relaxin-3 receptor, the Insulin-5 receptor, and the lactate receptor.

As part of this current investigative work, the team used tissue extracts to identify putative EBI2 activity in rat spleen and purified EBI2 ligands (compounds produced from within an organism that bind to a receptor site on a target protein, in this case, EBI2) from pig spleens through organic extraction followed by chromatographpy.

The team then isolated and tested more than 30 oxysterols for the activation of EBI2.

Comparing the retention times of the active oxysterols using the same chromatography method that was initially used to identify the ligand activities from the pig spleen extracts, the team next demonstrated by means of both in vitro and in vivo analyses in mice that oxysterols are specific endogenous ligands for EBI2 because they lack ligand activity for other GPCRs.  

Among the active ligands, the most potent ligand and activator observed was 7,25-dihydroxycholesterol (OHC), with a dissociation constant of 450pM for EBI2.  

Additional notes:

In vitro, 7,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells.

In vivo, EBI2-deficient B cells or normal B cells desensitized by 7,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen .  Blocking the synthesis of 7,25-OHC in vivo with clotrimazole reduced the content of 7,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice.

(Methodology Summary in brief:  Tissues were extracted using organic solvents followed by normal-phase HPLC.  Ligand activity was monitored by GTP-yS binding assay.  Structure analysis was done by mass spectrometry and NMR. GTP-yS binding, radioligand binding, cAMP accumulation and receptor internalization studies were used to characterize the receptor. In vitro and in vivo cell migration assays were used to test the chemotactic effect of 7a,25-OHC on various immune cells.)

Johnson & Johnson Pharmaceutical Research & Development, L.L.C.

Johnson & Johnson Pharmaceutical Research & Development, L.L.C. (J&JPRD) is headquartered in Raritan, N.J., and has facilities throughout Europe, the United States and Asia. J&JPRD is actively involved in drug discovery and development within a variety of therapeutic areas, including Cardiovascular and Metabolism, Central Nervous System, Immunology, Oncology and Virology, to address unmet medical needs worldwide. More information can be found at http://www.jnjpharmarnd.com

For more information on these published findings, please contact:
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J&JPRD
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Notes/References

1. Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G. & Kieff, E. Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors. J. Virol. 67, 2209–2220 (1993).
2. Pereira, J. P., Kelly, L. M., Xu, Y. & Cyster, J. G. EBI2 mediates B cell segregation between the outer and centre follicle. Nature 460, 1122–1126 (2009).
3. Pereira, J. P., Kelly, L. M. & Cyster, J. G. Finding the right niche: B-cell migration in the early phases of T-dependent antibody responses. Int. Immunol. 22, 413–419 (2010).
4. Gatto, D., Paus, D., Basten, A., Mackay, C. R. & Brink, R. Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses.Immunity 31, 259–269 (2009).
5. Goodnow, C. C., Vinuesa, C. G., Randall, K. L.,Mackay, F. & Brink, R. Control systems and decision making for antibody production. Nature Immunol. 11, 681–688 (2010).
6. Chan, T. D. et al. In vivo control of B-cell survival and antigen-specific B-cell responses. Immunol. Rev. 237, 90–103 (2010).
7. Schroepfer, G. J. Jr. Oxysterols: modulators of cholesterol metabolism and other processes. Physiol. Rev. 80, 361–554 (2000).
8. Bjorkhem, I. & Diczfalusy, U. Oxysterols: friends, foes, or just fellow passengers? Arterioscler. Thromb. Vasc. Biol. 22, 734–742 (2002).
9. Zhao, C. & Dahlman-Wright, K. Liver X receptor in cholesterol metabolism. J. Endocrinol. 204, 233–240 (2010).
10. Willy, P. J. et al. LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev. 9, 1033–1045 (1995).
11. Jin, L. et al. Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORc. Mol. Endocrinol. 24, 923–929 (2010).

SOURCE Johnson & Johnson Pharmaceutical Research & Development, L.L.C.