Title: Islet-infiltrating%20B-Cells%20in%20Nonobese%20Diabetic%20Mice%20Predominantly%20Target%20Nervous%20System%20Elements%20%20Jorge%20Carrillo,%20Maria%20Carmen%20Puertas,%20Aurora%20Alba,%20Rosa%20Maria%20Ampudia,%20Xavier%20Pastor,%20Raquel%20Planas,%20Nadal%20Riutort,%20Nuria%20Alonso,%20Ricardo%20Pujol-Borrell,
1Islet-infiltrating B-Cells in Nonobese Diabetic
Mice Predominantly Target Nervous System Elements
Jorge Carrillo, Maria Carmen Puertas, Aurora
Alba, Rosa Maria Ampudia, Xavier Pastor, Raquel
Planas, Nadal Riutort, Nuria Alonso, Ricardo
Pujol-Borrell, Pere Santamaria, Marta Vives-Pi,
and Joan Verdaguer
- Presented by Lei Lin and Aron Airall
2Structure of the Presentation
- Background
- Main Question
- Methods
- Figures and Tables
- Conclusion
3Background
- NOD mice develop diabetes similar to human type 1
diabetes - 8.3-NOD mice NOD mice whose B cells expressing
a transgenic diabetogenic TCR (from pathogenic
CD8 T cells that infiltrate islets in
pre-diabetic NOD mice) specific for beta cell.
So, these mice develop accelerated form of
autoimmune diabetes - Type 1 diabetes is characterized by selective
destruction of pancreatic beta cells by the
patients own immunity - Islet-infiltrating T-cells are major effectors of
beta-cell damage in type 1 diabetes - B cells also play a critical role in initiation
and progression of type 1 diabetes. They capture
beta-cell autoAgs via cell surface Igs and by
presenting these Ags to autoreactive T cells - Other islet cells may also be targets (autonomous
nervous system) of the autoimmune response
4Main Question of the Paper
- Question What is the antigenic repertoire of
islet-infiltrating B-cells in non-obese diabetic
mice (NOD mice) - Answer predominant B cell response against
nervous system elements
5Methods
- diabetes-prone (NOD) and diabetes resistant (NOR)
mice - NOD RAG-2-/- develop no diabetes or insulitis.
- 2 models (NOD X NOR) F1 (8.3-NOD X NOR) F1
- Pancreatic islets isolated cultured in media
- 12 hrs later, islet-infiltrating mononuclear
cells migrating into the culture media fused with
the NS-1 myeloma cell line - Fused cell lines cultured in HAT media for 2
weeks - Growing hybridomas screened for Ab production
- Immunofluorescence staining for Ab
- 33 Fusions,
- obtained 352 hybridomas,
- 74 were Ab-secreting
6Table 1
7Fig.1 A Table 2
- a-j RAG-2 -/- mice
- (no diabetes or insulitis)
- k-m Hep- 2 cell line n-o
Crithidi luciliae - (Presence of mAbs to nuclear Ags and native DNA
were confirmed by staining Hep-2 and Crithidia
luciliae ) - NOD 54 hybridomas, 12 of which Ab-secreting, 5
of which recognized pancreatic tissue components
other than beta-cells, 2 of which (d,e,f,
exocrine and i,extracellular connective tissue)
produced mAbs reacting to exocrine tissue/
extracellular matrix. 3 of the 5 produce Abs
specific for the same pancreatic tissue elements.
These elements react with the anti-peripherin,
anti-neurofilament 200, and/or anti-GFAP Abs.
Peripherin, neurofilament 200, and GFAP are
nervous system componentsgt specificity for
nervous system. - a) intraislet b) neuroendocrine c) mixed d-f)
exocrine g h) nervous system i) extracellular
connective tissue j) tissue -ve control k n)
nuclear anti-DNA l) nuclear dots staining the
nucleus m o) -ve control of Hep-2 and
Crithidia luciliae. - Secondary Abs Goat anti-mouse IgMGA labeled
with FITC
8Fig. 1A Table 2
- 8.3 NOD 151 hybridomas, 18 Ab-secreting, 12
secreted Abs specific for pancreatic tissues. 4
of the 12 produced mAbs specific for an islet Ag
that colocalize with peripherin, neurofilament
200, and/ or GFAP - gt suggesting reactivity against pancreatic
nervous system. - Remaining 7 of the 12 produced mAbs specific for
exocrine tissue, cell nucleus, or extracellular
connective tissue.
9Ag specificity of B cells recruited to pancreatic
tissue in non-diabetes-prone mice is also for
nervous system components of the pancreas
- In (NOD X NOR) F1 (8.3-NOD X NOR) F1 mice, the
largest proportion of Ab-secreting hybridomas
produced mAbs specific for nervous system Ags. - In (NOD X NOR) F1 mice, 11 of 17 Ab-secreting
hybridomas specific for pancreatic tissues, 9 of
the 11 produced Abs that colocalized with
peripherin, neurofilament 200, and/or GFAP, only
1 recognized pancreatic islets. - In (8.3-NOD X NOR) F1 mice, 24 of 27
Ab-secreting hybridomas produced mABs specific
for pancreatic tissues, 12 of the 24 recognized
nervous system elements, only 3 recognized
pancreatic islets. - gt indicated that predominant anti-nervous tissue
element B cell response in NOD mice also occurs
in mice in which islet beta cell destruction is
limited. - gt predominant recruitment of nervous tissue
specific B cells to pancreatic islets in
diabetogenesis is dissociated from islet beta
cell destruction
10Fig. 1 B C
Abs from the hybridomas are specific for
insulin, somatostatin, and glucagon A -
intraislet B - neuroendocrine C - mixed
(islet) H173- colocalize with Abs specific for
GFAP, neurofilament, and peripherin 2 nervous
system staining patterns H184- colocalize with
Abs specific for GFAP, neurofilament, and
peripherin H122- colocalize only partially with
Abs specific for GFAP
11Table 2
- -52 Ab secreting hybridomas tested, 20 produced
IgM mAb, 3 IgG1, 9 IgG2c, 19 IgG2b, and 1 IgA. - -IgM is the prevailing isotype for hybridomas
restricted to exocrine, nuclear, and
extracellular connective tissues gt they
originated from B cells producing polyreactive
natural Abs. - -Most islet and nerve specific hybridomas
secreted IgG2b, IgG2c, and IgG1 Abs gt they
derived from precursors that had undergone class
switch recombination - gt Most islet-associated, Ab-secreting hybridomas
specific for pancreatic islets and nervous system
derive from B cells that have undergone Ig class
switch recombination
12Antigenic specificity of B cells penetrating the
islet is not restricted to the pancreas
13Results
- Table 3 demonstrated that antigenic specificity
of B cells penetrating the islet is not
restricted to the pancreas. - Antibody-secreting hybridomas negative for
pancreas tissue was tested vs other NOD.RAG-2-/-
mice organs. - A total of 7/22 (31.8) pancreas were negative to
antibody secreting hybridomas directed against
several other moue organs. - In particular 3/7 (42.9) reacted with brain and
other tissues, whereas the 2/7 (28.6) were
exclusively nervous system elements (Carrillo et
al 2004).
14Results
15Results
- Figure 2 gives further evidence that antigenic
specificity of B cells penetrating the islet was
not restricted to the pancreas. - Cryosections of NOD.RAG-2-/- mice parotid, mixed
salivary, and bronchial glands, thyroid, brain,
stomach, and kidney were stained using a
monoclonal antibody delegate for each sectioning
pattern. - Monoclonal antibodies with exocrine or reticular
connective tissue staining properties reacted
with all tested tissues. - Similarly, monoclonal antibodies displayed
nervous system staining properties to most
tissues analyzed indicating that they were
directed vs endogenous antigens of the nervous
system. - In particular, monoclonal antibodies with ß cell
specificity cross reacted with only a few stomach
and kidney cells completely (Carrillo et al 2004).
16Results
17Results
- Figure 3 demonstrated that cross reactivity of
monoclonal antibodies to similar antigens in rats
and humans, indicating that the intrapancreatic
B-cell response that takes place during
initiation and/or progression of type 1 diabetes
is directed against antigenic determinants
conserved across species (Carrillo et al 2004).
18Discussion
- In mice, Th-1 humoral responses were dominated by
antibodies IgG3, IgG2a, and possibly IgG2c
isotypes. - Alternatively Th2 responses were dominated by
IgG1 and IgE antibodies, whereas Th3 responses
were dominated by IgG2b and IgA antibodies. - There was also evidence of autoreactive B-cells,
Th1 (IgG2c), Th2 (IgG1), and Th3 (IgG2b)
complexes penetrating islet reticular connective
tissue. - These results were consistent with the complex
cytokine profiles of islet-associated T-cells in
8.3-NOD, 8.3-NOR, and 8.3-F1 mice, indicating an
active B-cell response vs pancreatic nervous
system antigens at the earliest stages of
diabetogenesis (i.e., in diabetes resistant
mice). - It is beyond the scope of this paper as to
whether the intraislet immune response against
pancreatic nervous tissue elements also occurs in
human type 1 diabetic patients or whether this
response is key to diabetogenesis (Carrillo et al
2004).
19Discussion
- It is speculative to think that their study was
not a peculiarity of NOD mice. Moreover, an early
loss of islet sympathetic nerves during the
course of diabetes has also been observed in
BioBreeder diabetic rats (26). - To discover whether islet sympathetic nerves are
damaged during the autoimmune destruction of
islet B-cells, sections of pancreas from
BioBreeder (BB) diabetic rats were immunostained
using antibodies against vesicular monoamine
transporter 2 (VMAT2), a marker of sympathetic
nerve terminals. - They found a marked decrease in the
VMAT2-positive fiber area in the islets of BB
rats that had been diabetic for only 1-2 weeks
compared with their nondiabetic controls. - In contrast, there was no significant decrease in
the VMAT2-positive fiber area in the exocrine
pancreas in these early diabetic BB rats. - Furthermore, streptozotocin-diabetic rats showed
no decrease in VMAT2-positive fiber area in their
islets compared with controls.
20Discussion
- The classical diabetic autonomic neuropathy (DAN)
that eventually occurs in the heart was not
present in BB diabetic rats at this early stage
as evidenced by normal cardiac VMAT2
immunostaining and normal cardiac norepinephrine
content. - Also, in contrast to DAN, this islet neuropathy
did not worsen with duration of diabetes. These
data provided evidence of unrecognized early
sympathetic islet neuropathy. - Because eSIN occurs selectively in the islet,
rapid in onset, and associated with autoimmune
but not chemically induced diabetes, it is
distinct from DAN in location, time course, and
mechanism. - Nevertheless, cross reactivity of these murine
antibodies for both rat and human pancreas tissue
indicates the existence of conserved antigenic
determinants across species. - Presently, the molecular nature of these
antigenic protein determinants is unknown (data
not shown) (26).
21Clinical Implications
- Sera of 94 type1 diabetic patients were tested
for the presence of complement-fixing sympathetic
ganglia (CF-SG) antibodies. In cross-sectional
analysis (0-43 yr), 22 had detectable CF-SG
antibodies. - Participants at high risk for T1D were also
studied. Group 1 (4-64 yr) islet cell
antibody-positive (ICA) prediabetic, 10/19 (53)
were CF-SG group 2 (6-14 yr) ICA-prediabetic
(first-degree relatives of T1D with either
transient hyperglycemia, impaired OGT, and/or
first-phase insulin release after I.V GT
testing), 4/9 (44) were CF-SG (2/4 ICA- CF-SG
subjects have progressed to type 1 diabetes)
group 3 (1.5-43 yr) ICA T1D (lt or to 1 yr
duration) 6/10 (60) were CF-SG and group 4
(8-59 yr) ICA- T1D (lt or to 1 yr duration),
2/11 (18) were CF-SG.
22Clinical Implications
- Postural blood pressure and simultaneous CF-SG
antibody measurements were performed in 28 T1D
subjects. - The drop in systolic blood pressure was greater
in the CF-SG subjects (P less than .05), and the
frequency of CF-SG was greater in the mean to
-2SD group (P less than .03) when data were
analyzed within mean /- 2SD of the normal blood
pressure response (45).
23Clinical Implications
- There is also evidence that the immune system may
play a role in the pathogenesis of autonomic
neuropathy in T1D. - The presence of autoantibodies to sympathetic and
parasympathetic nervous structures and their
correlation with other typical autoantibodies in
well-characterised diabetic populations, with or
without diabetic neuropathy, and normal subjects
was investigated. - Indirect immunofluorescent complement-fixation
technique was used, with monkey adrenal gland,
rabbit cervical ganglia and vagus nerve as
substrates. - Patients with symptomatic autonomic neuropathy
33 were positive for at least one autoantibody
(20 anti-sympathetic ganglia, 10 anti-vagus
nerve and 13 anti-adrenal medulla).
24Clinical Implications
- The frequency of having one or more antibodies to
nervous tissues and the prevalence of
anti-cervical ganglia antibodies were
significantly higher in the neuropathic patients
than in the diabetic control subjects with
disease of similar duration and in the normal
subjects (p lt 0.05). - Patients without complications with diabetes of
shorter duration 33 were positive for at least
one autoantibody (13 anti-ganglia, 13
anti-vagus nerve and 13 anti-adrenal medulla).
No correlation was found with other tissue
autoantibodies, including islet cell antibodies. - They concluded that nervous tissue autoantibodies
are associated with symptomatic autonomic
neuropathy. Anti-sympathetic ganglia and
anti-vagus nerve antibodies seem to be more
disease-specific. - Therefore patients presenting with diabetes of
shorter duration testing positive for these
autoantibodies may represent pre-neuropathic
patients (47).
25Clinical Implications
- Yet still another group evaluated the incidence
of autonomic nervous system autoantibodies (ANS)
in nondiabetic family members of T1D diabetics. - 24 families, including 45 nondiabetic parents and
53 nondiabetic siblings of a T1D proband were
studied. - 101 nondiabetic population control subjects were
also studied. - Stored sera from nondiabetic family members and
control subjects were tested for
complement-fixing (CF) adrenal medullary
antibodies (CF-ADM), sympathetic ganglia
antibodies (CF-SG), and vagus nerve antibodies
(CF-V) by indirect immunofluorescence. - HLA-DR3 and -DR4 typing was performed on 42
nondiabetic family members and 104 diabetic
subjects.
26Clinical Implications
- One or more CF-ANS were in 45 of 93 (40)
nondiabetic family members compared to 2/70
(2.8) control subjects. - CF-SG were in 28/92 (30) family members compared
to 0/101 control subjects (P 0.0001). CF-V were
in 25 of 95 (26) family members compared to 0 of
76 control subjects (P 0.0001). - CF-ADM were in 10 of 83 (12) family members
compared to 2 of 70 (2.8) control subjects (P
0.056). - There was no HLA-DR3 or HLA-DR4 association with
ANS. - Subclinical autonomic dysfunction was
demonstrated in 3 of 4 family members with
autoantibodies compared to 0 of 4 family members
without autoantibodies (49).
27Clinical Implications
- To elucidate whether GAD-ab were associated with
diabetic autonomic neuropathy and/or complement
fixing antibodies against sympathetic ganglia,
adrenal medulla, and vagus nerve, another group
examined 133 diabetic patients (95 with T1D). - GAD-ab were determined by a radioligand binding
assay whereas sympathetic ganglia antibodies,
adrenal medulla antibodies, vagus nerve, and ICA
were evaluated by indirect immunofluorescence
assays. - Autonomic nerve function was evaluated by
objective tests (heart rate reactions to deep
breathing and to tilt). Out of 133 patients,
GAD-ab was detected in 36 patients, all of whom
had type 1 diabetes. The frequency of GAD-ab was
similar (38) in T1D with and without signs of
autonomic neuropathy (21/55 vs 15/40).
28Clinical Implications
- Also, there were no significant associations
between GAD-ab and autonomic nerve antibodies
GAD-ab were detected in 9/21 (43) of patients
with and in 27/112 (24) of patients without
sympathetic ganglia antibodies, in 5/15 (33) of
patients with and 31/118 (26) without adrenal
medulla antibodies, and in 5/15 (33) with and
31/118 (26) of patients without vagus nerve
antibodies. - The frequency of ICA, however, was significantly
increased in patients with sympathetic ganglia
antibodies compared with those without
sympathetic ganglia antibodies (10/21 48 vs
21/112 19 p lt 0.01). - In conclusion, GAD-ab were neither associated
with disturbed autonomic nerve function nor with
antibodies against autonomic nerve structures
(48).
29Conclusion
- Finally, in conclusion we describe a set of
antibody-secreting hybridomas from
islet-infiltrating B-cells of diabetes prone and
resistant mice. - Our presentation showed that most antibody body
secreting, islet secreting B cells recognized
antigens expressed by nervous cellular elements
of the pancreas and provide existence of an
active lymphocyte response against these cellular
elements early in diabetogenesis. - This work gives substantial evidence that nervous
system elements of islets of Langerhans are
important targets of the diabetogenic autoimmune
response (Carrillo et al 2004).