Immunosuppressive Drugs

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Immunosuppressive Drugs

• Jennifer Comber, Abigail Cooke, Daniel Pinch
  Helen Willicombe.

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Immunosuppressive Drugs Transplants

• Immunosuppressive drugs are used after allogenic
  transplants.
• Cyclosporin A (CsA) replaced dual treatment using
  steroids azathioprine.
• Allowed successful transplants prevented graft
  rejection.
• Frequently used after bone marrow organ
  transplants.
• But, problems with CsA, including nephrotoxicity,
  led to searches for new drugs.
• FK506 was discovered.

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Schreiber, S. Crabtree, G. (1992)
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Proposed Mode of Action in Bone Marrow-Derived
Mast Cells (BMMCs)
Adapted from Schreiber, S.(1991)
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Experimental Details

• Cell Culture
• Obtained from femurs tibias of mice.
• Activation of BMMCs
• IgE dependent activation.
• Washed centrifuged.
• Incubated with CsA, FK506 or vehicle.
• ß-Hexosaminidase histamine used to assess mast
  cell activation.
• mRNA Analysis
• Used to assess levels of cytokine mRNA in mast
  cells.
• Isolated total cellular RNA.
• RNA blots autoradiography allowed comparison of
  cytokine expression.

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Experimental Details

• Protein Blots
• Used to establish the presence of the binding
  proteins.
• SDS PAGE.
• Binding Elution of Immunophilins to Solid-Phase
  CsA
• Used to confirm CsA binding to CyP species.
• Calcineurin (CaN) Assay
• Used to compare CaN activity between drug
  treatment groups control.

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Results IC50 Values
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Results

• Release of cytokines in mast cells is not
  significantly inhibited by FK506.
• It was found that FKBP12 expression is reduced in
  mast cells.
• CsA inhibits calcineurin phosphatase activity
  with an IC50 of 8nM.
• But 1000nM of FK506 did not inhibit calcineurin
  phosphatase activity.

Adapted from Schreiber, S.(1991)
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Summary

• CsA
• Successful inhibition of cytokine expression in
  activated BMMCs.
• FK506
• Does not inhibit cytokine expression in activated
  BMMCs due to minimal amounts of FKBP12.
• Possible Clinical Implications
• A number of immune cell types with varying
  responses.
• Several immunosuppressive drugs available.
• Not all cell responses are inhibited by all drug
  types.

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Future Research

• 1. What happens in humans?
• Experiment should be repeated using human mast
  cells.
• Similar experimental techniques.
• 2. Use of combination drug therapy.
• Repeat the experiments using different drugs and
  different immune cell types.
• What effect does this have on cytokine
  production?
• Can this pathway be targeted at other points?
• i.e. cell proliferation.

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References

• Kang, C. et al (2008) FKBP family proteins
  immunophilins with versatile biological
  functions. Neurosignals 16 318-325.
• Kaye, R. et al (1992) Effects of cyclosporin A
  and FK506 on Fce receptor type I-initiated
  increases in cytokine mRNA in mouse bone
  marrow-derived progenitor mast cells Resistance
  to FK506 is associated with a deficiency in
  FK506-binding protein FKBP12. Proc. Natl. Acad.
  Sci. USA 89 8542-8546.
• Liu, J. et al (1991) Calcineurin is a common
  target of cyclophilin- cyclosporin A and
  FKBP-FK506 complexes. Cell 66 807-815.
• Schreiber, S.(1991) Chemistry and biology of the
  immunophilins and their immunsuppressive ligands.
  Science. 251 283-287.
• Schreiber, S and Crabtree, G. (1992) The
  mechanism of action of cyclosporin A and FK506.
  Immunology Today. 13 136-142.
• White, DJ. (1991) FK506 the promise and the
  paradox. Clin Exp Immunol 83 1-3.