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, - PowerPoint PPT Presentation

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To elucidate whether GAD-ab were associated with diabetic autonomic neuropathy ... Out of 133 patients, GAD-ab was detected in 36 patients, all of whom had type ... – PowerPoint PPT presentation

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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,

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

Structure of the Presentation
  • Background
  • Main Question
  • Methods
  • Figures and Tables
  • Conclusion

  • NOD mice develop diabetes similar to human type 1
  • 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

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

  • diabetes-prone (NOD) and diabetes resistant (NOR)
  • 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
  • Growing hybridomas screened for Ab production
  • Immunofluorescence staining for Ab
  • 33 Fusions,
  • obtained 352 hybridomas,
  • 74 were Ab-secreting

Table 1
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

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.

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
  • gt predominant recruitment of nervous tissue
    specific B cells to pancreatic islets in
    diabetogenesis is dissociated from islet beta
    cell destruction

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
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

Antigenic specificity of B cells penetrating the
islet is not restricted to the pancreas
  • 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).

  • 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
  • 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
  • In particular, monoclonal antibodies with ß cell
    specificity cross reacted with only a few stomach
    and kidney cells completely (Carrillo et al 2004).

  • 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).

  • In mice, Th-1 humoral responses were dominated by
    antibodies IgG3, IgG2a, and possibly IgG2c
  • 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
  • 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
  • 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

  • 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.

  • 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
  • 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
  • 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).

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
  • 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.

Clinical Implications
  • Postural blood pressure and simultaneous CF-SG
    antibody measurements were performed in 28 T1D
  • 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).

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
  • 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).

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
  • Therefore patients presenting with diabetes of
    shorter duration testing positive for these
    autoantibodies may represent pre-neuropathic
    patients (47).

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
  • 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

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
  • There was no HLA-DR3 or HLA-DR4 association with
  • Subclinical autonomic dysfunction was
    demonstrated in 3 of 4 family members with
    autoantibodies compared to 0 of 4 family members
    without autoantibodies (49).

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
  • 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).

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
  • 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

  • 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).
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