Gastroenterology and Hepatology From Bed to Bench.
©2015 RIGLD, Research Institute for Gastroenterology and Liver Diseases
ORIGINAL ARTICLE
Clinical and immunological relevance of anti-neuronal antibodies
in celiac disease with neurological manifestations
Giacomo Caio, Roberto De Giorgio, Alessandro Venturi, Fiorella Giancola, Rocco Latorre, Elisa Boschetti,
Mauro Serra, Eugenio Ruggeri, Umberto Volta
Department of Medical and Surgical Sciences, University of Bologna and St. Orsola-Malpighi Hospital, Bologna, Italy
ABSTRACT
Aim: To assess anti-neuronal antibodies (NA) prevalence and their correlation with neurological disorders and bowel
habits in celiac disease (CD) patients.
Background: Neurological manifestations are estimated to occur in about 10% of celiac disease patients and NA to
central nervous system (CNS) and enteric nervous system (ENS) are found in a significant proportion of them. Little is
known about the clinical and immunological features in CD patients with neurological manifestations.
Patients and methods: NA to CNS and ENS were investigated in 106 CD patients and in 60 controls with autoimmune
disorders by indirect immunofluorescence on rat / primate cerebellar cortex and intestinal (small and large bowel)
sections.
Results: IgG NA to CNS (titer 1:50 - 1:400) were positive in 23 celiacs (21%), being more frequently detected in those
with neurological disorders that in those without neurological dysfunction (49% vs. 8%, P< 0.0001). Of the 26 celiacs
(24%) with IgG NA to ENS, 11 out of 12 with an antibody titer > 1:200 had severe constipation. Only one patient with
cerebellar ataxia and intestinal sub-occlusion was positive for NA to CNS and ENS. NA to CNS and ENS were found in
7% and 5% of controls, respectively.
Conclusion: In CD the positivity of NA to CNS can be regarded as a marker of neurological manifestations. High titer
NA to ENS are associated with severe constipation. The demonstration of NA to CNS and ENS suggests an immunemediated pathogenesis leading to central neural impairment as well as gut dysfunction (hence constipation),
respectively.
Keywords: Celiac disease, Neurological symptoms, Anti neuronal antibodies.
(Please cite as: Caio G, De Giorgio R, Venturi A, Giancola F, Latorre R, Boschetti E, et al. Clinical and
immunological relevance of anti-neuronal antibodies in celiac disease with neurological manifestations.
Gastroenterol Hepatol Bed Bench 2015;8(2):146-152).
Introduction
1
Celiac disease (CD) is a chronic, multisystemic,
autoimmune disorder, triggered by gluten
ingestion, in genetically sensitive patients (1).
Further to the small bowel, the main target organ,
CD is recognized to be a systemic disorder
Received: 30 January 2015 Accepted: 6 March 2015
Reprint or Correspondence: Umberto Volta, MD.
Department of Medical and Surgical Sciences, Bldg #5,
University of Bologna S.Orsola-Malpighi Hospital Via
Massarenti 9, 40138 Bologna – Italy.
E-mail: umberto.volta@aosp.bo.it
involving many other tissues and organs, e.g. skin,
thyroid, liver, joints, muscle, bone, pancreas and
nervous systems (2). The reasons for the
multisystemic nature of CD is ascribable to the
widespread localization of the main autoantigen of
the disease, i.e. tissue transglutaminase type 2
(TG2) and related isoforms (TG3 and TG6) (2).
Idiopathic cerebellar ataxia (ICA), peripheral
neuropathy (PN) and different forms of epilepsy
represent the most common neurological
Gastroenterol Hepatol Bed Bench 2015;8(2):146-152
Caio G. et al 147
manifestations occurring in CD patients (3-5).
Other CD-associated neurological diseases include
multiple
sclerosis,
migraine,
multifocal
leukoencephalopathy, dementia, chorea, and
attention / memory impairment. Taken together,
neurological manifestations can occur in about
10% of CD patients.
The pathogenesis of neurological involvement in
CD is still debated. Previous pathogenetic theories
regarded mainly vitamin deficiencies (e.g.
thiamine, folic acid, cyanocobalamin, vitamin E),
due to intestinal malabsorption (6). More recently,
however, pathologic data on the CNS of patients
with neurological CD, indicated that immunemediated mechanisms can play a role by evoking
neuronal injury and dysfunction (7). In this line,
circulating anti-neuronal antibodies (NA) of the
IgG class have been demonstrated to target central
and enteric nervous systems (CNS and ENS,
respectively) in a significant proportion of
neurological CD patients (4).
This study has been designed to gain further
insights about clinical and immunological features
of CD patients with neurological manifestations.
In particular, we aimed to assess the prevalence of
NA to CNS and / or ENS. Another objective of
this study is to expand the knowledge of NA to
ENS and explore their association with gut
dysfunction.
neurological manifestations were also assessed. In
all cases, in addition to positive serology (antitTG2 and anti-endomysial antibodies), the
diagnosis of CD was always confirmed by
endoscopic duodenal biopsy revealing villous
atrophy (Marsh III lesion) (8, 9).
As a control group, 60 patients with immunemediated disorders (6 Crohn disease; 4 ulcerative
colitis; 15 autoimmune hepatitis; 15 primary
biliary cirrhosis; and 10 CREST syndrome) were
investigated. None of them showed any signs of
neurological manifestations. All patients and
controls gave their informed consent before
entering the study. Since patients were not
individually identified, a simplified International
Review Board approval by the Ethics Committee
of the St. Orsola Malpighi Hospital was obtained.
Detection of NA
NA to CNS and ENS were detected by indirect
immunofluorescence in all CD and control
patients. Cryostat sections (5 mm) of monkey and
rat cerebellum (for NA targeting the CNS) as well
as rat ileum and colon (for NA targeting the ENS)
(Astra srl, Milano, Italy) were used. Sera of
patients were tested at the initial dilution of 1:10
and, when positive, titrated to the end point.
Rabbit anti human IgG (Dako, Copenhagen,
Denmark) were used as secondary antibody at the
appropriate working dilution (1:60 and 1:100 on
rat and monkey tissue, respectively).
Patients and Methods
A total number of 106 non consecutive patients
with untreated CD (24 males, 82 females; age
range 17-59 years) were enrolled in this study. Of
these, 35 CD patients (5 males, 30 females; age
range
19-56
years)
had
neurological
manifestations (11 cases of ICA; 10 epilepsy
without cerebral calcifications at computed
tomography; 4 multiple sclerosis, MS; 5 attention /
memory impairment syndrome, AMIS; and,
finally, 5 PN). CD patients (n= 71; 19 males, 52
females; age range 17-59 years) without
Statistical analysis
The two-tailed Fisher's exact test was used to
compare the clinical findings and prevalence of
NA to CNS / ENS in CD patients with and without
neurological manifestations vs. autoimmune
disorder controls.
Results
The immunofluorescent pattern of NA to CNS
was characterized by an intense immunostaining
in the nucleus and cytoplasm of Purkinje cells
Gastroenterol Hepatol Bed Bench 2015;8(2):146-152
148 Antineuronal antibodies in celiac disease
along with positivity of granular layer neurons on
rat and monkey cerebellum sections (Figure 1).
Figure 1. Immunofluorescent pattern of NA to CNS on
rat
cerebellum sections.
Note
the
intense
immunostaining in the nucleus and cytoplasm of
Purkinje cells (arrows) along with positivity of granular
layer neurons (arrowheads) in a CD patient with
cerebellar ataxia. Original magnification 40x.
Figure 2. Immunofluorescent pattern of NA to ENS on
rat ileum sections. Note the bright staining in the
myenteric plexus (arrows) of the rat ileum observed in a
CD patient with severe constipation. Magnification 40x.
A bright staining in the myenteric and
submucosal plexuses of the rat ileum and colon
represents the typical pattern of NA to ENS
(Figure 2). NA to CNS were found in 23 (21%) of
the whole group of the 106 CD patients in
comparison with their positivity in 4 (7%) of the
60 controls with autoimmune disorders (P< 0.05
(Figure 3).
Figure 3. Prevalence of NA to CNS in the whole group
of celiac disease patients vs. autoimmune controls
(P<0.05, two-tailed Fisher’s exact test). Abbreviations:
CD: Celiac disease; CNS+: NA to CNS; CTRL:
controls.
NA to CNS were detected in 17 (49%) of the
35 patients with neurological CD vs. 6 (8%) of the
71 patients with non-neurological CD patients (P<
0.0001) (Figure 4). Of the 17 neurological CD
patients with NA to CNS, 7 had ICA, 4 epilepsy, 2
MS, 2 PN and 2 AMIS. None of the 4 controls
with immune-mediated disorders positive for NA
to CNS showed neurological manifestations.
NA to ENS were positive in 26 (24%) of the
106 CD patients vs. 3 (5%) of the 60 controls with
autoimmune disorders (P< 0.05) (Figure 5). NA to
ENS were not significantly different in
neurological vs. non-neurological CD, being
positive in 9 (26%) of the 35 vs. 17 (24%) of the
71 patients, respectively. A high antibody titre (>
Gastroenterol Hepatol Bed Bench 2015;8(2):146-152
Caio G. et al 149
1:200) of NA to ENS was detected in 12 (46%) of
the 26 CD patients and, notably, 11 of these 12
complained of severe, Rome III-defined
constipation (P< 0.0001) (Figure 6). Only one
patient with cerebellar ataxia and recurrent
intestinal sub-occlusive episodes was positive for
NA to CNS and ENS.
Figure 4. Prevalence of NA to CNS in neurological CD
vs. non-neurological-CD (P<0.0001, two-tailed Fisher’s
exact test). Abbreviations: CD neuro+: neurological
celiac disease; CD neuro-: non-neurological celiac
disease; CNS+: NA to CNS.
Figure 5. Prevalence of NA to ENS in the whole group
of celiac disease patients vs. autoimmune controls
(P<0.05, two-tailed Fisher’s exact test). Abbreviations:
CD: celiac disease; ENS+: NA to ENS; CTRL:
controls.
Discussion
The link between CD and neurological
disorders has been established since many years
and neurological impairment can be often the only
clinical manifestation for suspecting CD (2-7). A
CD antibody screening should always be sought in
any patients with ICA, PN and, especially, in
those cases with pharmacologically resistant forms
of epilepsy. The identification of gluten-sensitive
enteropathy and the subsequent strict gluten free
diet can often result into the improvement of
neurological impairment (3, 7). Published data
showed that ICA and PN are the most common
neurological disorders being found in 2-15% and
1.5-8% of CD patients, respectively (10, 11).
The present study was designed to test the
prevalence of NA to CNS and ENS in patients
with CD-related neurological manifestations. Our
results demonstrated that almost half of
neurological CD patients had circulating NA to
CNS mainly detected in patients with ICA. Also,
NA to CNS were found in patients with CDrelated epilepsy, PN, MS, and, finally, AMIS,
findings that confirm previously published data
(4). In contrast, likewise autoimmune disorders,
patients with non-neurological CD showed a very
low prevalence of these autoantibodies, thus
strengthening a significant association between
NA to CNS and neurological CD (4).
Further to NA to CNS, we found positive NA
to ENS in about a fourth of the total (n= 106) CD
group with a significantly higher prevalence than
that of autoimmune controls. Although NA to
ENS showed a similar prevalence in neurological
and non-neurological CD, their detection was
highly associated with gut dysfunction. In
particular, a subset (11 / 12) of CD patients
showing high titer (> 1:200) of NA to ENS had a
very severe form of chronic constipation as
established by Rome III criteria.
Gastroenterol Hepatol Bed Bench 2015;8(2):146-152
150 Antineuronal antibodies in celiac disease
Figure 6. Correlation between antibody titer of NA to ENS and severe constipation: 11 of 12 CD patients with
antibody titer > 1:200 showed severe constipation (P< 0.0001, two-tailed Fisher’s exact test). Abbreviations: "ENS
titre" refers to titer of NA to ENS.
Constipation is the prototype of functional
bowel disorders and its occurrence has been
estimated in 15-20% of the general population.
Based on this high prevalence one cannot
discard the possibility that constipation may be
just a coincidence in our CD patients. However,
the present data highlighted a strong association
linking CD to chronic constipation. Indeed, the
ENS is one of the major systems controlling gut
physiology. Hence any noxa (i.e. NA) perturbing
ENS morpho-functional integrity may cause
bowel dysfunction (dysmotility, altered secretion)
known to underlie constipation. Compared to CD
patients without NA to ENS, sera of CD patients
containing NA to ENS exposed to neuronal
cultures evoked apoptosis and neuronal loss (7).
Also, previous data demonstrated that NA may
alter the ascending reflex of small bowel
peristalsis and inhibit motorneuron excitability in
vitro (12). Taken together these results provide a
pathophysiological basis to the concept that
autoimmunity targeting ENS can be an important
mechanism operating in chronic constipation
identifiable in CD patients. Similarly to ENS, also
NA to CNS can exert a pathogenetic potential on a
wide array of different central and peripheral
neurons, thereby leading to neurological
manifestations. In support of this role, previous
pathological data showed an immune (humoral
and cellular) infiltrate in the CNS (mainly
cerebellum) of patients with neurological
impairment associated to CD (13).
The reasons to explain the immune mediated
targeting of enteric, peripheral and central neurons
are still partially understood. One possibility is
that tissue TG isoform expression may drive an
activation of the immune system in susceptible
CD patients. Indeed, one of these autoantigen can
be the TG6 isoform and anti-TG6 antibodies have
been identified in patients with ICA and PN with
or without underlying CD (14). Whether the NA
to CNS or to ENS may target or cross react with
TG6 (or other TG isoforms) remain to be
elucidated. Moreover, the presence of high titer
anti-gliadin antibodies (AGA) has been
demonstrated in patients with neurological
manifestations without a clear-cut intestinal
damage. The association between AGA and
neurological manifestations has been referred to as
gluten-sensitive ataxia / neuropathy (10, 15). AGA
can cross react with epitopes expressed by
Purkinje cells (16), thus expanding the spectrum
of autoimmune related mechanism operating in
neurological gluten-induced disorders.
Finally, neurological symptoms (i.e. headache,
foggy mind, leg / arm numbness, depression) are
Gastroenterol Hepatol Bed Bench 2015;8(2):146-152
Caio G. et al 151
also integral part of the clinical repertoire of non
celiac gluten sensitivity (NCGS), a further
manifestation of gluten-related disorders, and in
this condition AGA have been identified in as
many as 50% of patients (17,18). However,
whether AGA contributes to the symptom
generation in NCGS is still undeciphered and
awaits further elucidation.
Patients with minor intestinal lesions (i. e.
Marsh I and II) were not included in the present
study. In fact, minor intestinal lesions can be
expression of several pathological conditions
including potential CD, NCGS, autoimmune
disorders, immune-deficiency diseases, parasitic
infections and food intolerance. Independently
from diagnosis, a significant proportion of patients
with normal villous architecture, but with mild
duodenal inflammation, complain of bloating and
alternating bowel habit ranging from diarrhea to
constipation. Therefore, it would be interesting to
evaluate the presence of NA, in particular to ENS,
in patients with minimal lesions.
In conclusion, our study showed a high
prevalence of NA to CNS in CD-related
neurological disorders suggesting that these
antibodies could be a marker of neurological
involvement in CD. NA to ENS were associated
with severe chronic constipation in neurological
and non-neurological CD, thus leading to the
concept of an underlying autoimmune mediated
impairment of enteric neuron function. Taken
together our data contribute to define the clinical
and immunological features of NA to CNS and
ENS in neurological as well as non-neurological
CD and therefore pave the way for a better
management of these patients.
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