IMMUNOLOGICAL COMMLJNII:ATIONS,
7 ( 6 ) , 661-668
(1'3778)
Immunol Invest Downloaded from informahealthcare.com by Monash University on 01/06/15 For personal use only.
PIGEON IgA: A hAJOR AlITIGEli 11x1 P1GEOI.I SREF
DER'S DISEASE
J.Goudswocird, A.Naordzi j and J . W . E . Stoni D e p a r t m e n t o f Immunology cind S m a l l An.imo1 C l i n i c F a c u l t y of V e t e r i n a r y Medicine, U n i v e r s i t y of U t r e c h t Y a l e l a a n 1 , U t r e c h t , The N e t h e r l a n d s
A b stract P i g e o n IgA ha:: been i s o l a t e d f r o m crop-mi1.k and on a n t i s e r u m w ~ u s p r o d u c e d . E x t r a c t s o f t h e d u s t i n p i g e o n c o o p s contained d e t e c t a b l e a m o u n t s o f p.igeon IgA, b u t n o t IgG. The :;era o f p a t i e n t s w i t h pigeon b r e e d e r ' s d i s e a s e contained p r e c i p i t a t i n g antibody a g a i n s t p i g e o n IgA.
I n t r o d u c t i on ___o f ex-
P i g e o n b r e e d e r ' s d i s e a s e ( P B D ) i s a wel.1 d e s c r i b e d f a r m t r i n s i c a l l e r g i c a l v e o l i t i s (1,2).
P a t i e n t s wi.th PBD h a v e a n t i b o d i e s
i n t h e i r serum a g a i n s t v a r i o u s pigeon a n t i g e n s . These
antibodies
h a v e g e n e r a l l y been d e m o n s t r a t e d by i m m u n o p r e c i p i t o t i o n r e a c t i o n s i n a g a r g e l s (3). O r g a n i c c o m p o n e n t s i n p i g e o n g u a n o a r e b e l i e v e d t o have g r e a t e r a n t i g e n i c s i g n i f i c a n c e t h a n a n t i g e n s from
other
s o u r c e s , s u c h a s serum o r e g g s ( 4 ) . The u s e o f g u a n o e x t r a c t s cl i a g n a s t ic p r e c i p i t a
t i o n r e a c t i o n s , how eve ir , po s e s p r o b l ems
for of
i n t e r p r e t a t i o n b e c a u s e numerous a n t i g e n s , . i n c l u d i n g s e r u m p r o t e i n s , and n o n - s p e c i f i c a g e n t s may r e a c t and ever! c w 8 e 'Y'alse-psidve"
iests
(5-8). We h a v e t h e r l - f o r e t e s t e d t h e h y p o t h e s i s t h a t p i g e o n IgA m i g h t b e o n e o f t h e main a n t i g e n s i n v o l v e d i n PBD. I f s o , a p r e p a r -
661 Copyright 0 1979 by Marcel Delkker, Inc All Rights Reserved Neither thi, work n o r any part may he reproduced transmitted in any form or by any means, electronic o r mechanical. includlne photocopying. microfilming, arid recording, or by any informalion storage and retrieval system. without permission i n writing f r o m the publisher 01
662
GOUDSWAARD, N O O R D Z I J , AND STAM
o t i o n o f t h i s i m m u n o g l o b u l i n c o u l d be used f o r an easy and
un-
c o m p l i c a t e d i m m u n o p r e c i p i t a t i o n t e s t f o r t h e d i a g n o s i s o f PBD. R e c e n t l y , t h r e e c l a s s e s o f i m m u n o g l o b u l i n s have been i d e n t i f i e d i n t h e p i g e o n ( 9 ) . Pigeon IgA, on t h e f o l l o w i n g grounds:
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p i g e o n I g M and IgG; such 0 s b i l e ,
i s o l a t e d from b i l e ,
was
a) p o s s e s s i o n o f t h e same l i g h t c h a i n s as
b ) r e l a t i v e abundance i n e x o c r i n e
e g g - w h i t e and i n t e s t i n a l f l u i d ,
p r e s e n t i n p i g e o n serum;
identified
secretions
b u t o n l y s m a l l amounts
c ) o c c u r r a n c e i n t h e c y t o p l a s m of the mapr-
i t y o f t h e immunocytes f r o m t h e i n t e s t i n a l mucosa;
d) e l e c t r o p h o r -
e t i c m o b i l i t y ond m o l e c u l a r s i z e s i m i l a r t o t h o s e o f c h i c k e n I g A . I n addition,
cropmilk,
a s e c r e t i o n produced by o h o l o c r i n i c
i s r e l a t i v e l y r i c h i n IgA,
gland,
suggesting t h a t t h i s exocrine s e c r e t i o n
m i g h t be used f o r t h e i s o l a t i o n o f p i g e o n I g A ( 9 ) .
M a t e r i a l s and methods
I s o l a t i o n o f crop-milk
IgA.
Crop-milk
wos d i l u t e d t h r e e - f o l d
w i t h s a l i n e and ground i n a m o r t a r . A f t e r c e n t r i f u g a t i o n f o r 10 min a t 3000 x g,
t h e s u p e r n a t a n t was d e f a t t e d by t h e a d d i t i o n
v o l o f 10% d e x t r a n s u l p h a t e and 0.1 v o l o f 1 M CaC12. u g a t i o n a t 15.000 rpm, Tris-HC1
(pH 2.0)
o f 0.02
After centrif-
t h e s u p e r n a t a n t was d i a l y z e d a g a i n s t 0.01 M
c o n t a i n i n g 2% EDTA and 1% NaC1. The c l e a r super-
n a t a n t was chromatographed on a Sepharose
48
(Pharmacia) column
(40 x 2.5 cm) e q u i l i b r a t e d w i t h 0.01 M Tris-HC1 (pH 8.0) c o n t a i n i n g 1% NaC1. The I g A - c o n t a i n i n g f r o c t i o n was f u r t h e r p u r i f i e d by t r a t i o n through
LKB
U l t r o g e l ACA 22 (40 x 2.5
fil-
cm) i n t h e same b u f -
fer. Pigeon d r y f e a t h e r dust. greasy,
sebumlike m a t e r i a l ,
number o f p i g e o n coops, months.
Pigeon d r y f e a t h e r dust,
a white,
was c o l l e c t e d f r o m t h e i n s i d e t o p o f a
where i t had been p r e s e n t f o r a t l e a s t 3
The a n t i g e n s were e x t r a c t e d f r o m t h e d u s t by s i m p l e shaking
w i t h 1 v o l o f phosphate b u f f e r e d s a l i n e , by i m m u n o e l e c t r o p h o r e s i s .
pH 7.2,
and were a n a l y z e d
663
PIGEON IgA
Immunization procedures. Antisera to pigeon whole serum and an antiserum specific for pigeon IgA were raised in rabbits as described (9).
Sera of patients. Sera of patients with clinically proven
PBD,
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mostly the acute form, were kindly donated by Dr. L. Berrens, Utrecht University Hospital, these were stored in small aliquots at -7OOC until used. The sera were positive by immunodiffusion against
purified B antigens from guano extract ( 3 ) , except three samples, which were found positive only in a complement consumption assay. Ouchterlony and immunoelectrophoresis assays; reverse radial immunodiffusion. Ouchterlony and immunoelectropharesis assays
were
carried out in 1% agarose (Jndustrie Biologique Francaise) occoding to standard procedures. A reverse Mancini method in agarose contained 0.5 mg/ml pigeon IgA was developed. Specific
that
antibody
protein/ml of patient's serum was measured according to Weir (10). Each assay was performed in triplicate. The rabbit anti-pigeon IgA used as a standard in this quantitative reverse radial immunodiffusion assay had been purified after delipidotion with dextran sulphate and CaC12, by ammonium sulphate precipitation and affinity chranatography on Staphylococcal Protein A and purified pigeon IgA, successively. The protein content, as measured by the Lowry (11)
method
was 0.5 mg/ml.
Results
The purification procedure of crop-milk yielded purified as judged by immunae ectraphoresis (Figure
a). Although the
IgA, dry
pigeon feather dust had remained for many months in the taps of the pigeon coops, it still contained many antigens, as demonstrated in Figure lb. In this analysis, the amazingly intact antigens
were
developed with the serum of PBD-patient "Wal'. However, the pigeon
664
GOUDSWMI),
N O O R D Z I J , AND STMI
PIGEON
Immunol Invest Downloaded from informahealthcare.com by Monash University on 01/06/15 For personal use only.
h
PIGEON
t .Wa
(11)
Figure 1 .
Agarose g e l i m m u n o e l e c t r o p h o r e s i s . l a . Pigeon serum and pigeon IgA ( p u r i f i e d from c r o p m i l k ) d e v e l o p e d w i t h a n t i whole pigeon s e r u m ( A - w h ) . l b . A n t i g e n i c e x t r a c t of pigeon f e a t h e r dust. and of pigeon serum, developed w i t h t h e s e r u m of p a t i e n t "Wa" w i t k i PBD i n t h e upper g r o o v e and w i t h a n t i - - w h o l e pigeon serum ( l o w e r g r o o v e ) . Anode to the l e f t .
d u s t , o s t h e most p r o b a b l e immunizing a g e n t , was found n o t t o cont a i n IgG; t h e s t r o n g c a t h o d a l p r e c i p - t t a t i o n a r c of t h e d u s t e x t r a c t
w i t h t h e p a t i e n t ' s serum i n F i g u r e l b was n o t due t o pigeon IgG, a s judged by a n a l y s i s w i t h m o n o s p e c i f i c a n t i s e r u m a g a i n s t pigeon IgG ( n o t shown). Of t h e immunoglobulins, o n l y IgA was r e c o g n i z e d by t h e rabbit-anti-whole
pigeon a n t i s e r u m . The r e s u l t s of r o u t i n e immuno-
d i f f u s i o n t e s t s w i t h p u r i f i e d PbB-antigens from pigeon
droppings
( 3 ) a r e l i s t e d i n T a b l e 1 . F i f t e e n s e r a were employed, i n c l u d i n g c o n t r o l s of h e a l t h y pigeon Ibreeders. T a b l e I a l s o g i v e s t h e r e s u l t s of t h e O u c h t e r l o n y t e s t s ( F i g u r e 2) and of t h e q u a n t i t a t i v e r e v e r s e r a d i a l immunodiffusian assa:y w i t h pigeon IgA a s t h e a n t i g e n .
PIGEON I g A
665
TABLE I S e r o l o g i c a l e x a m i n a t i o n o f 15 smsrum sampl.es of p c i t i e n t s w i t h PBD and o f h e a l t h y p i g e o n - f a n c i e r s by d o u b l e d i f f u s i o n a g a i n s t d r o p p i n g a n t i g e n m i x t u r e PDB and a g a i n s t p i g e o n c:cop-nlilk IgA e v o l u a t e d quali t a t i v e l y . A l s o shown a r e t h e q u a n t i t a t i v e r e s u l t s o f r e v e r s e r o r i i a l i m m u n o d i f f u s i o n w i t h p i g e o n IgA. Immunol Invest Downloaded from informahealthcare.com by Monash University on 01/06/15 For personal use only.
Serum No
Clinical form
____---._-~.-
Double d i f f u s i o n PDB IgA
I
R e v e r s e i.ininundiffusi on antibody protein i n
w/ml
462
Acute
466
Sub o c lu t 6%
478
ChronicX
454
Acute
273
Acute
264
C h r o n i.c
175
Acute
173
Acute
169
Acute
143
AclJtF
117
Acute
054
Acute
039
S u bac: u t e
41 9"
-
42OXx
-
+ -
+ + -
0.24
+ +
+ +
iilot done
+ + + + +
+ + + +
+ + -
0.16 0
1.15 trace
1 .70 0.23 0.23 0.24
+ + +
0.51
-
0
-
0
0.24
0.14
A c u t e form 5 y e a r s pr'eviously; antisgenic c o n t a c t s a v o i d e d , a s y m p t o m a t i c a t t h e time o f b l o o d s a m p l i n g
xx
Controls
D i sc u s si o n A s shown i n Tcible I, t h e r e
'was a
good o v e r a l l c o r r e l a t i o n
between p r e c i p i t a t i n g a n t i b o d i e s r e v e a l e d w i t h 8 a n t i g e n s
from
g u a n o e x t r a c t and w i t h p i g e o n IgA. However, i n o n e p a t i e n t (No. 264)
GOUDSWAARD, NOORDZIJ, AND STAM
666
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Patient Nos
FIGURE 2
O u c h t e r l o n y a n a l y s i s of 13 s e r a o f p a t i e n t s w i t h PBD, 2 c o n t r o l s e r a , and s p e c i f i c a n t i - p i g e o n IgA d i f f u s e d a g a i n s t pigeon IgA i n the central w e l l .
t h e f o r m e r was p o s i t i v e b u t t h e O u c h t e r l o n y a n a l y s i s w i t h
pigeon
IgA was n e g a t i v e . I n r e v e r s e r a d i a l immunodiffusion t e s t s t r a c e s of a n t i - I g A a c t i v i t y c o u l d be d e t e c t e d i n t h i s serum. I n s e r a Nos 466 and 173 o n l y a n t i - I g A a c t i v i t y was d e t e c t e d . Ouchterlony a n a l y s i s f o r anti-pigeon
IgA ( F i g u r e 2) demonstrates
t h e i n t e r e s t i n g phenomenon i n a n u m b e r o f p a t i e n t ' s s e r a o f obvious s p u r r i n g w i t h t h e i m m u n o p r e c i p i t a t i o n l i n e between IgA and o u r anti-
667
PIGEON I g A
IgA p r e p a r a t i o n a s w e l l a s some o t h e r p a t i e n t ' s serum samples. f'orter and P a r r y ( 1 2 ) d e s c r i b e a p o s s i b l e a n a l o g u e of mammalian s e c r e t o r y component i n c h i c k e n i n t e s t i n a l c o n t e n t s . A p o s s i b l e exp l a n a t i o n f o r t h i . s s p u r formatiori c o u l d be t h a t a number of p a t i e n t s a l s o have a n t i b o d i e s t o pigeon s e c r e t o r y component. Immunol Invest Downloaded from informahealthcare.com by Monash University on 01/06/15 For personal use only.
The d e m o n s t r a t i o n of a n t i g e n i c a l l y i n t a c t IgA i n t h e d r y f e a t h e r
d u s t , t h a t had remained f o r many months i n t h e t o p s of t h e pigeon coops, was s u r p r i s i n g . Most p r o b a b l y , t h e a n t i g e n s had been p r o t e c t e d from d e g r a d a t i o n by t h e g r e a s y l a y e r envelop.ing t h e
dust
p a r t i c l e s . We found t h a t pigeon f e a t h e r d u s t i s v e r y r i c h i n u r i c a c i d . Consequently,
t h e IgA i n t h e d u s t i s most l i k e l y d e r i v e d from
pigeon guano. T h i s u l s o would e x p l a i n t h e p r e s e n c e
of a n t i b o d y
in
PBD-putients a g a i n s t e x t r a c t s of v a r i o u s segments of
the
pigeon
a l i m e n t a r y t r a c t (3). A s we e a r l i . e r p r e d i c t e d , Tebo,
F r e d r i c k s and
R o b e r t s (13) have r e c e n t l y p r o v i d e d i n d i r e c t e v i d e n c e f o r t h e i d e n t -
i t y o f t h e i r major a n t i g e n from pigeon dropp.i.ngs, PDE, as pigeon IgA. From t h e i m m u n o e l e c t r o p h o r e t i c a n a l y s i s of t h e p a t i e n t ' s and of t h e pigeon d u s t e x t r a c t we c o n c l u d e t h a t t h e r e p o r t e d
sera anti-
b o d i e s t o pigeon IgNG a r e most prclbably a n t i - l i g h t (chain a n t i b o d i e s
( 1 ) . The r e a l immunizing pigeon immunoglobulin a p p e a r s t o
be
IgA,
p o s s i b l y i n i t s s e c r e t o r y form.
Acknowledgement Thanks a r e due t o Dr.L.Berrens,
D i v i s i o n of E x p e r i m e n t a l Allergy,
l l t r e c h t U n i v e r s i t y I i o s p i t a l , f o r making a v a i l a b l e t h e p a t i e n t ' s s e r a used i n t h i s s t u d y . R e f erences -
1 . C h r i s t e n s e n , L.T.,
Schmidt, C.Ds.,
417,1975.
Robbi.ns, L.,
C l i n . A 1 1e rgy , 5:
il.Pepys, J . , i n The r o l e of immunological f a c t o r s i n i n f e c t i o u s , a l l e r g i c and autoimmune p r o c e s s e s , Raven P r e s s , New York,1977. 3 . B e r r e n s , L. and Maesen, F.P.V., 327,1972.
I n t . Arclh. A!.lerg.
appl.
Immun.2
668
GOUDSWAARD, N O O K D Z I J , AM) STAM
4.Edwards, 1970.
J.H.,
Barboriak,
J.J.
5.Faux, J.A., Holford-Strevens, Exp. Immun. 7:897,1970.
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6.Berrens, L. and Maesen, 259,1972. 7.Yang,
J.P.S.
8.Teb0,
T.H.,
V.,
F.P.V.,
and P u r t i l o , D.T., Moore,
V.L.
and F i n k , J.N. Wells,
I n t . Arch.
Clin.
Immunology, E : 7 2 9 ,
and Pepys, J.,
Allerg.
Immun.
g:
C l i n . A l l e r g y , 2:103,1977.
and Heremans, J.F.,
.
appl.
Clin.
Immun. Immunopoth. 4:46,1975.
and F i n k , J.N.,
9.Goudswaard, J., Vaerman, J-P. a p p l . Immun 53,~ 0 9 , 1 9 7 7 .
I.D.
,
I n t . Arch.
Allerg.
l O . W e i r , D.M., Handbook o f E x p e r i m e n t a l Immunology. a l a c k w e l l Scienti f i c P u b l i c a t i o n s , Oxfard,1973.
l l . L o w r y , O.H., B i o l . Chem.,
Rosebrough, N.J., E:265,1951.
1 2 . P o r t e r , P. and P a r r y , S.H., 13.Teb0, appl.
T.H., F r e d r i c k s , W.W. Immun. 2 : 5 5 3 , 1 9 7 6 .
F a r r , A.L.
and R a n d e l l , R.J.J.,
Immunology, =:407,1976. and Roberts, R.C.,
I n t . Arch.
Allerg.
7 ( 6 ) , 661-668
(1'3778)
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PIGEON IgA: A hAJOR AlITIGEli 11x1 P1GEOI.I SREF
DER'S DISEASE
J.Goudswocird, A.Naordzi j and J . W . E . Stoni D e p a r t m e n t o f Immunology cind S m a l l An.imo1 C l i n i c F a c u l t y of V e t e r i n a r y Medicine, U n i v e r s i t y of U t r e c h t Y a l e l a a n 1 , U t r e c h t , The N e t h e r l a n d s
A b stract P i g e o n IgA ha:: been i s o l a t e d f r o m crop-mi1.k and on a n t i s e r u m w ~ u s p r o d u c e d . E x t r a c t s o f t h e d u s t i n p i g e o n c o o p s contained d e t e c t a b l e a m o u n t s o f p.igeon IgA, b u t n o t IgG. The :;era o f p a t i e n t s w i t h pigeon b r e e d e r ' s d i s e a s e contained p r e c i p i t a t i n g antibody a g a i n s t p i g e o n IgA.
I n t r o d u c t i on ___o f ex-
P i g e o n b r e e d e r ' s d i s e a s e ( P B D ) i s a wel.1 d e s c r i b e d f a r m t r i n s i c a l l e r g i c a l v e o l i t i s (1,2).
P a t i e n t s wi.th PBD h a v e a n t i b o d i e s
i n t h e i r serum a g a i n s t v a r i o u s pigeon a n t i g e n s . These
antibodies
h a v e g e n e r a l l y been d e m o n s t r a t e d by i m m u n o p r e c i p i t o t i o n r e a c t i o n s i n a g a r g e l s (3). O r g a n i c c o m p o n e n t s i n p i g e o n g u a n o a r e b e l i e v e d t o have g r e a t e r a n t i g e n i c s i g n i f i c a n c e t h a n a n t i g e n s from
other
s o u r c e s , s u c h a s serum o r e g g s ( 4 ) . The u s e o f g u a n o e x t r a c t s cl i a g n a s t ic p r e c i p i t a
t i o n r e a c t i o n s , how eve ir , po s e s p r o b l ems
for of
i n t e r p r e t a t i o n b e c a u s e numerous a n t i g e n s , . i n c l u d i n g s e r u m p r o t e i n s , and n o n - s p e c i f i c a g e n t s may r e a c t and ever! c w 8 e 'Y'alse-psidve"
iests
(5-8). We h a v e t h e r l - f o r e t e s t e d t h e h y p o t h e s i s t h a t p i g e o n IgA m i g h t b e o n e o f t h e main a n t i g e n s i n v o l v e d i n PBD. I f s o , a p r e p a r -
661 Copyright 0 1979 by Marcel Delkker, Inc All Rights Reserved Neither thi, work n o r any part may he reproduced transmitted in any form or by any means, electronic o r mechanical. includlne photocopying. microfilming, arid recording, or by any informalion storage and retrieval system. without permission i n writing f r o m the publisher 01
662
GOUDSWAARD, N O O R D Z I J , AND STAM
o t i o n o f t h i s i m m u n o g l o b u l i n c o u l d be used f o r an easy and
un-
c o m p l i c a t e d i m m u n o p r e c i p i t a t i o n t e s t f o r t h e d i a g n o s i s o f PBD. R e c e n t l y , t h r e e c l a s s e s o f i m m u n o g l o b u l i n s have been i d e n t i f i e d i n t h e p i g e o n ( 9 ) . Pigeon IgA, on t h e f o l l o w i n g grounds:
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p i g e o n I g M and IgG; such 0 s b i l e ,
i s o l a t e d from b i l e ,
was
a) p o s s e s s i o n o f t h e same l i g h t c h a i n s as
b ) r e l a t i v e abundance i n e x o c r i n e
e g g - w h i t e and i n t e s t i n a l f l u i d ,
p r e s e n t i n p i g e o n serum;
identified
secretions
b u t o n l y s m a l l amounts
c ) o c c u r r a n c e i n t h e c y t o p l a s m of the mapr-
i t y o f t h e immunocytes f r o m t h e i n t e s t i n a l mucosa;
d) e l e c t r o p h o r -
e t i c m o b i l i t y ond m o l e c u l a r s i z e s i m i l a r t o t h o s e o f c h i c k e n I g A . I n addition,
cropmilk,
a s e c r e t i o n produced by o h o l o c r i n i c
i s r e l a t i v e l y r i c h i n IgA,
gland,
suggesting t h a t t h i s exocrine s e c r e t i o n
m i g h t be used f o r t h e i s o l a t i o n o f p i g e o n I g A ( 9 ) .
M a t e r i a l s and methods
I s o l a t i o n o f crop-milk
IgA.
Crop-milk
wos d i l u t e d t h r e e - f o l d
w i t h s a l i n e and ground i n a m o r t a r . A f t e r c e n t r i f u g a t i o n f o r 10 min a t 3000 x g,
t h e s u p e r n a t a n t was d e f a t t e d by t h e a d d i t i o n
v o l o f 10% d e x t r a n s u l p h a t e and 0.1 v o l o f 1 M CaC12. u g a t i o n a t 15.000 rpm, Tris-HC1
(pH 2.0)
o f 0.02
After centrif-
t h e s u p e r n a t a n t was d i a l y z e d a g a i n s t 0.01 M
c o n t a i n i n g 2% EDTA and 1% NaC1. The c l e a r super-
n a t a n t was chromatographed on a Sepharose
48
(Pharmacia) column
(40 x 2.5 cm) e q u i l i b r a t e d w i t h 0.01 M Tris-HC1 (pH 8.0) c o n t a i n i n g 1% NaC1. The I g A - c o n t a i n i n g f r o c t i o n was f u r t h e r p u r i f i e d by t r a t i o n through
LKB
U l t r o g e l ACA 22 (40 x 2.5
fil-
cm) i n t h e same b u f -
fer. Pigeon d r y f e a t h e r dust. greasy,
sebumlike m a t e r i a l ,
number o f p i g e o n coops, months.
Pigeon d r y f e a t h e r dust,
a white,
was c o l l e c t e d f r o m t h e i n s i d e t o p o f a
where i t had been p r e s e n t f o r a t l e a s t 3
The a n t i g e n s were e x t r a c t e d f r o m t h e d u s t by s i m p l e shaking
w i t h 1 v o l o f phosphate b u f f e r e d s a l i n e , by i m m u n o e l e c t r o p h o r e s i s .
pH 7.2,
and were a n a l y z e d
663
PIGEON IgA
Immunization procedures. Antisera to pigeon whole serum and an antiserum specific for pigeon IgA were raised in rabbits as described (9).
Sera of patients. Sera of patients with clinically proven
PBD,
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mostly the acute form, were kindly donated by Dr. L. Berrens, Utrecht University Hospital, these were stored in small aliquots at -7OOC until used. The sera were positive by immunodiffusion against
purified B antigens from guano extract ( 3 ) , except three samples, which were found positive only in a complement consumption assay. Ouchterlony and immunoelectrophoresis assays; reverse radial immunodiffusion. Ouchterlony and immunoelectropharesis assays
were
carried out in 1% agarose (Jndustrie Biologique Francaise) occoding to standard procedures. A reverse Mancini method in agarose contained 0.5 mg/ml pigeon IgA was developed. Specific
that
antibody
protein/ml of patient's serum was measured according to Weir (10). Each assay was performed in triplicate. The rabbit anti-pigeon IgA used as a standard in this quantitative reverse radial immunodiffusion assay had been purified after delipidotion with dextran sulphate and CaC12, by ammonium sulphate precipitation and affinity chranatography on Staphylococcal Protein A and purified pigeon IgA, successively. The protein content, as measured by the Lowry (11)
method
was 0.5 mg/ml.
Results
The purification procedure of crop-milk yielded purified as judged by immunae ectraphoresis (Figure
a). Although the
IgA, dry
pigeon feather dust had remained for many months in the taps of the pigeon coops, it still contained many antigens, as demonstrated in Figure lb. In this analysis, the amazingly intact antigens
were
developed with the serum of PBD-patient "Wal'. However, the pigeon
664
GOUDSWMI),
N O O R D Z I J , AND STMI
PIGEON
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h
PIGEON
t .Wa
(11)
Figure 1 .
Agarose g e l i m m u n o e l e c t r o p h o r e s i s . l a . Pigeon serum and pigeon IgA ( p u r i f i e d from c r o p m i l k ) d e v e l o p e d w i t h a n t i whole pigeon s e r u m ( A - w h ) . l b . A n t i g e n i c e x t r a c t of pigeon f e a t h e r dust. and of pigeon serum, developed w i t h t h e s e r u m of p a t i e n t "Wa" w i t k i PBD i n t h e upper g r o o v e and w i t h a n t i - - w h o l e pigeon serum ( l o w e r g r o o v e ) . Anode to the l e f t .
d u s t , o s t h e most p r o b a b l e immunizing a g e n t , was found n o t t o cont a i n IgG; t h e s t r o n g c a t h o d a l p r e c i p - t t a t i o n a r c of t h e d u s t e x t r a c t
w i t h t h e p a t i e n t ' s serum i n F i g u r e l b was n o t due t o pigeon IgG, a s judged by a n a l y s i s w i t h m o n o s p e c i f i c a n t i s e r u m a g a i n s t pigeon IgG ( n o t shown). Of t h e immunoglobulins, o n l y IgA was r e c o g n i z e d by t h e rabbit-anti-whole
pigeon a n t i s e r u m . The r e s u l t s of r o u t i n e immuno-
d i f f u s i o n t e s t s w i t h p u r i f i e d PbB-antigens from pigeon
droppings
( 3 ) a r e l i s t e d i n T a b l e 1 . F i f t e e n s e r a were employed, i n c l u d i n g c o n t r o l s of h e a l t h y pigeon Ibreeders. T a b l e I a l s o g i v e s t h e r e s u l t s of t h e O u c h t e r l o n y t e s t s ( F i g u r e 2) and of t h e q u a n t i t a t i v e r e v e r s e r a d i a l immunodiffusian assa:y w i t h pigeon IgA a s t h e a n t i g e n .
PIGEON I g A
665
TABLE I S e r o l o g i c a l e x a m i n a t i o n o f 15 smsrum sampl.es of p c i t i e n t s w i t h PBD and o f h e a l t h y p i g e o n - f a n c i e r s by d o u b l e d i f f u s i o n a g a i n s t d r o p p i n g a n t i g e n m i x t u r e PDB and a g a i n s t p i g e o n c:cop-nlilk IgA e v o l u a t e d quali t a t i v e l y . A l s o shown a r e t h e q u a n t i t a t i v e r e s u l t s o f r e v e r s e r o r i i a l i m m u n o d i f f u s i o n w i t h p i g e o n IgA. Immunol Invest Downloaded from informahealthcare.com by Monash University on 01/06/15 For personal use only.
Serum No
Clinical form
____---._-~.-
Double d i f f u s i o n PDB IgA
I
R e v e r s e i.ininundiffusi on antibody protein i n
w/ml
462
Acute
466
Sub o c lu t 6%
478
ChronicX
454
Acute
273
Acute
264
C h r o n i.c
175
Acute
173
Acute
169
Acute
143
AclJtF
117
Acute
054
Acute
039
S u bac: u t e
41 9"
-
42OXx
-
+ -
+ + -
0.24
+ +
+ +
iilot done
+ + + + +
+ + + +
+ + -
0.16 0
1.15 trace
1 .70 0.23 0.23 0.24
+ + +
0.51
-
0
-
0
0.24
0.14
A c u t e form 5 y e a r s pr'eviously; antisgenic c o n t a c t s a v o i d e d , a s y m p t o m a t i c a t t h e time o f b l o o d s a m p l i n g
xx
Controls
D i sc u s si o n A s shown i n Tcible I, t h e r e
'was a
good o v e r a l l c o r r e l a t i o n
between p r e c i p i t a t i n g a n t i b o d i e s r e v e a l e d w i t h 8 a n t i g e n s
from
g u a n o e x t r a c t and w i t h p i g e o n IgA. However, i n o n e p a t i e n t (No. 264)
GOUDSWAARD, NOORDZIJ, AND STAM
666
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Patient Nos
FIGURE 2
O u c h t e r l o n y a n a l y s i s of 13 s e r a o f p a t i e n t s w i t h PBD, 2 c o n t r o l s e r a , and s p e c i f i c a n t i - p i g e o n IgA d i f f u s e d a g a i n s t pigeon IgA i n the central w e l l .
t h e f o r m e r was p o s i t i v e b u t t h e O u c h t e r l o n y a n a l y s i s w i t h
pigeon
IgA was n e g a t i v e . I n r e v e r s e r a d i a l immunodiffusion t e s t s t r a c e s of a n t i - I g A a c t i v i t y c o u l d be d e t e c t e d i n t h i s serum. I n s e r a Nos 466 and 173 o n l y a n t i - I g A a c t i v i t y was d e t e c t e d . Ouchterlony a n a l y s i s f o r anti-pigeon
IgA ( F i g u r e 2) demonstrates
t h e i n t e r e s t i n g phenomenon i n a n u m b e r o f p a t i e n t ' s s e r a o f obvious s p u r r i n g w i t h t h e i m m u n o p r e c i p i t a t i o n l i n e between IgA and o u r anti-
667
PIGEON I g A
IgA p r e p a r a t i o n a s w e l l a s some o t h e r p a t i e n t ' s serum samples. f'orter and P a r r y ( 1 2 ) d e s c r i b e a p o s s i b l e a n a l o g u e of mammalian s e c r e t o r y component i n c h i c k e n i n t e s t i n a l c o n t e n t s . A p o s s i b l e exp l a n a t i o n f o r t h i . s s p u r formatiori c o u l d be t h a t a number of p a t i e n t s a l s o have a n t i b o d i e s t o pigeon s e c r e t o r y component. Immunol Invest Downloaded from informahealthcare.com by Monash University on 01/06/15 For personal use only.
The d e m o n s t r a t i o n of a n t i g e n i c a l l y i n t a c t IgA i n t h e d r y f e a t h e r
d u s t , t h a t had remained f o r many months i n t h e t o p s of t h e pigeon coops, was s u r p r i s i n g . Most p r o b a b l y , t h e a n t i g e n s had been p r o t e c t e d from d e g r a d a t i o n by t h e g r e a s y l a y e r envelop.ing t h e
dust
p a r t i c l e s . We found t h a t pigeon f e a t h e r d u s t i s v e r y r i c h i n u r i c a c i d . Consequently,
t h e IgA i n t h e d u s t i s most l i k e l y d e r i v e d from
pigeon guano. T h i s u l s o would e x p l a i n t h e p r e s e n c e
of a n t i b o d y
in
PBD-putients a g a i n s t e x t r a c t s of v a r i o u s segments of
the
pigeon
a l i m e n t a r y t r a c t (3). A s we e a r l i . e r p r e d i c t e d , Tebo,
F r e d r i c k s and
R o b e r t s (13) have r e c e n t l y p r o v i d e d i n d i r e c t e v i d e n c e f o r t h e i d e n t -
i t y o f t h e i r major a n t i g e n from pigeon dropp.i.ngs, PDE, as pigeon IgA. From t h e i m m u n o e l e c t r o p h o r e t i c a n a l y s i s of t h e p a t i e n t ' s and of t h e pigeon d u s t e x t r a c t we c o n c l u d e t h a t t h e r e p o r t e d
sera anti-
b o d i e s t o pigeon IgNG a r e most prclbably a n t i - l i g h t (chain a n t i b o d i e s
( 1 ) . The r e a l immunizing pigeon immunoglobulin a p p e a r s t o
be
IgA,
p o s s i b l y i n i t s s e c r e t o r y form.
Acknowledgement Thanks a r e due t o Dr.L.Berrens,
D i v i s i o n of E x p e r i m e n t a l Allergy,
l l t r e c h t U n i v e r s i t y I i o s p i t a l , f o r making a v a i l a b l e t h e p a t i e n t ' s s e r a used i n t h i s s t u d y . R e f erences -
1 . C h r i s t e n s e n , L.T.,
Schmidt, C.Ds.,
417,1975.
Robbi.ns, L.,
C l i n . A 1 1e rgy , 5:
il.Pepys, J . , i n The r o l e of immunological f a c t o r s i n i n f e c t i o u s , a l l e r g i c and autoimmune p r o c e s s e s , Raven P r e s s , New York,1977. 3 . B e r r e n s , L. and Maesen, F.P.V., 327,1972.
I n t . Arclh. A!.lerg.
appl.
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GOUDSWAARD, N O O K D Z I J , AM) STAM
4.Edwards, 1970.
J.H.,
Barboriak,
J.J.
5.Faux, J.A., Holford-Strevens, Exp. Immun. 7:897,1970.
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J.P.S.
8.Teb0,
T.H.,
V.,
F.P.V.,
and P u r t i l o , D.T., Moore,
V.L.
and F i n k , J.N. Wells,
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Clin.
Immunology, E : 7 2 9 ,
and Pepys, J.,
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C l i n . A l l e r g y , 2:103,1977.
and Heremans, J.F.,
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appl.
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Immun. Immunopoth. 4:46,1975.
and F i n k , J.N.,
9.Goudswaard, J., Vaerman, J-P. a p p l . Immun 53,~ 0 9 , 1 9 7 7 .
I.D.
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l l . L o w r y , O.H., B i o l . Chem.,
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