Transcript Immunological investigation in Czech patients with

Immunological investigation in Czech patients
with autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED).
A.Šedivá, 1J.Lébl , 1D.Čiháková
Institute of Immunology,
Second Faculty of Medicine and University Hospital Motol,
1Department
of Pediatrics,
Third Faculty of Medicine and University Hospital Královské Vinohrady,
Charles University, Prague, Czech Republic
APECED
Autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED), also known as autoimmune
polyglandular syndrome type 1 (APS1), is a rare
autosomal recessively inherited disease affecting
endocrine glands caused by the mutations of the gene
known as autoimmune regulator – AIRE.
chromosome 21
q22.3
AIRE
The AIRE protein functions as a transcription factor or as a
transcriptional co-activator that might have an important role in
the control of immune recognition.
AIRE is expressed in the thymus, lymph node and fetal liver ,
tissues that have important roles in the maturation of the immune
system. AIRE expression was described also in monocytes and
dendritic cells.
The impaired expression of AIRE in the thymic medulla and
antigen presenting cells may cause the breakdown of the
processes of tolerance and induction of autoimmunity.
APECED and autoimmunity
APC
The process of autoimmunity in
APECED may be the result of
impaired processes of both central
and peripheral tolerance –
defect of expression of organ
specific antigens in the thymus
The mutations leading to APECED and defective
AIRE production may contribute to the shift of
immune balance towards preferential Th2
response.
HLA
antigen
TCR
T lymphocyte
IFNg
Th1
Th0
IL 4
Th2
- IFNg
IL 10-
IFNg
IL 2
NK
makrofage CD8
cellular immunity IgG2
IL 4,5,6
plasmocyte
antibodies
genetic analysis of 24 APECED patients of
Eastern and Central European origins
From 48 analysed APECED chromosomes eight mutations
were detected, four (T16M, W78R, delE2-4, 156179ins23bp) of which being novel.
The most prevalent reason
for APECED in these
populations was the
occurrence of R257X (36
chromosomes) that has
been described earlier as
common and reccurent
mutation in several other
populations.
Analysis of autoantibodies of 24 APECED
patients of Eastern and Central European
origins
The analysis of humoral immunity to
steroidogenic P450 cytochromes by
immunoblotting of E. coli expressed antigens
showed that 65%, 55% and 55% of the
Eastern and Central European APECED
patients had autoantibodies to P450c17,
P450c21 and P450scc, respectively.
Czech Republic, Hungary, Slovenia, Croatia, Serbia, Russia.
Patients and methods
Four girls with APS1 diagnosed
in Czech Republic, their
siblings, parents and aged
matched controls were included
to the study of immune
functions. The age of girls was
7,12, 17 and 22 years.
Patient
Age (years)
Genotype (mutations in AIRE gene)
Clinical symptoms
1
17
R257X / R257X
HP, MC, AD, CH
2
22
R257X / R257X
HP, MC, AD, AL, KC, CL
3
12
not known
HP, MC, AD, AL, CH, VI, SJ
4
7
R257X / W78R
HP, AD, AL
Age, genotype and clinical symptoms in four female patients with APECED.
HP - hypoparathyroidism; MC - mucocutaneous candidiasis; AD - Addison disease; VI vitiligo; AL - alopecia; ED - ectodermal dystrophy; KC - keratoconjuctivitis; HT hypothyroidism; CH - chronic active hepatitis; CL – cholelithiasis; SJ – Sjögren
syndrome.
The immune parameters included immunoglobulins, panel of
autoantibodies, cellular immunity and levels of cytokines IFNg, IL-4 and IL10 measured in the supernatants of PHA and LPS stimulated lymphocyte
cultures.
Autoantibodies:
ANA, ANCA, ENA, AMA, ASMA, LKM, GPCA, EMA, anti-gliadin.
Results of immunological investigation in APECED
patients
INFy
IFNg
1600
1400
1200
pg/ml
1000
INFy
800
600
400
200
f4
co
nt
ro
l
p4
m
4
f3
s3
m
3
f2
p3
m
2
f1
p2
s1
m
1
0
p1
Low values were found in all affected girls,
indicating that APECED can be connected
with low production of this cytokine and
possible shift to Th2 type of immune
reactivity.
The significance of the difference between
the girls with APECED and controls was
tested using Man-Whitney test. The result
showed clear trend, but did not reach
statistical significance. The borderline pvalue is surely influenced by low number of
patients.
1800
Man-Whitney test
IFNg
mean (pg/ml)
patients
455
standard deviation
191
(4)
controls
(14)
p-value
0.0559
910
406
Results of immunological investigation in APECED
patients
immunoglobulins
IgG
IgA
2
2
er
fat
h
er
oth
m
pa
pa
tie
n
t2
IgM
CD4
80,00
60,00
40,00
CD4
tien
mo t 2
the
r2
fath
er
2
pa
tien
t1
sis
ter
mo 1
the
r1
fath
er
1
20,00
0,00
pa
%
Besides low IFNg values, the patients
did not follow unified immunological
pattern and present themselves with
individual values. However, 2 girls with
homozygous mutations R257X show
very similar results with marked
elevation of IgM and high numbers of
CD3+CD4+ lymphocytes.
20,00
15,00
10,00
5,00
0,00
tie
nt
1
sis
ter
m 1
oth
er
1
fat
he
r1
g/l
IgM, CD4+ T lymphocytes
The changes in heterozygous members of the
affected families
Interestingly, all fathers, but not sick girls, had elevated levels of IgA and
activated T lymphocytes according to the number of CD3 HLA DR+
lymphocytes.
IgA
CD3+HLADR+
6
18
5
16
14
12
IgA
3
%
g/l
4
2
10
CD3+HLADR+
8
6
f4
ntr
ol
co
f3
p4
s3
2
m
1
p1
0
m
co
ntr
ol
4
f4
p4
m
3
f3
s3
m
p3
2
f2
p2
m
m
1
2
f1
0
s1
4
p1
1
In 2 girls we found the positivity of autoantibodies, once against
gastric parietal cells, the result shared with her mother, second
time against smooth muscle.
Conclusion
• Low IFNg production was found in all investigated APECED
patients.
• The frequency of pathological immune parameters among
heterozygous members of affected families is noteworthy.
• The details of the development of organ specific autoimmunity
are still to be investigated.