Stefanie Helm, Diplom-Biochemikerin 
Tel. 0621-383-2983
Dieter Koenig-Gressel, BTA, cand.biol. Uni Tübingen             
Tel. 0621-383-2983
Dr. rer. nat. Patrick Pankert, Postdoc               
Tel. 0621-383-3770
Dr. sc. hum. Lisa Dietz, Postdoc
Tel. 0621-383-3770
Patrick Illgen, cand. biotech. FH Mannnheim
Tel. 0621-383-3770
Sven Kinzebach, cand. biotech. FH Mannnheim
Tel. 0621-383-2983
Annika Jakob, cand. med. Uni Heidelberg MedMa
Tel. 0621-383-2983

In humans, allergic contact hypersensitivity towards the heavy metal Nickel (Ni) represents the most common form of contact allergy, affecting about 15-20% of the population in industrialized countries. Both Ni-reactive CD8+ and CD4+ human T cells, effector as well as regulatory T cells, have been analyzed and characterized in detail by various laboratories. Sensitization and/or secondary Ni-specific T cell reactions may not only be induced by Ni-containing jewellery, but possibly also by implanted Ni-releasing biomaterials like heart instents or orthopaedic prostheses. However, despite increasing knowledge on contact hypersensitivity, several molecular processes involved in the sensitization phase as well as in the elicitation phase remain to be elucidated. Neither the precise mechanism of Ni2+ ion transport by potential metalloproteins to antigen presenting cells, e.g. Langerhans cells in human skin, nor the antigenic determinant(s), which lead(s) to Ni-specific T cell activation, is/are known.
With the aim to identify new cellular allergen-interacting and allergen-induced proteins, which might be involved in “Ni-epitope processing” in human nickel allergy and/or may function as metallochaperones, we started a new subproteomic approach and differential proteomic expression analyses from human keratinocytes, B cells, dendritic cells and T cells.  

Using modern proteomic technologies and different types of allergens, current and future data may help to give a better basic understanding of molecular processes involved in pathogenesis, diagnosis and therapy of human contact allergies, as well as to develop new in vitro assays for testing allergens.

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking.
Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least 9 of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin.
Conclusion: The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses. Further investigations: Analysis of other immune cells, including primary kerationcytes and dendritic cells.

Proteomic alterations in diseases such as allergen induced contact hypersensitivity or asthma bronchiale (skin and respiratory sensitization & elicitation) may occur in a variety of ways that are not predictable from genomic analysis, and it is clear that a better understanding of these alterations will have a substantial impact in diagnosis, prediction and therapy of such diseases. The opportunities as well as the challenges facing disease proteomics are remarkable. Promising areas of research include: i) the identification of altered protein expression at the whole-cell level  (e.g. keratinocytes, dendritic cells and/or T-cells); and ii) at the tissue level (or organ culture, 3-D models). Such alterations could be reflected in quantitative differences in protein expression, qualitative differences in protein processing and/or differential protein interaction profiles. To compare allergic diseases with other inflammatory diseases differntial protein expression is analyzed e.g. by DIGE technology, mass spectrometry and bioinformatics. 

Nickel contact hypersensitivity involves the activation of HLA-restricted, skin homing, Ni-specific T cells by professional antigen-presenting cells (APC). However, knowledge concerning molecular details of the delivery of metal ions to APC during the early sensitization phase and their interactions with HLA and T cell receptors (TCR) is still fragmentary. This study investigates the role of human serum albumin (HSA), a known carrier for Ni2+ and an often disregarded, major component of skin, in these processes. We show that Ni-saturated HSA (HSA-Ni) complexes can replace equimolar concentrations of Ni salts in stimulating human T cells, but that neither HSA itself nor its Ni-binding N-terminal peptide are involved in determining the specificity of antigenic determinants. In fact, HSA could be replaced by xenogeneic albumins exhibiting sufficient affinity for Ni2+ as determined by surface plasmon resonance using a Biacore®-instrument or atomic absorption spectroscopy. Moreover, despite rapid internalization of HSA-Ni by APC, it was not processed into HLA-associated epitopes recognizable by Ni-specific T cells. In contrast, the presence of HSA-Ni in the vicinity of transient contacts between TCR and APC-exposed HLA molecules appeared to facilitate a specific transfer of Ni2+ from HSA to high affinity coordination sites created at the TCR/HLA-interface.

To support application and development of modern proteomic technologies at the Medical Faculty Mannheim, University of Heidelberg, an European Proteomics Reference Laborartory has been established in collaboration with  

FUJIFILM EUROPE Life Sciences  (Laser Scanner)
http://www.fujifilm.eu/products/life_science/  

BRUKER Daltonics (Spot picker)
http://www.bdal.com/  

DECODON   (Delta 2D software)   
http://www.decodon.com/

M. Schnoelzer, DKFZ, Heidelberg
S. Gibbs, University of Amsterdam, NL
A. Anel, Zarragoza, E
S. Martin, University Freiburg
F. Lottspeich, MPI for Biochemistry, München
Franz, EMBL, Heidelberg
N. Guerreiro, Novartis, Basel, CH

European Union, 6th Framework, 2 Projects (Immunology & Proteomics) in IP Project, Novel Testing Strategies for In Vitro Assessment of Allergens (www.Sens-it-iv.eu),
LSHB-CT-2005 – 018681;Workpackage leader Proteomics. 

Landesstiftung Baden-Württemberg, Programm Allergologie, Subproteomisches Projekt LS-AL26. 

BMBF, Projekt in Klinischer Forschergruppe: "Pathomechanismen der allergischen Entzündung". 01GC97.

1. Schledzewski K, Géraud C, Arnold B, Wang S, Gröne HJ, Kempf T, Wollert K, Straub B, Schirmacher P, Demory A, Schönhaber H, Gratchev A, Dietz L, Thierse HJ, Kzhyshkowska J, Goerdt S. Deficiency of liver sinusoidal scavenger receptors stabilin-1/-2 causes glomerulofibrotic nephropathy via impaired hepatic blood clearance J Clin Invest. 2011, pii: 44740. doi: 10.1172/JCI44740. ). In press.

2. Thierse HJ, Budde P, Dietz L, Ohnesorge S, Eikelmeier S, Conde M, Zucht HD, Schulz-Knappe P. Proteomic identification of allergen-regulated proteins and allergen-protein interaction networks in assisting biomarker and assay development Progress Towards Novel Testing Strategies for in Vitro Assessment of Allergens., Eds. Roggen EL, Weltzien HU, Helma H, Transworld Research Network, Kerala, India, 2011, 145-166.

3. Gibbs S, Thierse HJ, Corsini E. Multifunctional role of keratinocytes in sensitization; in Progress Towards Novel Testing Strategies for in Vitro Assessment of Allergens, Eds. Roggen EL, Weltzien HU, Helma H, Transworld Research Network, Kerala, India, 2011, 35-53.

4. Gibbs S, Martin S, Corsini E, and Thierse H-J. Identification of contact allergens by in vitro cell culture based methods. Book: Kanerva’s Occupational Skin Diseases, 2nd Edition; Eds T. Rustemeyer, P. Elsner, S.M. John and H. Maibach; 2011, Chapter 116; Springer, New York, USA. (http://www.springer.com/medicine/dermatology/book/978-3-642-02036-0). In press.

5. Martin S., P. R. Esser, S. Schmucker, L. Dietz, D.J. Naisbitt, B.K. Park, M.Vocanson, J.-F. Nicolas, M. Keller, W. J. Pichler, M. Peiser, A. Luch, R. Wanner, E. Maggi, A. Cavani, T. Rustemeyer, A. Richter, H.-J. Thierse and F. Sallusto. T cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays. Cellular and Molecular Life Sciences, 2010, 67:4171-84.

6. Dietz, L., P.R. Esser, S.S. Schmucker, I. Goette, A. Richter, M.Schnoelzer, S.F. Martin and H.-J. Thierse. Tracking human contact allergens: From mass spectrometric identification of peptide-bound reactive small chemicals to chemical-specific naive human T cell priming. Toxicol Sci, 2010, 117:336-347.

7. Löhr JM, Faissner R, Koczan D, Bewerunge P, Bassi C, Brors B, Eils R, Frulloni L, Funk A, Halangk W, Jesnowski R, Kaderali L, Kleeff J, Krüger B, Lerch MM, Lösel R, Magnani M, Neumaier M, Nittka S, Sahin-Tóth M, Sänger J, Serafini S, Schnölzer M, Thierse HJ, Wandschneider S, Zamboni G, Klöppel G: Autoantibodies Against the Exocrine Pancreas in Autoimmune Pancreatitis: Gene and Protein Expression Profiling and Immunoassays Identify Pancreatic Enzymes as a Major Target of the Inflammatory Process. Am J Gastroenterol, 2010, 105:2060-71.

8. Kalenka, A, Gross B, Maurer MH, Thierse H.-J., Feldmann RE 2010. Isoflurane anesthesia elicits protein pattern changes in rat hippocampus. J Neurosurg Anesthesiology, 2010, 22:144-54.

9. Dietz, L., A. Bosque, P.Pankert, P.Merz, S. Ohnesorge, A.Anel, M.Schnoelzer and H.-J. Thierse. Quantitative DY-maleimide-based proteomic 2-DE labeling strategies using human skin proteins. Proteomics, 2009, 9:4298-4308.

10. Weltzien HU, Corsini E,  Gibbs S, Lindstedt M, Borrebaeck C, Budde P, Schulz-Knappe P, Thierse H-J, Martin SF and EL Roggen, Safe cosmetics without animal testing? Contributions of the EU Project Sens-it-iv. J of Consumer Protection and Food Safety, 2009, published with open access at Springerlink.com. 1-8.

11. Vossmerbaeumer, U, Ohnesorge S, Kuehl, S, Haapalahti H,  Kluter H, Jonas JB, Thierse H.-J.^* and K. Bieback^*, 2009. Transdifferentiation plasticity of human mesenchymal stem sells into ocular neuroectodermal lineage. Cytotherapy, 2009, 11.177-188. (^*Equal last authorship)

12. Thierse, H.J. and K. Heiss et al. 2004-2009. InterAct Database - Reviewed Entries: 33 binary protein interactions with nickel. http://www.ebi.ac.uk/intact/; InterAct Protein Database, European Institute of Bioinformatics, EMBL

13. Thierse, H.-J., H. Helm, P. Pankert. Metalloproteomics in the Molecular Study of Cell Physiology and Disease. Meth. Mol. Biol., 2008, 425.139-147.

14. Adams V, Mangner N, Gasch A, Krohne C, Gielen S, Hirner S, Thierse H-J, Witt CC, Linke A, Schuler G, Labeit S. Induction of MuRF1 Is Essential for TNF-alpha-Induced Loss of Muscle Function. J. Mol. Biol., 2008, 384.48-56.

15. Thierse, H.-J., H. Helm, M. Pink and H. U. Weltzien. Novel Fluorescence Assay for Tracking Molecular and Cellular Allergen-Protein Interactions. J Immunol Meth, 2007, 328.14-20.

16. Martin, S.F., Merfort I., and H.-J. Thierse. Interactions of Chemicals and Metal Ions with Proteins and Role for Immune Responses. Mini-Reviews in Medicinal Chemistry 2006, 6:71-89.

17. Weltzien H.U., Gamerdinger K. und Thierse, H.-J. Nickel presentation to T cells in contact hypersensitivity. In Handbook of Dendritic Cells - Biology, Diseases and Therapies, edited by M.B. Lutz, N. Romani, A. Steinkasserer, Wiley-VCH Verlag, Weinheim, 2006, Vol 3:1047-1058.

18. Thierse, H.-J., H. Helm, P. Pankert. Krankheitsbezogene Proteomics: Allergen-Protein Interaktionen in der Allergieforschung. BioSpektr, 2006, 12:512-514.

19. Heiss, K., C. Junkes, N. Guerreiro, M. Swamy, M.M. Camacho-Carvajal, W.W.A. Schamel, I.D. Haidl, D. Wild, H.U. Weltzien and H.-J. Thierse.  Subproteomic analysis of metal-interacting proteins in human B cells. Proteomics, 2005, 5:3614-3622.

20. Thierse, H.-J., K. Gamerdinger, C.Junkes, N.Guerreiro and H. U. Weltzien. T cell receptor interaction with haptens: metal ions as non-classical haptens. Toxicology  2005, 209:101-107.

21. Thierse, H.-J., C. Moulon, Y. Allespach, B.Zimmermann, A. Dötze, S. Kuppig, D. Wild, F. Herberg, and H. U. Weltzien. Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation. J Immunol. 2004, 172:1926-1936.

22. Weltzien,H.U., A. Dötze, K. Gamerdinger, S. Hellwig,  and H.-J. Thierse. Molecular recognition of haptens by T cells: more than one way to tickle the receptor. In: Immune Mechanisms in Allergic Contact Dermatitis (ed. A. Cavani) Landes Bioscience, Georgetown, TX, USA (www.eurekah.com), 2004, 14-27.

23. Weltzien H.U., K. Gamerdinger, H.-J. Thierse. T cell receptor interaction with haptens. Allergologie  2004, 27:138.

24. Moulon, C., Y. Choleva, H.-J. Thierse, D. Wild, and H. U. Weltzien. TCR transfection shows non-HLA-restricted recognition of nickel by  CD8+ human T cells to be mediated by ab T cell receptors. J Invest Dermatol. 2003, 121:496-501.

25. Lange H, Kiesch B, Linden I, Otto M, Thierse H.-J., Shaw L, Maehnss K, Hansen H, Lemke H.Reversal of the adult IgE high responder phenotype in mice by maternally transferred allergen-specific monoclonal IgG antibodies during a sensitive period in early ontogeny. Eur J Immunol. 2002, 32:3133-41.

26. Thierse, H.-J., C. Moulon, D. Wild, M. Juergens, and H. U. Weltzien. Analysis of nickel-binding proteins in human T cells. In International Proceedings. Monduzzi Editore, Bologna, Italy, 2001, pp. 727-732.

27. Thierse, H.-J., C. Moulon, D. Wild und H.U. Weltzien, Erkennung von Hapten-Peptidkomplexen durch T-Zellen, Suche nach allergenen Epitopen bei Nickelallergie, in Allergie 2000: Probleme, Strategien und Konsequenzen, Herausgeber J.Ring und U.Darsow, Dustri-Verlag Dr.K.Feistle, München-Deisenhofen, 2001, 100-104.

28. Seeger M, Thierse H.-J., Lange H, Shaw L, Hansen H, Lemke H. Antigen-independent suppression of the IgE immune response to bee venom phospholipase A2 by maternally derived monoclonal IgG antibodies. Eur J Immunol. 1998, 28:2124-30.

29. Thierse, H.-J., Aktivierung und Hemmung der Phospholipase A2 (PLA2) und der NO-Synthase (NOS) in humanen und murinen Zellen des Immunsystems und im Tiermodell der Adjuvans-Arthritis, Reihe Biochemie, Shaker Verlag, Aachen, 1997, 1 -188.

30. Tibes U, Scheuer WV, Thierse H.-J., Burgermeister E, Schramm S, Friebe WG, Dietz E. Role of cytosolic PLA(2), secretory PLA(2) and nitric oxide synthase in inflammation, in W.Uhl, TJ.Nevalainen, MW.Büchler (eds.), Phospholipase A2 Basic and Clinical Aspects in Inflammatory Diseases, Prog Surg. Basel Karger, 1997, Vol.24:153-167.

31. Thierse H.-J., Friebe WG, Scheuer W, Voelter W, Tibes U. Evidence for activation of cyclooxygenase-1/-2 by endogenous nitric oxide in adjuvant arthritic Lewis rats. Adv Exp Med Biol. 1996, 416:343-8.