Role of therapeutic blockade of CCL2 in a mouse model of SLE and lupus nephritis [Elektronische Ressource] / vorgelegt von Onkar P. Kulkarni

Kulkarni, Onkar

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• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 76
• Poids de l'ouvrage: 2 Mo

Extrait

Aus der Medizinischen Poliklinik – Innenstadt
der Ludwig-Maximilians-Universität München
Komm. Direktor: Prof. Dr. med. Martin Reincke










Role of therapeutic blockade of CCL2 in
a mouse model of SLE and lupus
nephritis





Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München




vorgelegt von
Onkar P. Kulkarni
Solapur, India

2010










Mit Genehmigung der Medizinischen Fakultät
der Universität München







1. Berichterstatter: Prof. Dr. med. Hans-Joachim Anders
2. Berichterstatter: Prof. Dr. Dr. h.c. Thomas Ruzicka

Mitberichterstatter: Prof. Dr. Hartmut Wekerle

Mitberichterstatter: Priv. Doz. Dr. Udo Kummer

Dekan: Prof. Dr. med. Dr. h. c. M. Reiser, FACR, FRCR

Tag der mündlichen Prüfung: 16.09.2010
























Onkar Prakash Kulkarni, M. Pharm.

Med. Poliklinik, Klinische Biochemie,
Ludwig-Maximillians University (LMU),
Schiller straße-42, Munich- 80336,
Germany
[e-mail]
.

DECLARATION

I here by declare that the present work embodied in this thesis was carried out by me
under the supervision of OA PD Dr. Hans Joachim Anders, Internist-Nephrologe-
Rheumatologie, Medizinische Poliklinik-Innenstadt Klinikum der Universität
München. This work has not been submitted in part or full to any other university or
institute for any degree or diploma.






Onkar P. Kulkarni

Date:










ACKNOWLEDGEMENTS

I can not resist myself from expressing my heart felt deep sense of gratitude and
respect for my PhD supervisor PD Dr. Hans-Joachim Anders, for his keen interest in
my research, constant encouragement, concrete suggestions and meticulous guidance
that helped me at each and every step of my research work during my PhD. Above all
his kindness and support to me through out my tenure at Klinische Biochemie, LMU. I
feel myself extremely lucky to be one of his students.
I would like to acknowledge Prof. S. Klussmann and Dr. D. Eulberg (Noxxon Pharma,
Berlin) for providing me experimental drug molecules for the research work carried
out and constructive discussions during my PhD work.
My sincere thank goes to Dr. Bruno Luckow and Dr. Peter Nelson for their constant
encouragement of my research work and constructive suggestions throughout my stay
at Klinische Biochemie.
I wish to express my profound gratitude to Ewa Radomska, Dan Draganovici and Jana
Mandelbaum for providing skillful technical assistance to carry out the research work
successfully.
I am really grateful to all my friends who always cared for me and made my stay a
delightful and helped me at every stage of my PhD. To name the few which I really
hold close to my heart Rahul, Anji, Sufyan, Julia, Mi, Stephie, Pati, Anil, Anela,
Maciej, Lilly, Murthy. The list is long but for those who are missed I would like to
mention that you will always be close to my heart.
I wish to express my heartiest thanks to my lab colleagues for their delightful and
stimulating companionship during my stay at Klinische Biochemie, LMU.
I would like to take this opportunity to mention here few of the best pals during my
stay in Munich, who were and are always there whenever I called them for any kind of
help and support namely, Pandu, Pallavi, Jas, Ebru, Ravi, Saritha, most importantly
Shiva (my roomy who could bear me for these long years).
There are no words to express my feeling, love and affectionate gratitude to my
parents, my brother Nilesh, sister in-law Ashwini and family members for their faith,
love, inspiration, selfless sacrifices and constant encouragement throughout my life.
My special thanks to Supriya for inspiring me to do better in my life.





Date: Onkar P. Kulkarni
Place: München






Dedicated to

‘My loving parents’

Without whom i would have not reached so far

Table of content

1. Introduction 3
1.1 Lupus nephritis 3
1.1.1 Histology 4
1.1.2 Pathogenesis of lupus nephritis 5
1.1.2.1 Predisposing factors associated with lupus nephritis 6
1.1.2.2 Apoptosis 8
1.1.2.3 Role of T cells 9
1.1.2.4 B 11
1.1.2.5 Anti-DNA antibodies 12
1.1.2.6 Nucleosomes 14
1.1.2.7 Complements 15
1.1.2.8 of cytokines 16
1.1.2.9 Other inflammatory mediators of chronic inflammation 18
1.1.2.10 Role of chemokines 20
1.1.3 Biology behind therapy of lupus nephritis 22
1.2 Spiegelmers-Next generation aptamers 26
lpr/lpr1.3 MRL mice- mouse model of lupus nephritis 30
2. Research hypothesis/objectives 31
3. Materials and methods 32
3.1 Materials 32
3.2 Methods 36
3.2.1 Methods part-I 36
3.2.2 part-II 51
3.2.3 Mehtods part-III 55
4. Results 58
4.1 Results part-I 58
lpr/lpr4.1.1 Pharmacokinetics of anti-CCL2 Spiegelmer in MRL mice 58
4.1.1.1 Bioavailability 58
4.1.1.2 Distribution 59
4.1.2 Survival rate 60
4.1.3 Renal parameters 61
4.1.3.1 Albumin/creatinine ratio 61
4.1.3.2 Glomerular filtration rate 62
4.1.3.3 Renal histology 63
lpr/lpr4.1.4 Extra renal autoimmune tissue injury in MRL mice 68
4.1.4.1 Skin lesion 68
4.1.4.2 Lung histology 69
4.1.4.3 Splenomegaly and lymphadenopathy 70
4.1.5 Systemic parameters 71
4.1.5.1 Plasma IgGs
4.1.5.2 aCCL2 72
4.1.6 Emigration of monocytes from bone marrow 72
4.1.7 RT-PCR analysis 74
4.1.8 Body weight development 74
4.1.9 Immunostimulatory effect of Spiegelmer (in vitro) 75
4.2 Results part-II 76
lpr/lpr4.2.1 Pharmacokinetics of anti-CCL2 Spiegelmer in MRL mice 76
14.2.2 Renal parameters 77
4.2.2.1 Renal histology 77
lpr/lpr4.2.3 Extra renal autoimmune tissue injury in MRL mice 82
4.2.3.1 Lung 82
4.2.3.2 Splenomegaly and lymphadenopathy 84
4.2.4 Plasma cytokines 85
4.2.4.1 Plasma IL12p40 and TNF-  85
4.2.4.2 aCCL2 86
4.2.5 Body weight development 86
4.3 Results part-III 87
4.3.1 Splenomegaly and lymphadenopathy 87
4.3.2 Effect on blood bone marrow 89
4.3.3 CFU-GM colony assay 90
5. Discussion 91
6. Summary and Conclusion 98
7Zusammenfassung 101
8. References 104
9. Abreviatons 114
Appndx 117
Resume 120
21. Introduction
Systemic lupus erythematosus (SLE) is characterised by the production of antibodies
to components of the cell nucleus in association with a diverse array of clinical
manifestations. The basic pathological features of SLE are that of inflammation and
blood vessel abnormalities, which include band or occlusive vasculopathy, vasculitis,
and immune complex deposition. The best characterised organ pathology is in the
kidney. By light and immunofluorescence microscopy, renal biopsies in patients with
SLE display mesangial cell proliferation, inflammation, basement membrane
abnormalities, and immune complex deposition, with immunoglobulins and
complement components (Tumlin JA.2008). On electron microscopy, these deposits
can be visualised in the mesangium and the subendothelial or subepithelial surface of
the basement membrane. Current therapeutic regimen for lupus nephritis mainly
comprised of medications which target abnormalities of immune regulation e.g.
immunosuppressant B and T cell targeting drugs. Corticosteroids, antimalarial drugs
and other therapies are being practiced along with immunosuppressants (Houssiau FA,
Ginzler EM.2008).
Even though we are yet to narrow down the exact aetiology of lupus pathogenesis,
immunologists have enough idea about how the end stage organ damage happens?
Researchers have been able to identify some of the inflammatory mediators which
play important role in chronic tissue inflammation in lupus nephritis. With this
information, we assume that reducing inflammatory mediators derived tissue
destruction along with low exposure to immunosuppressants; can be an excellent
approach to lupus nephritis treatment. In this study we evaluated the effectiveness of
blocking CC-chemokine ligand 2 (CCL2) in a murine model of lupus nephritis using a
novel tool to neutralize CCL2 in vivo. Before going in to details of the study, we will
try to summaris