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Pregnancy Specific Glycoproteins

Pregnancy Specific Glycoproteins. Binding of Pregnancy –Specific Glycoprotein 17 to CD9 on Macrophages Induces Secretion of IL-10, IL-6, PGE2 and TGF- b 1. Introduction. Definition of Pregnancy specific glycoproteins.

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Pregnancy Specific Glycoproteins

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  1. Pregnancy Specific Glycoproteins Binding of Pregnancy –Specific Glycoprotein 17 to CD9 on Macrophages Induces Secretion of IL-10, IL-6, PGE2 and TGF-b1

  2. Introduction • Definition of Pregnancy specific glycoproteins. • Discussion of pregnancy and the role of pregnancy specific glycoproteins in the outcome of pregnancy. • Discussion of macrophages and cytokine expression. • Role of macrophages and cytokines in our immune system and in survival of a pregnancy. • Importance of receptor mediated signaling and discussion of the receptor used to mediate PSG signaling (CD9). • The importance of certain regulatory molecules such as PKA in mediating cytokine expression in response to PSG-CD9 binding.

  3. Questions Being Addressed • Determine the pattern of cytokines secreted by primary macrophages and macrophage cell lines derived from wt and CD9-deficient mice following PSG17N treatment. • Elucidate the signaling molecules involved in the CD9-dependent response to PSG17N. • Assess whether CD81, a molecule that is closely related to CD9 can compensate for the absence of CD9 in macrophages.

  4. Figure 1: Is the biological Response to PSG17 Mediated by CD9? • Human and other murine PSG • induce expression of • anti-inflammatory Cytokines • such as IL-10. • LPS induces secretion of cytokines • by macrophages. • CD9 (tetraspanin) identified • as the receptor in Mf for PSG17. • PSG 17N contains only the • N-terminal region of PSG17— • This is the biologically active portion.

  5. Figure 1: Is the biological Response to PSG17 Mediated by CD9? • METHOD • Peritoneal Mf were isolated from • wt and CD9-Deficient mice treated with control or PSG17N. • ELISA assay is used to detect IL-10, IL-6, TGF-b and IL-12. • RESULT: • In wt Mf PSG17N induces • secretion of the anti-inflammatory • cytokines, IL-10, IL-6 and TGF-b. • CD9 KO cell show no difference in cytokine expression as compared to the control. • CONCLUSION: • PSG 17N mediated cytokine secretion is blocked in the absence of CD9.

  6. Figure 2:Is CD9 necessary for PSG-Mediated cytokine expression? • CD9-deficient mice have very small litters and the female animals are infertile. • As a result, it is difficult to obtain enough macrophages from these animals to • perform these experiments.

  7. Figure 2:Is CD9 necessary for PSG-Mediated cytokine expression? • METHOD: • Isolated bone marrow from wt and CD9-deficient mice. • Infected with J2 virus to generate immortalized bone marrow macrophage cell lines. • Treat cells with various doses of PSG 17N and analyze for cytokine secretion by ELISA. • RESULTS: • IL-10, IL6 and TGF-b secretion is induced only in wt cells. • In CD9 KO cells, cytokine secretion remains unchanged compared to the control. • CONCLUSION: • Again, this result indicates that CD9 is necessary for PSG17N mediated cytokine expression in J2 transformed bone marrow macrophages. • Other tetraspanin family members such as CD81 do not compensate for the absence of CD9. • Immortalized Mf respond to PSG17N in a similar manner to that of peritoneal Mf.

  8. Figure 3: How Does PSG 17N-CD9 Binding Lead to Cytokine Secretion in Macrophages? • PGE2 is known to have a role in • regulating the balance of pro- • inflammatory and anti-inflammatory • cytokines. • It has been shown to regulate IL-6 • And IL-10 by activated Mfs. • Can PSG17N induce PGE2 • to regulate cytokine secretion? • METHOD: • RAW cells or BMDM are • treated with PSG17N. • PGE2 synthesis was • Measured by ELISA. • COX-2 Expression was • Detected by Western Blotting.

  9. Figure 3: How Does PSG 17N-CD9 Binding Lead to Cytokine Secretion in Macrophages? • RESULT: • RAW Cells respond to PSG 17N • with increased PGE2 expression. • Only BMDM from wt mice showed • Increased PGE2 expression following • PSG17N treatment. • Cox-2, an enzyme involved in the • synthesis of PGE2, is also only present • in wt BMDM. • Ns-398 (a COX-2 inhibitor) prevents • PGE2 synthesis. • CONCLUSION: • CD9 is required to produce PGE2 • or Cox-2 in response to PSG17N • treatment.

  10. Figure 4: How Does PSG 17N-CD9 Binding Lead to Cytokine Secretion in Macrophages? • PGE2 and Cox-2 seem to be necessary PSG17N mediated CD9 cytokine expression. • Phosphorylation of Protein kinase A is • known to induce expression of target genes such as cytokines. • What is involved in synthesis of PGE2? • METHOD: • Examine the possible involvement of cAMP-dependent PKA pathway in PSG • Mediated cytokine expression. • BMDM cells were treated with the • PKA inhibitor, KT5720, and control or PSG17N. • IL-10, IL-6 and TGF-b secretion were • analyzed by ELISA.

  11. Figure 4: How Does PSG 17N-CD9 Binding Lead to Cytokine Secretion in Macrophages? • RESULTS: • PSG17N mediated induction of • IL-10 and IL-6 is decreased in the • presence of both the Cox-2 and PKA • inhibitor. • TGF-b secretion is not decreased. • CONCLUSIONS: • IL-10 and IL-6 secretion triggered by PSG17N signaling requires both Cox-2 • and PKA. • TGF-b secretion by PSG17N does not • Require PKA or Cox-2. TGF-b is regulated via another pathway.

  12. Figure 5: Through what signaling pathway is PSG17N mediated TGF-b secretion regulated? • CD9 is known to also interact with • Protein kinase C (PKC). • Is PKC necessary for secretion of • TGF-b? • METHOD: • Pre-treated RAW cells with Go6938, an inhibitor that blocks most forms of PKC. • Treated cells with control or PSG17N and analyzed cytokine expression by ELISA. • RESULTS: • All cytokines were decreased in the presence of the inhibitor.

  13. Conclusions • Anti-inflammatory cytokines are induced by PSG17N treatment, pro-inflammatory cytokines are not. • PSG17N induces secretion of IL-10, IL-6 and TGF-b in various types of macrophages. • This cytokine expression is mediated through the PSG receptor, CD9 and other family members do not compensate for the absence of CD9 in macrophages. • PGE2 and Cox-2 are necessary PSG-mediated cytokine expression. • PSG17N-CD9 binding activates PKA-cAMP pathway to trigger IL-10 and IL-6 secretion. • The PKA-cAMP pathway does not have a role in TGF-b secretion. • PSG17N-CD9 binding activates PKC pathway to trigger IL-10, IL6 and TGF-b pathway.

  14. Critical Analysis • PSG17N mediated IL-6 and IL-10 secretion seem to be regulated through the PGE2-Cox2-PkA pathway. • TGF-b secretion is not regulated through this pathway. • Problems with inhibitor studies – toxic effect of many inhibitors produces false results. (Data shown in Fig. 5)

  15. Future Directions • The data shown in the paper begins to address the biological pathways triggered by PSG-CD9 binding in macrophages. • Further investigate the pathway(s) used for PSG17N-CD9 mediated cytokine expression. • Investigate the phosphorylation of the transcription factor cAMP (known to activate IL-10 and IL-6 expression). • Identify other transcription factors that are involved in regulating PSG-mediated cytokine expression. Additional inhibitor studies. • Identify other pathways that may be involved in mediating cytokine expression via CD9-PSG17N binding. • Develop a better understanding of the role of PKC in mediating cytokine expression.

  16. References • It is essential that you give credit where credit is due! • Be sure to include every reference or source that you used in preparing your presentation.

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