Substance P Mediate Activation of Mast cell by Allowing Nerve-mast Cell interaction

1. Substance P Mediate Activation of Mast cell by Allowing Nerve-mast Cell interaction California State University, Los Angeles Bio 520: Advanced Immunology Seminar Abby Yilma: 2/2/09

2. Background • Neurotransmitters • Substance P • Receptor molecule • Mast cells • Adhesion molecule • Nerve-mast cell interaction

3. Neurotransmitter • Chemicals that transmit nerve cell impulse from one cell to another cell • Can be neuron to neuron or neuron to other cells such as immune cells • The release of chemical is driven by • Arrival of action potential at the synapse • Effect of a neurotransmitter usually depends on • Chemical properties of the receptors • Stimulatory or inhibitory Neurotransmitter in side vesicles Vesicles fuse and release their contents into the synapse Synaptic cleft Bind to a receptor on the postsynaptic membrane www.nature.com/.../fig_tab/nm0907-1018_F1.html

4. Class of Neurotransmitter Molecules

5. Substance P (SP) • Discovered by Von Euler and Gaddum in 1931 • Extracts of horse brain and intestine contained a hypotensive and spasmogenic factor • Later found to be protein • Isolated and characterized by Leeman’s group in 1971 • Bovine hypothalamus O’Connor et al., 2004

6. Structure of SP • Polypeptide synthesized in the ribosome and then cleaved into undecapeptide (11 aa) • SP: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 • Belongs to tachykinin family • Share common carboxy-terminal sequence • Essential for receptor interaction and activation O’Connor et al., 2004

7. Where is SP Found? • Widely distributed in the sensory neurons (afferent neuron) • Physical stimulation such as touch, vision and hearing • Chemical simulation such as capsaicin • Active component of chilly peppers & produce sensation of burning • Native to the gastrointestinal, respiratory tracts and genitourinary tract • Effect mediated by specific receptor called neurokinin-1 receptor (NK-1R) • Expressed in nervous cells and immune cells O’Connor et al., 2004 & Bulut et al., 2008

8. Neurokinin-1 Receptor (NK-1R) • Interaction via the third cytoplasmic loop • Cytoplasmic carboxyl terminus becomes phosphorylated • Cause desensitization of the receptor in response of repeated application of agonist • Glycoprotein (407 aa, 46 kDa) • 7 α-helical transmembrane loops • Many serine and threonin residue at cytoplasmic carboxyl terminal O’Connor et al., 2004

9. SP’s Role in Regulating Body Functions • In the central nerve system • Participates in regulating neuronal survival and degeneration • In the spinal cord • Participates in neurotransmission of pain • Immune cells (?) mast cells? • Proinflammtory mediator • Induces inflammation • Contributes to chronicity of inflammatory response O’Connor et al., 2004

10. Mast cells • First describe by Paul Ehrlich in 1878 • A resident of several types of tissues • Contains many granules • Visible by light microscope • Rich in histamine • Involves in local immune response • Acts as neurotransmitter • Located in close proximity to nerves • Skin, intestinal mucosa Human skin mast cell grown on human skin fibroblast feeder layers www.aad.org/education/students/mastcellsfig1.htm Ito et al., 2006

11. Activation of Mast Cells Degranulating Mast cells • Direct injury • Physical or chemical • Cross-linking of immunoglobin (IgE) • Through Fc epsilon RI (FcєRI) • Complement protein • Produced in response to evasion • Substance P (?) http://instructional1.calstatela.edu/eporter/BIOL520/

12. Molecules Released By Mast Cells into the Extracellular Environment • Preformed mediators from the granules • Histamine • Heparin (anticoagulant) • Serine (phosphorylated by kinase during cell singling) • Newly formed lipid mediators from cell membrane • Prostaglandia D2 (inhibitor of platelet aggregation) • Leukotrien C4 ( act on inflammatory response) • Cytokines • Critical for development and function of innate and adaptive immunity • Example: IL-3, TNFa, and GM-CSF • GM-CSF stimulates stems cells to produce granulocytes and monocytes • Molecules that can influence neuronal activity upon interaction with nerve cells (?) Bulut et al., 2008

13. Molecules from Mast Cells That Act on Nerve Cells • Tryptase • Granule-derived serine proteinase • Used as marker for mast cell activation • Histamine • Released as a neurotransmitter • Regulating physiological function in the gut • Binds to proteinase- activated receptors 1 and 2 Ito et al., 2006

14. Nerve-mast Cell Interaction • Is a true chemical synapsis formed during the communication? Ito et al., 2006 & Furuno et al., 2005

15. Adhesion Molecules Common to Nerves and Mast cells • In 2003, Ito and colleagues discovered • Synaptic cell adhesion molecules (SynCAM) or spermatogenic immunoglobulin superfamily (SgIGSF) • In later work showed • SgIGSF/SynCAM is localized on both side of most synapses • Functions as a homophilic or heterophilic adhesion molecule • Depending the binding partners avialable • Can span the synaptic cleft • Drives synapse assembly Ito et al., 2006 and Furuno et al., 2005

16. Hypothesis • Mast cell activation requires not only a direct cell to cell interaction between nerve fibers and mast cells but also substance P, which is the main transmitter of nerve fibers

17. Research Articles • In support of the hypothesis • Proximity between nerve and mast cells • Importance of MC/NC crosstalk in MC-driven inflammatory skin responses • Mast cells activation in the presence of Adhesion molecule • Refuting the hypothesis • Receptor-mediated SP activation of mast cells event • Activation of mast cells by substance P in vitro without nerve cells

18. In support of the hypothesis

19. Importance of MC/SN crosstalk in MC-driven inflammatory skin responses • Mice were anesthetized • Surgically removed the sensory nerves from the ear skin • Employed histochemical/immunohistochemical detection technique using SN marker proteins (SP) • To test the importance of nerve function, denervated and control skin sites were treated with capsaicin and vehicle (control) • Capsaicin induces strong cutaneous neurogenic inflammation in normal skin • Measure increase in ear thickness at different time points using caliper

20. Denervated Ear Skin Fail to Develop Inflammatory Reaction in Response to Capsaicin Control d = dermis dp = dermal papilla e = epidermis sg = sebaceous gland p < 0.05 ; *** p < 0.005 n = 3 Nerve fiber presented at physiological site Suggests the importance of MS/SN crosstalk Denervation results in loss of functional skin nerves Siebenhaar et al., 2008

21. To examine the Proximity of Mast to Nerve cell • Mast cell distribution and proximity with nerve elements in urinary bladder and stomach • Normal tissue (+/+) from mouse bladder and stomach • Performed histochemical/Immunohistochemistry • For mast cells (stained with toluidine blue) • For nerve cells detection (neuron-specific enolase (NSE) polyclonal antibody) • Examined the submucosa layers in both tissues

22. The Bladder and Stomach Contained Nerve Cells in Higher Proximity to Mast Cells Mast cell = black overhead Blood vessels = black arrow Nerve elements = white overhead Blood vessels play a role in virtually every medical condition The distribution of the mast cell and its contact with nerve element is varied in both tissues Andrea et al., 2005

23. The role of SP on mast cells activation in vivo study • Female mice aged 8 weeks maintained for 1 week • Psychological stress (PS) and foot shock stress (FS) were administered • Killed the mice by decapitation • Analyze the skin portion using histochemistry /immunohistochemistry • Counted degranulated mast cells outside of the cell membrane and calculated the ratio of the degranulated mast cell to all mast cells • Determined SP-positive nerve fibers

24. Degranulated Mast Cells and SP-positive Nerve Fibers Increased with Stressed Mice Control After stress was administered , the ratio of degranulated of mast cells increased significantly following FS and PS SP-positive nerve fibers were detected in the upper and mid dermis After FS and PS was administered, the number of SP-positive nerve fibers per/skin area was increased significantly *** p < 0.001 Substance P-positive nerve fibers in the skin Kawana et al., 2006

25. Examine whether SgiGSF adhesion molecule involves in Mast cell degranulation • 8 to 10-week-old mice • Remove the mesentery portion • Surrounds part of small intestine • Stimulated mesentery part with capsaicin and electric pulse • The electric pulse applied at their nerve root • Employed histochemical/immunohistochemistry • Stained with alcian blue and nuclear fast red • Counted degranulated mast cells

26. Capsaicin Caused Mesenteric mast cell Degranulation Degranulated stage = overheads arrow Arrows = stable stage 1/3 of mast cells were degranulated after capsaicin , which suggest that capsaicin caused degranulation This doesn’t rule out the possibility that capsaicin might directly activate mast cells, so the stimulated mesenteric nerve at their root Ito et al., 2007

27. SgIGSF Mediated Attachment of Mast Cell to Neuron * p < 0.05 n = 7 Electric simulation will induce mast cells degranulation through releasing neurotransmitters from nerves >1/3 degranulated Both group failed to activate mast cells Mi wh = lack SgIGSF mivit = express less SgIGSF SP = nerve impulse transmitted Degranulation restored Ito et al., 2007

28. Refuting the Hypothesis

29. Mast cells dependent on NK-1R to develop relationship with nerve cells • 10-to-12 week-old mice lack NK1-R (NK1-R-/-) and mast cell with no expression of NK1-R (NK1-R BMR kit) • Removed tissue from bladder mucosa • Employed histochemistry/immunohistochemistry • For mast cells stained with toluidine blue • For nerve cells detection (neuron-specific enolase (NSE) polyclonal antibody) • Determined mast cells distribution • Assessed the contact between mast and nerve cells

30. NK1-R Expression Affect the Contact Between Mast and Nerve Cells Mast cell = black overhead Blood vessels = black arrow Nerve elements = white overhead Suggests the contact between mast and nerve cells is mediated through NK1-R NK1-/- BMR kit = mast cell with no expression of NK1-R Ercan et al., 2006

31. Substance P activates mast cells without nerve cells in Vitro • Culture human peripheral blood-derived CD34+ (HuMC) and LAD2 mast cells overnight • HuMC is primary cell line and LAD2 isolated from tissue • Degranulation assay • Cells were sensitized overnight with myeloma (cancer cells) IgE • Cell were preincubated overnight with IgE and then stimulated with anti-IgE and SP • FcєRI mediated activation • Measured β-hexosaminidase and cytokines release into supernatant

32. LAD2 Stimulated with SP Released More β-hexosaminidase Potent activators of LAD2 SP activated LAD2 to degranulate Kulka et al., 2007

33. SP Activated Mast cells to Produce Large Cytokines N = 3; p< 0.01 LAD2 cells HuMC cells SP induced LAD2 cells to produce large quntities of TNFα, GM-CSF and IL-3 SP stimulated HuMC cells to produce small quantities of TNF GM-CSF and IL-3 Suggests that SP can activate mast cells without nerve cells Kulka et al., 2007

34. Substance P fail to activate mast cells in human intestine • Human mast cells isolated from normal intestinal tissue were cultured for 1-2 weeks • Mast cells were challenged for 1 h with different Neuropeptide, including SP, mAB29C6, and ionomycin • mAB29C6 and ionomycin induces IgE receptor crosslinking used as positive controls • Measured histamine and sulphidoleukotriene (sLT) released in cell-free supernatants using radioimmunoassay

35. SP Fail to Activate Mast cells Significantly ** p < 0.001; n = 2 Positive controls Positive controls Control Control Not only SP but also other neuropeptide fail to activate mast cells Bischoff et al., 2004

36. Summary: Supporting the Hypothesis • Siebenhaar et al., 2008 • Importance of MC/SN cross talk to develop inflammatory reaction • Andrea et al., 2005 • Nerve cells are in higher proximity to mast cells • Kawana et al., 2006 • Activation of mast cells by SP and mast-nerve cells interaction • Ito et al., 2007 • Importance of adhesion molecules for mast-nerve cells interaction Capsaicin Degranulation

37. Summary: Refuting the Hypothesis • Ercan et al., 2006 • Receptor-mediated SP activation of human mast cells event • Questions the direct interaction • Kulka et al., 2007 • Substance P activates mast cell in vitro without nerve cell • Arguing nerve cells are not required for mast activation • Bischoff et al., 2004 • Failure of SP to activate mast cells • Suggests that SP is not required for mast cells activation Capsaicin Degranulation X NK1-R X

38. Follow up Experiment • Using human model to characterize the presence of SgIGSF expression on other immune cells Inflammation response in reddish SP Nerves SgIGSF Capsaicin treated skin tissue www-biology.ucsd.edu/news/article_030603.html

39. Conclusion • The different results presented from different research group on SP’s role and MC/SN crosstalk on mast cell activation suggests further investigation in this area, specially using human model • The conflicting results can place mast cells at the border of potential critical host-pathogen interactions

40. Study on Proinflammatory Effects of SP in Immune Cells • Involves observation of abnormal level of SP, SP-nerve fibers, NK-1R in disease tissue • Knockout experiment in animal model and coculture appraoch O’Connor et al., 2004