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,

1
Islet-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

2
Structure of the Presentation

• Background
• Main Question
• Methods
• Figures and Tables
• Conclusion

3
Background

• 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

4
Main 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

5
Methods

• 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

6
Table 1
7
Fig.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

8
Fig. 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.

9
Ag 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

10
Fig. 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
11
Table 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

12
Antigenic specificity of B cells penetrating the
islet is not restricted to the pancreas
13
Results

• 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).

14
Results
15
Results

• 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).

16
Results
17
Results

• 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).

18
Discussion

• 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).

19
Discussion

• 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.

20
Discussion

• 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).

21
Clinical 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.

22
Clinical 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).

23
Clinical 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).

24
Clinical 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).

25
Clinical 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.

26
Clinical 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).

27
Clinical 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).

28
Clinical 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).

29
Conclusion

• 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).