Dec 21, 2014 - 1Arête Discoveries, Inc., Weston, MA; 2Department Rheumatology, Immunology, Brigham and Women's Hospital, Harvard Medical School, ...
The Role of Mast Cell Degranulation in the Pathogenesis of Multiple Sclerosis Jack Cowie1, Michael Gurish2, Tanuja Chitnis3 4
Arête Discoveries, Inc., Weston, MA; 2Department Rheumatology, Immunology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Partners Multiple Sclerosis Center, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 4Pediatric Multiple Sclerosis Center, Massachusetts General Hospital, Boston, MA 1
3
BACKGROUND Multiple Sclerosis (MS) is an inflammatory, autoimmune, demyelinating disease of the central nervous system. Patients are currently diagnosed using the McDonald 2010 standard which includes clinical presentation of symptoms, exclusion of any other diagnosis, and the presence of one or more lesions located on the optic nerve, two areas of the brain, and the cervical spine, separated by space and time. Currently there is no known cause, diagnostic biomarker, nor a predictor. While geographic and environmental factors appear to play a role in MS diagnosis frequency, research is needed to fully understand the pathogenesis of the disease.
Method-II Adoptive transfer EAE in C57BL/6 mice This method demonstrates detection of mast cell activity factors and biomarkers in a mouse model of multiple sclerosis. A single group of four C57BL/6 female mice, aged 8 to 9 weeks at Day 0 (day of transfer) were used. Typical study length is 16 days (from Day 0 until Day 15). Fully encephalitogenic cells, cultured 14 days in donor mice, were transferred into three recipient mice, where they induced EAE (effector phase of EAE).
In the past, studies1,2 have shown evidence of the involvement of mast cells (MC), and mast cell metabolites (MCm), in MS lesions and the cerebral spinal fluid (CSF) of people living with multiple sclerosis. Mast cells produce a plethora of mediators, including biogenic amines (histamine and serotonin), cytokines [interleukin (IL)-1 to IL-6, leukemia inhibitory factor, tumor necrosis factor (TNF) α, interferon-γ, transforming growth factor-β, and granulocyte-microphage colonystimulating factor], enzymes (acid hydrolyzes, chymase, phospholipases), rat mast-cell protease I and II, and tryptase), lipid metabolites (prostaglandins, leukotrienes, and platelet-activating factor), ATP, neuropeptides (vasoactive intestinal peptide), growth factors [nerve growth factor (NGF)], nitric oxide, and heparin. It should be kept in mind that while mediator release via degranulation is a very rapid process, longer lasting activation results in the release of de novo formed mediators. Mast cells as a class are heterogeneous, and no single mast cell makes all of these substances. Their immune regulatory role includes participation in switching off IgE by B cells and the release of chemoattractants that recruit eosinophils and monocytes3.
Adoptive Transfer Method - Expected results EAE will be consistently induced in 90-100% of the mice, with onset of paralysis between 9 and 14 days after immunization, and maximum clinical score of 3.0 to 3.5 in most mice.
Urine
# mice
Mean day of onset ± SD
MMS of 1st wave ± SD
% EAE relapse
MMS of relapse* ± SD
1
15
13.3 ± 5.6
3.2 ± 0.5
87.0 %
2.8 ± 0.6
2
15
11.9 ± 0.6
2.7 ± 0.3
80.0 %
2.9 ± 0.6
3
10
12.3 ± 1.7
3.3 ± 0.9
80.0 %
2.7 ± 0.7
* Only for mice which relapsed ** For all mice in the group
MMS of relapse period (Days 2142)** ± SD
End score (Day 42) ± SD
Disease incidence
1.7 ± 1.2
100 %
2.3 ± 1.3
1.4 ± 1.3
100 %
2.7 ± 0.9
1.5 ± 1.0
100 %
2.7 ± 0.9
18
Serum
16 Urine
14
Serum
12 10 8 6 4 2 0
Naïve
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Naïve Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 13
Day 13
Urine
Serum
Serum
Concentration of Histamine in B6 Mice
Concentration of PGDM in B6 Mice
100 20 ng/ml (+/- SEM)
80 ng/ml (+/- SEM)
Data are from three independent groups in a single experiment. Immunization used Hooke Kit™ MOG35-55/CFA Emulsion PTX (EK-2110), with 11-13 week old female C57BL/6 mice (Taconic Biosciences). Pertussis toxin from 3 vials was pooled before administration. Similar results are obtained using C57BL/6 mice from The Jackson Laboratory, as well as with MOG1-125/CFA Emulsion PTX (EK-2160) using the recommended protocol.
60 40 20
Adoptive Transfer
Exp.
# mice
Mean day of onset ± SD
MMS of 1st wave ± SD
% EAE relapse
MMS of relapse* ± SD
MMS of relapse period (Days 21-42)** ± SD
1
15
13.3 ± 5.6
3.2 ± 0.5
87.0 %
2.8 ± 0.6
2.7 ± 0.9
1.7 ± 1.2
100 %
2
15
11.9 ± 0.6
2.7 ± 0.3
80.0 %
2.9 ± 0.6
2.3 ± 1.3
1.4 ± 1.3
100 %
10
12.3 ± 1.7
3.3 ± 0.9
80.0 %
2.7 ± 0.7
2.7 ± 0.9
1.5 ± 1.0
100 %
End score (Day 42) ± SD
Disease incidence
References 1 Secor VH, Secor WE, Gutekunst CA, Brown MA (2000) Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 191:813–822. CrossRef Medline 2 Toms, R., H.L. Weiner, and D. Johnson. 1990. Identification of IgE-positive cells and mast cells in frozen sections of multiple sclerosis brains. J. Neuroimmunol. 30:169–177. 3 Hongquan Dong, Xiang Zhang, Yanning Qian 2014 Mast Cells and Neuroinflammation. Med Sci Monit Basic Res. 2014; 20: 200–206. Published online 2014 Dec 21. doi: 10.12659/MSMBR.893093, PMCID: PMC4282993 4 Paresh Thakker, Michael W. Leach, Wen Kuang, Stephen E. Benoit, John P. Leonard and Suzana Marusic. IL -23 Is Critical in the Induction but Not in the Effector Phase of Experimental Autoimmune Encephalomyelitis. J Immunol 2007; 178:2589-2598; doi: 10.4049/jimmunol.178.4.2589
15 10 5 0
0
* Only for mice which relapsed ** For all mice in the group
Exp.
Concentration of PGDM in SJL Mice 20
Urine
EAE in C57BL/6 mice Urine and serum were collected from the naïve and EAE mice on day 1 post-cell transfer. Histamine/N-methylhistamine and PGDM were measured. A significant increase in urine histamine/N-methylhistamine level was detected, as compared to the level in the naïve mice. PGDM was measured in urine and serum. A significant increase in urine PGDM level was detected, as compared to the level in the naïve mice.
3
Data are from three independent experiments. Immunization used Hooke Kit™ PLP139-151/CFA Emulsion (EK-0120), with female SJL mice. Similar results are obtained using PLP139-151/CFA
100 90 80 70 60 50 40 30 20 10 0
ng/ml (+/- SEM)
ng/ml (+/- SEM)
Concentration of Histamine in SJL Mice
OBJECTIVE To demonstrate mast cell activation resulting in degranulation of metabolites are the present in the earliest stages of disease inception, resulting in experimental autoimmune encephalomyelitis (EAE). the murine model of human multiple sclerosis, and may be critical in triggering subsequent inflammatory response composed of T-cell, B-cell and macrophage activation. METHOD-I EAE in SJL mice This method demonstrates detection of mast cell activity factors and biomarkers of CNS inflammation in a mouse model of multiple sclerosis. A single group of 23 mice were SJL mice, females aged (Day 0) 8 to 9 weeks were scored for signs of paralysis (EAE) daily starting on Day 9 after immunization. Three mice were sacrificed and tissue collected on Days 4, 5, 6, 7, 8, 9 and five on day 13. EAE was induced via subcutaneously injection (0.05 mL) at four sites with PLP139-151/CFA. Outcomes Primary outcomes were to find elevated N-methylhistamine and tetranor-PGDM levels in the urine and serum collected early in the study timeframe after disease initiation, and, well prior to clinical presentation of symptoms. EAE Induction by Active Immunization in SJL Mice Typical results
RESULTS EAE in SJL mice Urine was collected from the naïve mice and from the EAE mice on day 3, 4, 5, 6, 7, 8, 9 and 13 post-immunization. Serum was collected from the naïve mice and the EAE mice on day 4, 5, 6, 7, 8, 9 and 13 post-immunization. A significant increase in urine histamine/N-methylhistamine levels, as compared to levels in naïve mice, was detected at day 3 and day 5 (63.6 ng/ml, 53.7 ng/ml). A significant increase in serum histamine levels, as compared to levels in naïve mice, was detected at day 5 and day 7 (30.6 ng/ml. PGDM was measured in urine and serum. A significant increase in urine PGDM levels, as compared to levels in naïve mice, were detected at day 3 (15.764 ng/ml). A significant increase in serum PGDM levels, as compared to levels in naïve mice, were detected at days 5-9 (8.950 ng/ml).
Naïve B6
Adoptive Transfer
Naïve B6
t-test comparison of EAE group to naïve group (p
