rARC H IVES
FISHERIES AND NARINE SERVICE .
Tr.anslation Sertes No. 4141
The immune response in agnaths and in iawed fishes.
of the immunoglobulins
Structure
by Y. Carton
Original title: La réponse immunitaire chez les agnathes et les poissons.
Structure des immunoglobulines
From:
Ann. Biol. 12(Fasc. 3-4): 139-184, 1973
Translated by the Translation Section
Department of the Environment
Department of the Environment
Fisheries and Narine Service
Biological Station
St. John's Nfld.
1977
45 pas!es typescript
r— ci-
ORIGINAL: FRENCH
'.1'088914
139
*
THE IMMUNE RESPONSE IN ACNATHS AND IN JAWED FISHES. STRUCTURE OF THE IMMUNOGLO•
•
BULINS.
by Yves Carton*
SUMMARY
INTRODUCTION
PHYLOGENY OF THE FISHES
THE CYCLOSTOMES
1. Family Myxinidae
2. Family Petromyzontidae
THE SELACHIANS
1. Immune response
2. Immunoglobulins in sharks
3. Immunoglobulins in rays
THE CHONDROSTEANS
THE HOLOSTEANS
THE TELEOSTS
1. Immune response
2.Immunoglobulin structure
THE DIPNOANS
CONCLUSIONS
INTRODUCTION
The last fifteen years have seen a great increase in research on the
immune response in malimals and on the biochemical structure of mammal
antibodies. The antibodies that have been described are all
*
evolutionary genetics laboratory, Conseil national de recherches scientifiques, Gif-sur-Yvette department
of biomedicine, University of Paris XIII,
Bobigny.
140
•1
immunoglobulins (1g), which are serum proteins. There are'several classes
of immunoglobulins, the best known of which . are IgG, IgM and IgA. They are
distinguished by physical, biochemical, antigenic and genetic features.
Immunoglobulins are composed of equal numbers of heavy (H) chains and light
(L) chains. The heavy chains are different for each class:IgG has y chains
(molecular weight 53,000), IgM p chains (Mid: 72,000) and IgA oc chains
(MW: 64,000). The light chains, with molecular weights of 20,000 to 22,000,
are common to all classes, though two types are distinguished: kappa (K) and
lambda (X). IgG thus has the molecular formula''Y'RK2 or yiX2 while IgM with its rentamer
structure has the formula (p 2K 2. ) 5 or (P 2 X 2 ) 5 .
.
In the immune response in mammals, the immunoglobulins appear in a certain
temporal seqtience. After the first injection of antigen, the organism synthe- .
sizes IgM, which reaches maximum titer around the fifth day and then decreases.
IgG then appears, and remains for a longer period. Reinjection brings a secondary response in which only IgG appears, and it does so much sooner and at
a greater rate; this is called an anamnestic response. The antigen-antibody
reaction is noteworthy for its specificity: an antibody formed after immunization reacts only with a particular antigen combining site.
The main problems of immunology are connected with the immune response
sequence and the remarkable immunological "memory" that goes with it. Much
work has been done on the nature of the antigen-combining site of the antibody
and its strict specificity, which seems to depend on the primary structure
of the H and L chains, or at least their variable areas. Much work has also
been done on the genetic aspect of the 'synthesis of these variable areas.
And recently the attention of researchers has been drawn to the problem of the
origin and phylogeny of the immunoglobulins, and several hypotheses have
been put forward (Lennox and Cohn, 1967; Hilschmann, 1967; Hill, Delaney,
Fellows and Lebovitz, 1966).
At the same time, immunologists have become interested in other vertebrates.
It was hoped that less evolved organisms like the fishes would provide less
elaborate systeMs to study. It does appear that the complexity and potential
of the immune response increases as one climbs the zoological ladder. Work
on immunity in fish bas therefore increased . in the last few years; three
quarters of the items in the bibliographyare,less;.than four,yeara old; A
141
summary of the latest results wi ll be of use sinc e . the most recent published
overviews are already dated (Good and Papermaster, 1964ç Smith and Miescher,
1966; Fougereau, 1968; Grey, 1969; Clem and Leslie, 1969).
There are several reasons why the fish results are extremely interesting
and useful. We now have a lot of data on their immune response. Like other
vertebrates, fish are capable of forming antibodies against a wide variety
of antigens. They are of the IgM class but in some cases thereare signspointing
toward the appearance of IgG or at least precursors.of IgG. Some support can
be found for hypotheses on the molecular phylogeny of the immunoglobulins,
though the hope entertained by some--that the original immunoglobulin would
be found--has not been-fulfilled. Fish, or at least those species studied
so far, have an immune response comparable in many ways to that of mammals.
Immunè processes probably first appeared in very early vertebrates which
have now completely disappeared.
As far as-we know, an immune process with
antibody formation is not found in invertebrates (Carton, 1968).
Before turning to the latest results, it will be useful to see where
the species that have been studied appear in a classification of the fishes
(see table I). The organisms usually called fish are little more than a
group of animals sharing a certain habitat. The superclass of fishes is
divided into five classes', two of which are completely extinct (Acanthodii
and Placodermi). The others are the Cyclostomi (also called Agnatha or
Ostracodermi), the Chondrichthyes (cartilaginous fishes) and the Osteichthyes
(bony fishes). The amphibians, reptiles, birds and mammals represent the
other classes of vertebrates. Since most of the work referred to here
seeks to clarify the phylogeny of the immune response in fish, it is necessary to know ho w the taxonomists treat fish evolution (Baer, 1965;. Grassé, 1958;
Lehman, 1959; Romer, 1962).
•PHYLOGENY'OF THETISHES
Fish were the first vertebrates. They have always lived in an aquatic
environment, whether freshwater or saltwater, and the stability of this environment has perhaps contributed to preventing their early disappearance.
The
water has certainly been better in this respect than the land environment,
which has varied enormously over the geological ages.
This stability makes
it easier to understand how some very early forms have survived--"living fossils"
like Polyodon, Aida, Lepisosteus, Protopterus and Latimeria. They are the
last extant representatives of intermediate groups that developed extensively
in certain geological periods.
Because this group of vertebrates is so old, study of the extant forms
is essential to the solution of any problem of evolution, for they are the
descendants of organisms from which the other classes of vertebrates developed.
The cyclostomes or agnaths (jawless fishes) lived at the end 012 the
Silurian and inthe Devonian, after which they seem to have almos Lotally
disappeared. The modern cyclostomes "appeared" at the end of the. Tertiary
are the only living vertebrates with a fibro-cartilaginous skelton and
no jaw. All other fishes have a jointed jaw (superclass Gnathostomi), a
feature which first appeared in the placoderms--numerous in the Silurian and
Devonian but now completely extinct. The cartilaginous fishes (Chondrichthyes)
and the bony. fishes (Osteichthyes) Are generally assumed to have both aris e
directly fro.m the placoderms. The evidence does not seem to point to derivatLon
14 2-4
s
Table I
List of fish species studied for immune response
For each species there is given the Latin name, the common English name,
the common French name and the period or epoch when it appeared (column I).
The studies (referred to by their number,in the bibliography) are classified
into six subject areas: biochemical structure of immunoglobulins (column II);
- study of immunoglobulins by electron microscopy (column III); immune response
sequence (column IV); natural immunity (column V); role of complement (column
VI); immunocompetent tissue (column VII).
Legend
1
class
2
family [Myxinidael
3
subclass
4
order rP1eurotremata7
5 superorder
=
• It l•
t
1,
•
.• •
• r.
•
PoSiet de redierelies.
Lisle des esees
t. .
•
•••
te non, coninitintn8
tarèp(1‘1111;
Pr el•-■ •.tque capèt-4.1 6:LIU rent k nOril Iin. k ;loin :orneuen
‘,;•orter àij li‘te bibliographiqu.r) sons
non ,111 vent,: (colonne I). Les divers t ervaux (chaque nurn ■mo
inimunogloisulin2e
Ii) ; étude
classes en six rultriques struetute binchinlique
••-• •••.,nitaire (colonne IV) ; ininyunité
; .létoldernen; de la
cr, microscopic eleetronique (colonne
(eulonne
naturelle (colonne
; tide du complérnent (co:urine Vi) ; k tistut j j
•
,
.•':
,
.
•.. • •
• .
•
AC. NAlli
.
r:
(elesse), lawless fishes.
-
eles Myxinids.
hagtish, /Ilyeine
californienne.
(5)
—
39. 53. 5-5
\
Fsrnille de.ca Pctrornyzontid,e.
»itemized el-dui:1w, lat-nprey. Lump/Die.
S. 7
22 , 3 4. 5 2 . 64,
107, III, 112,. 115.
, 1
3. 1 4.
1 4,
7. 3 7
ts,i3,&I
1 12a.
•
' GNATHosTomEs
te!. -
CIMNIMICIrell seVS (:t) ou Elasreohrunehii (Sh.tklike tishes or
cartilaginous tishex, Poissons csttilegincux).
„
.
.
.
."
•
,
1.1. Stu,enit (sous-c1àss.1).
f
Pieurotttnatei (Re.
a. 1 .1. sir:.
twins nu Squelc-a- ).
1,1.1.1. Fernille r12r. lieterrxSontielec.
lirkrediceidit fteetistil, horned s11.1r1r, Jurassique
zupe
Requin.
t.t.t.2. Famine ace isuridx..
Crérece
C.d.rràtteimd t.rildaridtt,
inf.*
men-eater. ll.oun blene.
reazi:crel-shatii, Ctézeci
.
Itiewt
.
'
67, In, ley, no
y.
50
;0
•
I.i.t .5. Fluriille ace Cercharhinidee.
Murkier
•
ernoozh
Oligocène 1,9
47
Chien è..2 mer.
Triakit ten:fascia:a, leoperd Shark,
Ceitecil
Requir,1,:ut,,serel.
emtarlica, gurniny sherk,
dNaptimt brevirearit, lemon shark,
Requin.
Ca/es-tab eerier', leopa«1 shark.
hull shark,
Cartharrhinta
Requin.
2 9.
55 , 8 1, 10 5
28 , 1 9.
74. Soo
Si
118,
14.
Éoe,lne
Çioeèn
1; 1 9
8,
17, 35. 4 0, 79. 80,
ay, toy,
87
126
_
•
.
.
.
.
•
1
1,
In
•
•
1 .13
Titill.P.A I • 1
•,"
•
3.
•
/...e/t//r. )
; • '
1
1
;111 1
11
I
i
v
Iv
t
•
•
des
1.1.r...4. Famille
Scyliorhinid4e.;
est shark, l':ous-1
Selina Jidher,
.4
1
t
.
.
•
.
1
t
Crètete
-' i se
1
t
Orecto1obida-4.1
.
Giàuljertelk.wa
Cl/ L,
1turee-1 Jurassiquel 17, 26, 47, 40, 62. 79, 1
)49, log, 112, 124., 41> i
shetk, Etc.-pain.
,
ales
Sphymid4r. t
t.t.I.6. Fumille
I
I
SAyrea iibera,
homier shark,1 Creme,: 1112
1.
i
sup'
1 •
Rceivin marteau.
I
R.hinobstide.1
1.1.1.7. F21214410
d.n
I
....--tte.
1.1.1.1. Famillc
• •
vzi •
7
des
Rh:we-1i/4i prvai.ylms, guiturtishd Cretacé I
Poisson 4)-nitsre.
!;
:
I
I
1
4.1.3. Serie dos ilypocrement (raj.= 1
.
ou torpilles).
1.1.1.1. Famine des D2syatirla%
:
D4.901i1 ante (item 1, stingray, Raie, i Cretace
17, 36 , 62, 124
•.
66,73
I
'
I
•
.
42, 109 , 112, 121142
•
I
4.
!, 3
1
(sous-classe).
1.2. B11.31/Yupoe-rrr
I
1
Higher
a. OacirrirEs (classe)
bony fishes, Poissons osseux.
1
•
(cous-c)asse),.
2.1. ACTIN0p-re8ycnt
Rey finned fishes.
•
'
(1)
C14mmeost81 (supt.-r-ordre),I
Famillc des Polyodootirke. I
Pc•,5ou stmlbet, paddleiish, Spam- Co.:lace
1
2.1.1.
sup!
laity.
2.1.2, HoL6)sre.1 (341;.-..er-ordm).
Famille des Atniid:e.
.4reits
bcp.etin, grinclle, mud- Ëocène
tin.
1.1.2.2. Famine des I.c-pisosteidre.
1...epiraerms orreaa. lonxnoze gt, Crerace
Lei-41509re.
Leplioneer
6, 40, 13, 16, 18, 36..16
37, wt.., tog, 440, 1381
Lepisoste.
garpike, Cteracc
surir
1,63
•
I
23, 86, 105, 106, 110,15
128
6.
le,
37, 4 1 , 109, 1 6
112
ple.yrhynew
13, 22 , 30. ve 6 3
2 6 . 44, ¶ 1 .
(1
9. 89, 891
•
I
.;
1
1
66
!.
i
26. 54, 10.1 . 51, 57 ;
66
f
•
Tr.LOS r
(sur,er-ordre).
2.1.3.1. Ordre des Clupeiformea.
2.1.3.
des Salmon id,.
S:.•uroon.
1.s.fiocne
rsinbort troue, iNticeene
truite arc•en-cicl.
•
2.1.3.1.1. F41014110
S.ilpto
S.Vmo
•
Oncothyadmi tarka,
Saurnott.
2.1.3.2. Ordre
sockeye salmon,
—
la,
93, 414, 117.
TF. 1T4j414
39 , 77. 9 2,
120 •
91
.
.
9 1,
.
•
•
des
Cyprinifortnes.
2.1.3.2.1. Famille des Gyninoridre.
Elect ropi.orgs e le clricief cicarric eel,
•
.
•
112
Gymnotc.
2.1.3.2.2. F2111ille
des Catottornide.
bigmouth buf-
Ictianis yprinellia,
falo, Suceur.
.a.
: 63
1
•
■
••
/•)
1
•
•
\
.1 •
'
•
1.1
i rfcti•it•
.
VW Milk tic> Cyp i" iflitle.
..11rea.ttow.ikeett typ-indla,
t ....live. •
• Tiara Illter+. tench. Tanche.
Canif.ries
..m.r.lia.r,
t jes',
.
•
'
c.up,1 M-et,c-ène .38. 78,
• 135
10 1
, 112 .
it
il
4's 173 , 9 8
11 9.
.
,Oligocencl
goltitish,: i'itoceste '33, 43, too, 339 •
.
'
1
f 'atassul.
.3.3.3.1.4., ramilk des lia grid's'
..-leirre,•■ •es my•Ids.. hurdle:1d, Pt tisson- ..Olizocène '. 31:
.
!=4
1
I
1
dret.
hiaillria .m.vali.r. freshwater catfish,:
—
1
;
•
.
I
—
.,6, 37. 112
1
:3,
21
I
1
1109
1,.
I
!
!
1
•
l
i
•
1 65
.4
'
-
•
..-..
'3.63
33 •
i
Poiseon-char.
•
laaberis K.-ha/via, catfish, Poiseon-:
chat.
'.
2. 3.3.1.3. Rrnille cics Inotoshi:c.
7i.:JettleletZr ialtulanaf, AUS ■ faliun fresh- .
water ei:ttish.
dma :\ nyusillifOrrnes.:
2.1. ,,..3.1. Famine dcs Angude.;
73, 102,
•
;
Poisson-char.
itiukrte.t p4welaimr. channel cartish„.
129
•
•
1 e , zie
•
3.1 °rein:
'•
1.
1
;
'. 130. 7 3 7 . 1 3 2
!
l'amillc dcs Conyeidat.
:
Cônser fee!, con,;er ccl, Congre. 'Ac-zeuic
. .1.3.4. Ordre des i'erciformcs.
2.3.3.4.1. Fan-iilie des :2K-en:ethic.
i
.t1(itlensi,:e/4,. t'zitteri, gi• ■ •wpci.,..\ fir:coo.'
t
..Epiireplhilles itaiara, gisnt grouper,: i-...:ocene i 31, 83, 89, 96, 309!
Mérou.
i
2.1.3.4.1. Farnilte cles Lutianiclat.
griwef,
napper.
:ccealc. :89, 109
09111Wf CbryfIerei,
yellow tail.
-snapper,
Farnille des l'omadasichc.1
Fle.fintloti dbera, relargatc, grunr,:
t 26, 29, 104, 109
.eln,v(illa a.ygni/ 4r, cel. Anguille.
.:70
2.14.5.2.
1'.12rm•-•.1(2.
49
•'
1 19, 26, 104
Centriirchidee.
blacithass,', Miocene •
Alirroptereir .taletelairs,
l'erchst.
2.7.3.4.3. Fzmille des. &disc hire.
Ball.feri ccpri.t.w, trigi;er fish, i.
2.7.3.5. Ordre deis 'Men roctectieorrnes..
F:zmillet des Pleutonccridie.:
Pleurtmecia plalaAI, plaice.,
1
1
4.3
DI I'NELSVI (sous-classe.), Fleshy:
finned fishes.
Fantille dee Lepidosit•cnid.c.
Probe/err,'
et/jot:tic/es,
A irican , Oligocene :z3, 118, toy, 110, 128
Lungtish, Protopteec.
1
11:W4-m11 ,d/a forsirri,
Australian
—
46.
•
Lungeish.
•
.
Lepidorirceparedocett, Lungfish.
. Miocène .1 rz
2.2.
3.3. (.1ll)SS.1)PTERYGII
•
•
;
•
•
•
.49
145
of the bony from the cartilaginous fishes, as some have thought on the basis
that, ontogenetically, a cartilaginous structure appeared first and so could
be considered more primitive than a bony structure. In reality, the
Chondrichthyes appear to be more evolved than some Osteichthyes with respect
to other features, such as the urogenital structure. We must assume that
these two classes separated very early on (in the Silurian) and have since
followed distinct evolutionary paths. There has hardly been any reduction
in the number of cartilaginous fish since the beginning of the Carboniferous,
most of the extant genera having appeared in the Jurassic or the Cretaceous.
The class includes the Selachii (sharks, rays, skates) and the Holocephali
(chimaeras). It is very hard to tell which of these is the most evolved.
It would appear they are equally ancient and have followed divergent evolutionary paths.
While the. bony fishes did not appear at a later date, their structure
has evolved further. Their morphological features can be seen as postembryonic developments on a juvenile stage common to all gnathostomes, a stage
beyond which the cartilaginous fishes did not advance.
The appearance of
the bony fishes (very early on--in the Devonian) was a decisive step in the
history of the vertebrates. The group evolved through the ages: the axial
skeleton, once cartil'aginous, gradually ossified, this process reaching its
greatest development in the teleosts and some extant holosteans. The class
Osteichthyes has continued to expand since the Cretaceous and its modern
representatives constitute the great majority of living fishes.
146
The bony fishes are divided into two groups sometimes called the
and the Sarcopterygii (Romer, 1962). The forffier are characActinoperyg
terized by the absence of internai nares and by a fin structure with rays._
The latter have internal nares and paired fins in the form of fleshy plates.
There are many other distinguishing features and the taxonomic division
seems to reflect a phyletic reality. Paleontological data suggest that the
two separated very early in geological time.
Other fishes, the Sarcopterygii in particular, began to give way to the
Actinopterygii starting in the Carboniferous. The chondrosteans (Palaeonisciformes) were the first to flourish but they almost completely disappeared
at the end of the Secondary. Survivors are the sturgeons and paddlefishes
and, according to some writers, the African lungfishes.
The first holostean
appeared in the middle of the Secondary. This seems to be when the group
gradually penetrated the marine environment, though some must have
remained in fresh water as evidenced by such modern representatives as the
bowfin and the gar. The teleosts, which developed from the holosteans, appeared
around the middle of the Cretaceous. Other groups rapidly gave way to them
and they gradually penetrated allparts of the . aquatic environment,-with some
ràturning.to fresh water. The teleosts can be seen as having developed from
the level of the Malacopterygii, with fins lacking spiny rays, up to the
Acanthopterygii, with spiny rays. The former appear in three orders:
Clupeiformes, Cypriniformes and Anguilliformes, the first of these containing
the most primitive teleost species. The latter appear in two orders:
Perciformes and Pleuronectiformes.
However it is the Sarcopterygii that are of most interest for understanding the transition from fish to amphibians. It is quite certain' that the
terrestrial vertebrates descend from this group, or at least from some
representatives of it, in the subélass CroSsopterygii.
Some anatomical
features of terrestrial vertebrates are already present in these fishes.
The Crossopterygii became extinct around the end of the Primary, except for
theCoelacanth, which has survived to'elrepresent.
The other subclass, the
Dipnoi, with three extant genera, is in many ways analcgous to amphibians
both morphologically and developmentally. They may not be in the direct
evolutionary line leading to the terrestrial vertebrates but they are nevertheless one of the most useful links in the chain, as far-as understanding
vertebrate evolution is concerned.
Figure 1 (aftet Romer, 1962) shows the phyletic tree for these various
groups.
147
Figure 1 Phyletictree for agnaths and jawed fishes (afte.r Romer, 1962). The tree shows genera that have been studied for immune response.
Legend
1 agnaths
2
amphibians
•
•
I;
•\•
•
l'
‘
•
..i .
rie11tilOpteryg
\
. .:_.,.i:. : ',. . . ; .f. :.:.....7 .
CIPRINti; 7,..:7'..«.;, f
ÇAr:ASS:US i,. ' . ......1
■
Al.liRU
e/7
.-
.
,
• fe
E LEOSTEI
•
M
-
•
-•
h:11
•
..,, •7i.. EPISOST EUS.
ezr
OLCSèLJ
4
.
NF.00E.,ZATODUS
PRO TOP TERUS
51 r,7N—
t
, c'•-•
•
eSA5;CC;:-) T'..-71;RYG11
e'rem
DAsyAris,
EPTAT It n'us
■
v
,
•
Fig.
—
e
. e.\‘
r",,-;:rnr
^
Arbre phylogénique des Agnathes et des Poissons (d'après RomER, 196z).
Les genres ayant fait l'objet de recherches sont mentionnés..
.
148
THE CYCLOSTOMES*
1.
FAMILY MYXINIDAE
The earliest work on the California hagfish Eptatretus stoutli did not
reveal formation of circulating antibodies after antigen stimulation (protein,
virus or bacteria). It was thought that this lack of immune response resulted
from the absence of clearly differentiated peripheral lymphoid tissue (21).
However more recent work on the possibility of immunoglobulin synthesis in
this species has given significant results.
Subcutaneous injection of a bacterial suspension (EMB-1, a gram-negative
bacillus from the gut of the American Spiny Lobster Palinurus) results in
appearance of bactericidal activity in the blood 48 hours later. Maximum
titer is reached on the sixth or seventh day (4). A second injection gives
a more rapid response though the titer is not greater than in the primary
response (39). Bactericidal activity disappears irreversibly if the antiserum
is heated at 50 ° for 20 minutes, and it is not restored by addition of fresh
hagfish serum. The authors conclude that this neutralization of baeteria
does not involve complement, though certain complement components (CD are
present (39, 60, 61). When the antiserum is filtered on Sephadex 0-200 it
separates into three protein fractions one of which (an excluded peak indicating high molecular weight) shows anti-EMB-1 activity.
This fraction has the
same elution position as mammal IgM and on ultracentrifugation it reveals a
major component with a sedimentation coefficient of 20S and two minor components, with coefficients of - 13S and 9S. A denaturing agent such as urea or
guanidine-HC1 dissociates the molecule and its antibody activity disappears
(39). Biochemical analysis of the 13S antibody has not yet been Undertaken;
it would be of interest to know the number and nature of its polypeptide chains.
The action of natural (54) and acquired (53) anti-sheep erythrocyte agglutinins has been studied in hagfish. AgglutinatimY activity is low in the
natural state but increases by a factor of 103 or 10- after immunization (six
injectionsinseventy days). The activity is always found, after filtration on
Sephadex 0-200, in the excluded fraction of high molecular weight.
*
Abbreviations used:
0
sedimentation coefficient;
Ig: immunoglobulin; MW: molecular weight; S20m:
heavy chain; L: light chain.
H:
The items in the bibliography are diided into three groups: 1. general
articles 2. articles on the higher vertebrates (a to n in the text)
3. articles on the agnaths and jawed fishes (1 to 132 in the text).
149
The sedimentation coefficient obtained for this agglutinin was about
23.8S. Note that the natural antibody is thermolabile while the immune antibody, highly specific, is thermostable. Immunoelectrophoresis
reveals a
difference between this agglutinin and mammal IgM in addition to the already
mentioned difference in sedimentation coefficient.
Injection of soluble antigen (hemocyanin from the mollusk Megathura)
results in the appearance of highly specific antibodies; as evidenced by
passive hemagglutination and also by a precipitin reaction(54). Thus the
most primitive of chordates can synthesize precipitins. An increase in antibody titer after three immunizing injections (at 14 day intervals) gives
some reason to posit a degree of immunological wemory. The anti-hemocyanin
activity is found only in the héavy fraction (Sh u,= 28S at a concentration
of 5 mg/ml) of the antiserum after elution on Septadex G-200, This fraction,
with a glucide concentration of 3.42 per cent, may be assigned to the IgM
class despite a slightly higher electrophoretic mobility at the anodal end
of the distribution. The antiserum also has another fraction of lower molecular weight which is antigenically related to the first but has no antibody
activity. The authors want to study it more before identifying it as a 7S
IgM. It should be mentioned that natural (low titer) anti-hemocyanin activity
is found in the fish when they are caught. This may be explained by the
necrophagous diet of these benthic species, which almost certainly feed on
limpets--mollusks living in the same areas.
Thus a humoral immune response is indeed found in the "primitive" hagfish,
though this response does not exhibit all the features found in the higher
vertebrates. The first work with this species produced negative results but
we have seen that if certain precautions are taken (54) with respect to the
aquarium conditions under which the fish are raised (temperature of 18 0 ),
feeding (by tube) and selection of antigen, the hagfish can produce an immune
response. The chemical nature of the antibody fraction has not yet been elucidated, in particular its precise polypeptide chain composition and mode of
linkage. The extreme lability of the antibody activity of this immunoglobulin must be emphasized (54); non-covalent interchain bonds may be the source
of this instability, as in the immunoglobulins of the Petromyzontidae.
2.
FAMILY PETROMYZONTIDAE
The first work (5, 7, 22, 27, 73) on the lamprey Petromyzon marinus
showed that this agnath exhibited no immune response to molecular antigen
150
stimulation (bovine serum albumin, hemocyanin, bovine gamma globulin,
Escherichia cou iendotoxin).
However there is a positive response to a viral antigen (T 9 bacteriophage)
(2, 5). And an injection of bacterial antigen (strain of dead .Prucella abortus)
causes the appearance of agglutinins (13, 27). This work suggests that the
lamprey does have an anamnestic response. The serum fraction exhibiting the
agglutination reaction migrates under electrophoresis like the alpha globulins
in mammals; under immunoelectrophoresis this fraction gives only a single precipitation arc (22). Its sedimentation coefficient is 9S and its molecular
weight 150,000 - 200,000 (27). The lamprey has a natural hetero-agglutinin
for human red blood cells (15, 64, 52). The existence of a non-specific
agglutinin has been demonstrated, as well as of a specific anti-H agglutinin (14)
which does not agglutinate A y B or Bombay red cells (111). .Immunization with
red blood cells of group 0 causes a clear increase in the specific agglutinin
only.
The natural agglutinin has a sedimentation coefficient of 46S. The
acquired agglutinin has an electrophoretic mobility comparable to that of
Yi-globulins (equivalent to (3 2 -globulins). The slowest moving protein fraction (with a mobility comparable to the y,-globulins, that is IgG) is in fact
a transferrin (64) which'some writers (22, rightly say has no anti Brucella
action. Hemolysis has not been demonstrated in lampreys; its serum contains
no complement (3, 81).
-
There have been several studies of these immunoglobulins in recent years
and there are fundamental differences in the interpretation of the chemical
structures involved. A synthesis of the results not being possible at this
time, each study will be considered separately.
One analysis (2) concerns the nature of the antibodies formed after
injection of F. bacteriophage. Anti-phage activity is found in two protein
fractions of tfie antiserum, having sedimentation coefficients of 6.6S and
14S, and blood concentrations of 0.3 and 0.1 mg/ml respectively. Only the
6.6S fraction was studied. A simple denaturing agent (urea, acetic acid or
propionic acid) dissociates the molecule into its various polypeptide chains
which are revealed by chromatography to be of two types: light (ld: 24,000)
and heavy (MW: 70,000). The L chains behave quite heterogeneously during
electrophoretic migration while the H chains behave like the IgM chains of the
higher vertebrates. Since no reducing agent (2-mercaptoethanol) is needed
to split the antibody molecule into its chains, it can be assumed that the
bonds between the H chains and the L chains are non-covalent (and do not
consist of disulphide bridges). On the other hand it can be assumed that
there are disulphide bridges linking the H chains to each other and the L chains
151
to each other. That would explain, according to the authors, why filtration of the 6.6S fraction (without prior exposure to 2-mercaptoethanol)
shows some L chains with a MW of 40,000: the molecules are dimers. After
alkylation-reduction and then filtration of the 6.6S fraction, the L chains
represent 30 per cent of the initial protein mass and the H chains 70 per
cent. This means the 6.6S antibodies have two light chains and two heavy
chains. The molecular weight of the fraction, determined by filtration,
varies between 88,900 and 115,000, depending on the solvent and gel used.
This is less than what would be expected for this structure (188,000). It
could be explained by the instability of this protein in solution. From the
properties of the H chain, the fraction belongs to the IgM class, but it has
certain peculiarities. There is no degradation when papain is introduced
and degree of dissociation depends on dilution. The L chains have nothing
in common antigenically with the native immunoglobulin; their secondary
structure changes when they are separated from the H chains. Immunological
study has shown the 14S fraction does have antigenic properties in common
with the 6.6S fraction; it is therefore thought to be a polymer of the latter.
As we have already seen, injection of group 0 red blood cells or of
Brucella causes the appearance of agglutinins. Study of these antibodies
(107, 111) has shown a clear specificity: the anti-0 agglutinin does not
react with A, B or Bombay red cells. Activity is found in two of the antisebum fractions, one of low molecular weight and the other of higher weight
(S20m.; 9S; MW: 320,000). Only the 9S fraction has been studied (107); its
activity is four times greater than that of the lighter fraction. It is
very unstable and its antibody activity lasts only a short time, which makes
it very difficult to study. The molecule divides spontaneously into subunits
of molecular weight 90,000. But total reduction followed by alkylation in
the presence of guanidine does not cause further degradation (except for a
small polypeptide fragment whose molecular weight is much lower than that of
the L chains). The authors therefore suggest that this immunoglobulin has
four subunits (MW: 90,000), joined by non-covalent bonds, each of which
has a small peptide fragment linked to the rest of the chain by disulphide
bonds. It should also be noted that most of the polypeptide chains constituting this immunoglobulin have an a helix secondary structure while in
other vertebrates they always have the S pleated sheet form.
The problem arises of the origin of antibodies in the lamprey because
152
the presence of immunocompetent tissue comparable to that found in the
higher vertebrates has yet to be demonstrated.
The theory is that immunocompetent cells are found in an organ identified as a primitive spleen which
is located in an infolding of the gut; the periphery of this infolding"(27)
contains clustem of small round cells taken to be lymphoid tissue; similar
cells are found in the epithelium of the peripharyngeal gutter, which evaginates from the pharyngeal floor and is identified as a thyroid.
The concentration of immunoglobulins remains low in the serum of immunized
animals, about 0.4 mg/ml, while the total protein content is 38 to 60 mg/ml
(15, 52, 64).
The exact chemical structuee of the antibodies remains uncertain, there
being wide'differences of opinion from one study to another. The problem
appears to arise from method, details of which cannot be given here (see 2
and 107). That can be said though is that lamprey immunoglobulins are quite
distinct from those found in other vertebrates, particularly as regards
their secondary and tertiary structure (113, 128).
THE SELACHIANS
1..
IMMUNE RESPONSE
There are numerous studies on this group, and especially on the chemical
structure of the immunoglobulins.
Work on the immune response sequence remains difficult to interpret as
far as comparison with mammals is concerned. Heterodontus franciscil, a
very primite selachian, can clear hemocyanin and phages (T 2 bacteriophage)
injected into the blood (5). The antibodies show up in passive agglutination
or neutralization reactions; no precipitins ever appear. The immune response
is found only in some individuals. The antibody titer is higher after a
second antigen injection, which is interpreted by researchers as an anamnestic
response.
Two interesting studies have been made on the leopard shark Triakis
semifasciata (29, 74), the immune response in which was compared to that of
the mouse. With similar antigen injections (T 2 bacteriophage); the antibody
titer on the fortieth day is ten to twenty times higher in the mouse than in
the shark. Over the following sixty days, the titer in the shark remains
stable despite repeated injections; around the 130th day, the titer increases
once more. The antibodies are found in the 6.7S and 17.4S fractions of the
antiserum. However, in the initial months only the 17.4S antibody
153
is present; it is only around the sixth to eighth month that the 6.7S fraction
appears. A sample taken on the 380th day reveals an equal quantity of the
two. There is a similar response when the leopard shark is immunized with
hemocyanin (from Negathura crenulata)(29). The antibodies formed, highly
specific precipitins (no cross reaction with hemocyanin from Limulus), are
in two fractions of the antiserum: a 17S and a 7S fraction, with three times
more activity in the former. The slowness of the immune response in this
species is worth special note.
In the lemon shark Negaprion brevirostris, a single intradermal or
intraperitoneal injection of T 2 bacteriophage causes the appearance of
neutralizing antibodies (38). The response•is slow, the antibodies not
appearing until the fifth day and increasing slowly until the fourteenth day.
Reiniection on the 42nd day of sharks already immu•ized bY four injections over
28 days provokes no anamnestic response. Similar results were obtained for
immunization with bovine serum albumin (8).
The immune response in the lemon shark and in the nurse shark Ginglymostoma
cirratum (26) to viral antigen has been studied by the passive hemagglutination
method. Five weekly injections are made of PR 8 virus (type A human cold
virus). The agglutination titer increases slowly, reaching a maximum around
the thirtieth day; just after that it tends to decrease despite a further
injection on the thirtieth day. Injections on the 100th, 141st and 283rd
days bring no further increase. There is thus no evidence of immunological
memory in these two species. Comparable results are obtained when the virus
is first killed with Formol, though the maximum titer reached is lower. Study
of the antibodies formed (see below) shows two types, one of high and one of
low molecular weight. In the initial months after immunization, the heavy
fraction clearly predominates. It is only around the fourth montfi that the
two types are found in equal quantities and around the sixth month*there is
slightly more of the light fraction.
Other studies of the nurse shark have shown (68) that the concentration
of the two fractions varies with the time elapsed after immunization. At the
end of the immunization period, the 19S fraction clearly predominates, according
to these studies.
In neonatal nurse shark immunized with two injections of type A cold virus,
the concentration of immunoglobulins was extremely low (fifty times lower
than in the adult) .and ail were of the heavy form (72). Only on the 24th
day after immunization did the light form appear. On the 48th day the two
fractions were present in equal quantities. But the concentration was
still half that of the adult.
■
154
Injection of nurse shark with bovine serum albumin Caused the appearance,
after a latent period of several days, of antibodies (revealed by passive
agglutination). Their titer tended to decrease slowly after reaching a
maximum around the fortieth to sixtieth day (17).
The stingray Dasyastis americana, when immunized with Salmonella typhosa
or human red blood cells, exhibits an immune response similar to that found in
lemon shark: slow synthesis of antibodies, maximum at the twenty-first day,
and no anamnestic response.(42).
2. IMMUNOGLOBUL1NS IN SHARKS
An extremely large amount of information has been gathered on selachian
immunoglobulin structure and it cannot be described in detail here. For
references, see table I; for a summary, see table II. The results for various
types of shark agree and it is therefore possible to state for this group
the general features of the immunoglobulins having an antibody function.
0
There are two molecular forms, one of high molecular weight (S-a.0,1v : 17S - 19S)
which I will call 19S Ig, and one of lower molecular weight (S9D,eu : 6S - 7S)
were tempted to
which I will call 7S Ig. The discoverers
mammal
immunoglobulins
IgM and IgG.
two
classes
of
identify these with the
In adults, at the end of the immunization period, these immunoglobulins
represent half of the total protein weight in the serum (68, 72). In neonatal
shark they are almost non-existent, accounting for only 1 to 2 per cent of the
proteins (72).
The two forms in fact belong to the same class; native 19S and 7S are
antigenically identical (9, 29, 67). In double diffusion, antiserum to the
19S fraction is used up on 7S Tg and gives no precipitation arc with 19S Ig.
Their glucide contents (table II) and amino acid compositions are quite
similar (1). .They are composed of equal numbersof heavy and light chains
(see percentage of protein mass, table II).
The H chains of the two forms have the same electrophoretic mobility and
double diffusion reveals they are antigenically identical (8). Their peptide
maps, obtained by electrophoresis or two-dimensional chromatography after
tripsin digestion, are quite similar (35, 67). Also, their amino acid
compositions are identical (1). The same analyses performed on the L chains
also reveal identity of the two forms.
, 155-6
Table II
Summary of known facts on the biochemical structure of the immunoglobulins
in the main fish species studied
Lesend
1
native immunoglobulin
2
heavy chain
3
light chain
4
suggested tertiary and quarternary structure
5
antibody activity
6
genus
7
molecular weight
8
percentage of glucides
9
percentage of mass
10
present or absent
11
antigen used
12
Brucella and red blood cell
13 hemocyanin
14
bovine serum albumin
15.
activity not studied
16
Salmonella. H
17
Salmonella 0
18
human IgG and hemocyanin
19
bovine IgG
20
red blood cell
21
present
22 F(ab)
2
of 16.1S Ig
■ 11t..•,
e À
•••-•
I.
•.-• • • •
• • • , '!'t
strumar
Tahleau ri:capiiulaIy des condaisAaà.es sin.
des intinunoendines des principales espèces de Poissons étadiées.
Il
C.1.115ificarion ;
.
•
Activiré
i
anticorps
' Immunoglobuline i
Cliaine lourde (lame légere
des immnnonacire
::,,3„0,
.
globulines
. p ropo,,,,1
:
'
:,
pour la
'
.
i
• strucrunt
'
.
I.
r --...•.,../.•-•,,•\ : ,/.;•,;•\ .terciaire
tr-....„.\
./,•11
.
\....-i
1.,(.: ,*
l_9 ; <-3 ) • ■ (-,1
U-'''
cite'te'•• ,.. . 1-'''-•''
•
C...,/
'
,....:•' .!
.er'5
naire
p-rer..en- • •'n.
nti- :
; Poids '.% da; Poids ! '-',:,, de ' Poids °;„ de
la
`
te
ou
moie•
gène
glu.
an,71é.
la
mole'
,
•
Genre : S,,°
,absente unlisé '
: ''''' . culaire i' cicles ; culaire i masse • cuiaire • m asse •
.
1
:
.
•
.
i
.
.
••
.
•
:
.
.
:
.
••
i
•
1 on
I
.0.900- I
•
•, 6.6s i. 1 z 1.000 I
.
'Pelrom),-,--.
.AGNATH' S
9S = 320.000 .
.
:
•
.
•
1 .S
'''
.
-
.
.
'
. 9,3 ; 68.5 ,00
'11,9 68.100
' 7,2S .
7.3
23.000
/5
2
8,7 !71.600
73
7,6 .73.40,0
. 68
'..1befil67.1' . 6,7S : 198.000!
.
.
17,1S : 982.000 i
•
• .-
.
•
_
. --
-•
'
1 1 ,4 '
6 7S •
''
■ T riakir
•
:
.
Yee- •
.. p.
0
.
urella 107
t
1
II
-,.--• et-nude•
25
L251-2
-1-
20.100
27
(1, ,,,, 2) s
' Brure//a :67
20.300
32 '41...,.
i
I. 9
, -i-.
' hérno-
• —
. cya_e,
nine ,,,...1.;
prior?
•
•
....
•.
•
' •
19S • 830.000 • ?„67171,000 • 74
3,51 /Loa) • 74
;TS • 163.0oo
22.500
26
(1.,2 / 2)5
22.100
26
1..2;h2
•
; + : hémo- 29,
aeya- ,103
.
;nine ec
•
phage '
:
.-1• séntin. 8
—
alhu - ,.••
.
117)
, :zravine
'
•
.
.
•
5 ,000
.
:77.00,7
.76.3 oo •
•
•
•
1
26 '(L23.4),.
•-t-
phage z
.
I
bELACHII
.
.
•
Heterw • IS,5S ':
' rezraus
'
:
.
< 9S •
r
(polypep:
peptid.._.)41:
* absente
•abScritc
a
i ±
.
•
•
Lei-,
30
,70.000 , 70 24.000
•
!
I
Crier - .• ; .„9,1:
elyenn. . ''''''
3,14.7Q.coo
12
22.000
.1,b0 70.000
72
.12.•Doo
Jima
_........_._ .
' 17,1S •
.
—.
(1..
..,v...0 4
.2.3
2g
1-,1;)..,
_........_ _ _
2 3. 00 0 .
. 9.3 ;7 0. 000 . 75
25
(ileds
•1
•
:
• 950.000
I
:
CHONDRO- ipoowd,„!
S TE1
•
•
1
.1,71wiendh,
42
.
•
;72.100 ! 74
,
• 14,25 630.000 • 6,8 ; 0.000
' séru m- I - .
aibu- .
, mine
bovine
hettlaric
•
•
,
,
1 11,0S 355.0c,› -
—
- --
: 7S (?).
Daealii
.."
75
21 .906
26 (1-2;•11
.21.000
25
(1...',../.2)à
acrivité non (-.K.tr
recherchée C)
-- .
.t.e/i-
6,
maella
..
...--,.
• 87o.oee •
75•3 00 :
.
-
'
2 3.5 00
.
—
ii.-ocella
Val.
emarila
ID
.
.
,
'
%
(A .
Inine un tyatohu line
,.' -,
III, • l
"1.1..1
.
A
.
.
i I -- .
.,
• ...,,...
r.,
iltilIC ■ epe
:
4,-. ■ Allle i0111‘..0 t.tmila it.è.,•::rt, ,. •
.,.1rodè,tu I .,;!•: immuno, !
i
i.:7.
pi.„,,,,,,,i•
....v.iuliteliner.
1,
C.113•Silir-âtiOrl i
•
•
I
GCMG
.
'
•
'
Sw ,
.
.
!:
1
.
.
•
•
•
I
1
1
i
!
.
I
I
;Ref,
•
:-.",..:.•';
: "..-!-'•
Lepli- •
''''
li-i-...)
pewrella !
_
à; 97$ ! 6so.000 • 4,8618,000
HOLOSTEI
7
i
a3.00o
Anti-L,
7
Sel- ;4z
!
•
'
;
Goo.000- •10,7 .6t.oco-; 58 ,-14.000:•':::,i2 .; .(1,..,1.8„),;
900.000 .
70...).7,7 •
• '...;:!.
,
.'.:•'
:
9,1 52.c,e,o ;
: ;•;•''
..1..;,:e2
1.
16S !
;
70.000 1,
to
.
1
"' •
,
!
,I
■
'•,`..
-
: A':::-
.
--,•.':;....
I
1
;22.000 , Yli. ,(1..,,V-.),
,
....2.•.,•
: Deniella ..10.1.
ph:age
—
.
•
•
et
.189
• 11.*::no••
• clot; ,••
I
•
19 S
!
•
•
•
•78.600
•z,•3 00
.
Carat-
?;':
• .'"...i1'
I- S.:!..'.
:ii:...:. 7
si.rs
•1 / 9
•
htirruzie:ci
ci
I
.1
l/err.o-• ;
"
cyetine ,
•
•
.
- t
.
61o.000 . 3,3 ;70,000 :
t
125.000
•
■
1
1
PicKro- , 144.S 1.
•
-.•:, 1,
i
i
8 ,8
1
I
i* .•;:•;:.
'...'-:::.'
(pré; m.trite)
.
i
1 Geratc) ,•••., .
'•:.1'4;1- ,IS l'
._. \----'
.
•
pierps
..1 0.000
:
I
0,cm° : •
;
•
,4s
5.9s • 125.000
7c).zU0
37.95 0
i
z'.;
7$
":
•
i (L.4S.. •
•
a3.01 ,3 ••••••••
•
:7:: •
)
(1e
'
''.7:77C'.7r'''T.j. • .
1
1
19
Soh
f
e.ropella !
!
.•
.
;.
'
!
"-•.:•••".
1.'
I
.
i
5,5 70.000
70 '22.000 H.„`.i10
.,
,: a....,,I
. 1
, •:-. 27
: , ; IgG
.
8 3.
, t 1 4o.oco 1, 38 ..12.000 '• , .4.1 iii (ab), de! .‘,,,•-r. boelne . 96
!
i9S
01
.
. I0,1., ;700.000
3) 1 /20.'100
pbeler ;
;
t
1
I
gen.:
I
1
,
s.nti- 6
•
I
!
I
.
---,
Ide- '
Di 1, NRuslri t
117
r1.--:G ,o.i .
://://,..ine-48,
) :':..'
:Cypriffief
Tr.L17.0S'll
:
• aini. •69
e:mc
1.
•
•
nikei
•
—
U.
ç'-'•.':
I
•
•
.
.
r pour le i .".., ..
:
•-••••';' •*!'groctuee ..
,,.
t
•
1
:
.
t t..
I./nixie/ et;
1
;
'1
:
?,: eioutet, 1 ....'
• '
Poid8 '.`, (lc; Po1,18 ! %de l'oids ! °•',:, de :'. in,„•are
ii)(t, SCIF.
.‘1111. ;
rook:- • glu• ' mol./.• :
ix
nko1é.- ., '',I_..t Yr.' . ,1 te,ou
gene :
: .
;
•
i
et/Lure I ctd.csi.entxtre mucsc eulairc! nr..N..., ':'-'", •
l
• ....
.
.'
1
•
1
,
:
I '' ....
•
. ..::,
' :
,-
1
}le. M '' , "• . ...43
''''':' ).;
................,.•_.
;.•
,tos
. '88
1
1 46
non
recherchè.:
.acttè
•
•
157
However there is no relation between the H and L chains, as regards
antigenicity (8, 9), electrophoretic mobility (1, 8, 9, 42), amino acid
composition (1, 42) or peptide maps, except that in horned shark there seems
to be some similarity in peptide map.
There is no antigenic relationship (17), or only a partial relationship
(9, 29), between the native immunoglobulin (19S or 7S) and its constituent
chains, but the method of extracting heavy and light chains (partial or total
reduction) does appear to be very significant (17), which would explain the
differences from one set of results to another.
The authors suggest that 7S Ig is composed of two H chains and two
L chains, such a structure conforming to what is known about the molecular
weight and the percentage of the weight of the native molecule represented
by each type of chain. The chains are linked by disulphide bridges. However
non-covalent bonds also figure prominently in the tertiary structure (17, 67).
Non-covalent interactions between H and L chains remain after partial reduction,
alkylation and acid gel filtration, unlike in mammals. Only by complete
reduction using denaturing agents (guanidine, urea) can full dissociation
be obtained (8, 9, 67).
The 19S Ig is thought to be the pentamer form (1, 9, 19). After partial
reduction (125) it splits into subunits (S9 c,w
7S) which in some cases
continue to exhibit antibody activity (67).
Comparison of the molecular weights
of 19S and 7S Ig lends support to the pentamer interpretation, which has been
nicely confirmed by electron microscope study of 19S 1g (42, 47). In leopard
shark (87), a light chain different from the L chains has been found in 19S Ig
only; it has a molecular weight of 20,000 and an electrophoretic mobility
greater than that of the L chains. It is linked to the molecule by disulphide
bridges which prove to be far more stable than those of the L chains. The
writers think this polypeptide is homologous to mammal J chains. A similar
phenomenon has been observed in nurse shark.
It would thus appear that selachian 19S and IS Ig belong to the same
class of immunoglobulins, and one which seems to be homologous to mammal IgM.
The 19S Ig has a high molecular weight and glucide content, like IgM (table
II). Its H chain resembles the mammalp chain in many respects: molecular
weight (table II), amino acid composition (1) and electrophoretic mobility
(1). In leopard shark the N-terminal amino acid sequence in the H chains is:
Glu - . Ile - Leu - Thr Glx - Val - (28)
158
This same sequence is found in the L chains of the species, with some
variations in residues 1 and 4. The sequence is identical to that of human
kappa chains (K111 subgroup). Recent work on the leopard shark (55, 85) shows
that a high proportion of H chains (60 per cent) and L chains (75 per cent)
have no free NH 2 group, the N-terminal amino acid being pyrrolidonecarboxylic
acid. Some H chains and some lambda chains in mammals have this terminal
acid and also a comparable sequence for positions 3, 4, 5 and 6 (see
table V).
The L chains behave heterogeneously under electrophoresis, as with mammals
(8, 9, 28, 35, 67).
Thus we
described as
mammals, and
two forms is
find among selachians two molecular forms that are to be
19S IgM and 7S IgM. The latter has recently been discovered in
in man in particular (d e, f); however, the antigenicity of the
not identical (a), as it is in selachians.
,
Several writers have attempted to distinguish the two forms in selachians
in terms of a differing sensitivity to reducing agents (5, 8), but a difference
is not always found (29, 84). This characteristic can however be useful,
provided it is combined with others (17, 26).
The antibody activity of the two immunoglobulins is quite different.
In
general, 19S IgM exhibits between four times (29) and one hundred times (62)
as much activity as 7S IgM at equal concentrations. Also, the degree of affinity
for antigen increases during the immunization process (62, 124).
Upon introduction of antigen, the two forms do not appear siMultaneously.
In measuring them, we must know whether the method depends on concentration
or on affinity for antigen. Only by total isolation (by immunoadsorption for
example) can their precise concentration in the antiserum be known. Measurement
through serum reaction (passive hemagglutination, precipitation) gives the
antibody titer, which depends not only on concentration but on affinity for
antigen.
Generally speaking, 19S IgM appears first, one to six months after
immunization, depending on the species. It is then gradually replaced by
7S IgM (17, 20, 26, 29, 78). This is also observed in neonatal Sharks (72).
It has however been shown that 19S IgM remains throughout the duration of the
immune response (84). A more detailed pictUre has been obtained through a recent
study on nurse shark, for which the two immunoglobulin forms have been isolated
and measured. Initial antigen injections cause a small increase in 19S IgM
but a great increase in 7S IgM. There follows a general decrease, more pronounced
159
in the 7S form. After three months, 19S IgM clearly predominates. But after
further injections the initially high titer of this form tend S- to decrease,
then increase again one or two months later. Thus there appears to be a
cycle in the immune response: 19S --> 7S---> 19S.
The question arises whether 7S IgM is the precursor of 19S IgM. The
association constants are similar (82); they are related not only structurally
but also in their biological activity. However after injection of labelled
7S IgM (40, 67, 79), serum was not found to contain labelled 19S IgM formed
by polymerization of 7S IgM. The opposite process, beginning with labelled
19S IgM, also gave negative results (79) so there is little basis for assuming
that the 7S form is a degradatien product of the 193 form. And in fact,
despite their antigenic identity (29), some differences have been found
between 7S IgM and the subunit 7S (obtained in vitro by degradation of
19S IgM), in particular differences in valence (17).
Immunoglobulin valence has usually been deduced from sérum reactions,
positive hemagglutination with 7S IgM tending to show that it is bivalent
In one study however (82)
(8, 17, 29), which would make 19S IgM decavalent.
it has been shown by equilibrium dialysis that 73 IgM is monovalent and
19S IgM pentavalent. There appears to he a contradiction here. Study of
•
rabbit IgM (b) has shown that five of the combining sites have an antigen
affinity one hundred times greater than that of the five other sites. Also,
the 7S subunits of Waldenstrom IgM (c) are potentially bivalent but functionally
monovalent. The same could apply to selachians. It may be hypothesized that
selachian 7S IgM affinity for antigen changes over the period of the immune
response, as with 19S IgM (62). The rate of synthesis and half life are
shown on table III (79): the properties of the two immunoglobulins are quite
similar. Finally, some work has been done on their respective locations (40,
79): 19S IgM prefers intravascular spaces while 7S IgM is also found in
extracellular fluids.
It is difficult in the current state of knowledge to state preeisely
the relation between the two forms. For some writers (40) the-observed differences
in their modes of formation and their functions are sufficient to consider them
independent. For others (82), the similarities make it possible to consider
7S IgM as a subunit, precursor or degradation product of 19S IgM.
160
Table III
Biological characteristics of the two immunoglobulin forms in two species of •
shark
1
lemon shark
2
nurse shark
3
half life (in days)
4
concentration in serum (in mg/ml)
5
rate of synthesis (in mg/kg body weight/day)
3. Immunoglobulins in Rays
The main results of studies on the stingray Dasyatis americana are given
in table II. They are similar to the shark results (42). However one recent
study (121) has shown the existence > of a protein having the structure of an
inummoglobulin (though of unknown antibody activity) and according-to the
study it is a dimer of which each subunitlas two H chains and two L chains.
The writers also discovered, in the serum of unimmunized rays, a small
quantity of an immunoglobulin of high molecular weight, which almost certainly
corresponds to the 17.1S ig described in (42). After immunization, its
concentration may well increase sharply and it would be easy to detect. A
laresuantity of it has been obtained and its study undertaken (42) (table
ii).
THE
CHONDROSTEANS
Study of the paddlefish Polyodon spathula, found, in the rivers of North
America, has been concerned mainly with the nature of the immunoglobulins.
In the phylogeny of the fishes, it is customary to say that the members of
the superorder Chondrostei were the first of the Actinopterygii, though the
origins of this latter group are far from resolved. The two genera of
chondrosteans (or three if the African lungfish Polypterus is counted)
have some of the characteristics of selachians, which suggests common ancestors
in the now extinct class Placodermi (Romer 1962).
.
'
•
•
• ..
Cd/ 1i1 .1. i.Cli ■pir.e /1/ •
....il&
.‘
.•
If;
.1, •■
//if I
irle////Mi0:.;11/1•11111/11r.e
•
•
•
•••
•
.
(79).
elaIN gWre.l. Ehili/i0branCbe$
•
•
'
.
.
•
t.\
•i;ApitioN
GINGLYAIOSTOM.,-1
(-;)
•
•
.
l'el 7S
Igàf r9S
IgiNI 7S
.
•
Ig111 t9S
.
,
(«D '1)etni-duree de vie (en
itourS)
•Concentraton
al.• ■■ ,...", l
dans
....clame i e
•
serurn (vil Ingtrni) . .1
:-.
,,,.., ' Taux de synthèse (en'.
mszikg de poids corpo-1
re our)
•
.
i
.
..,...
..,
••41.tvgi
t‘...
■
. •
4
.-.
•
.*
.•
•.
5
8 5
)
Io°
I
12 - ty
4
i
6
G,-7 7
,
•
50
8 ,5
.
70 . .
•
6
C
.
3 0 1..ES IMMUNOGLOBULINES CHEZ. LES HYPOTRIMES
r
Les principaux résultats obtenus chez la Raie Dasiatis sont consignés
clans k tableau II. Ils sont conformes à ceux obtenus chez les Pleuro-•
trèmes (42). Cependant un travail récent (x zi) a montré l'existence d'une
protéine à structure d'immunoglobuline (sans en avoir recherché l'activité anticorps); elle serait constituée d'un dimère, chaque sous-unité •
étant formée de deux chaînes 1-1 et de deux chaînes L. De plus, ces auteurs
ont décelé l'existence d'une faible quantité, dans le sérum de ces animaux •
non humunisés, d'une immunoglobuline de haut poids moléculaire qui;
correspond très certainement à l'Ig 17,1S précédemment .décrite
On peut concevoir qu'après immunisation la concentration de cette
ig r7,rS augrnente fortement, et qu'elle est alors facilement décelable; obtenue en quantité appréciable, son étude a pu être entreprise (42)
(tableau II).
.•
.•
LES CHONDROSTÉENS
L'étude de Poirdon eathula, présent dans les rivières d'Amérique du •
Nord, a principalement porté sur la nature de ses immunoglobulines.
Dans la phylogénie des Poissons, il est de règle de placer le super-ordre
de Chondrostéens à la base des Actinoptérigiens, bien que l'origine de
ces derniers soit encore loin d'être résolue. Il convient de rappeler que
les deux ou trois genres de ce groupe (si on y inclut le genre africain
Poblveras) présentent des caractères qui les rapprochent des Sélaciens;
ce qui suggère l'existence d'ancêtres communs totalement disparus e9H,
regroupés dans la classe des Placodermes (RomEa, 1962).
"•
.
.
161
The paddlefish displays a-primary immune response after antigen
stimulation and a secondary response appears following reinjection (30,
73. , 22). The antigen may be a protein (BSA, hemocyanin), a virus (T2
bacteriophage) or a cell (red blood cell, Brucella). The species has a
well developped thymus, peripheral , lymphoid tissue and a family of plasma
cells.
Antibody activity is found in only one protein fraction of the antiserum
having a sedimentation coefficient of 14.2S (18, 36). Writers differ on
the molecular weight: 630,000 (6, 18) or 870,000 (10)--we will return to
this important matter later. The concentration in the serum of immunized
paddlefish is 17.2 mg/ml, which is half of the protein weight (36). The
glucide content is 6.8 per cent (6, 36).
After complete reduction (using urea .or guanidine), the molecule
dissociates into several polypeptide chains of two types which can - be
identified with mammal H and L chains. Fractionation shows them to be
present in equal numbers (36). Non-covalent bonds play a large role in
the secondary and tertiary structure and in the combining site: antibody
activity is not stopped by simple reduction without use of denaturing agents
(10). There are 70 &sulfide bridges (6), a result obtained by the first
hour of incubation; in mammals (g) 90 disulfide bridges are cleaved after
six hours. The L chains have a molecular weight of about 21,000 (10, 18, 36).
Writers differ on the molecular weight of the H chains: 72,000 - 75,000
(10, 18) or 58,000 (36). The first result is obtained by column filtration,
the second by sedimentation equilibrium. Those who claim the H chain weight
is 58,000 say the total Ig molecular weight is 630,000. The Ig must consist
of four subunits (each with two H and two L chains), as is shown by electron
microscopy (6), and this tetramer structure would give approximately the total
weight claimed:
630,000
4(2(58,000) 4- 2(20,000))
The N-terminal amino acid sequences are identical in the H and L chains
(1 0) :
Asp - Ile - Val -
Ile
(Leu)
Thr -
It appears that a high proportion of the L chains (75 per cent (10) or 100
per cent (18, 36)) have no free amino acid because pyrrolidonecarboxylic acid
162
is present.
The structures are:
-- H chains:
Asp - Val -
Val
- Thr - (18, 36)
(Leu)
-
L chains:
(25 per cent)
Asp - Ile -
Val
(Leu)
-
L chains:
(75 per cent)
PCA Ser Glx Glx Thr - (18)
Thr - (10) (see table V)
There may be two types of L chains comparable to mammal kappa and lambda
(10). There are certainly strong analogies with human L chains and some human
H chains (some human p, a and y chains have been shown to have a free N-terminal
amino acid). •
A study of the amino acid composition (6, 36) or the peptide map reveals
clear differences between the H and L chains in paddlefish. Also, there
is no J chain (37) in the 14.2S Ig, which must continue to be seen as in many
ways untypical (16).
It is thus hard to identify paddlefish 14.2S Ig
classes of mammal immunoglobulin. However the amino
N-terminal amino acid sequence of the H chain enable
reservations, to the p chains in higher vertebratès.
be assigned to the class IgM.
with any of the known
acid composition and the
us to compare it, with
- •14'..2S-Ig:mob1d then
It is claimed (106, p 753) that one (unnamed).chondrostean has a
protein of low molecular weight with an immunoglobulin structure but no
antibody activity of any kind. It would be good if this could be confirMed
by publication of the data.
THE HOLOSTEANS
Most work on the superorder Holostei concerns the genera Lepisosteus
(gars) and Amia (bowfins), both of which are found in the fresh waters of
North America.
In Lepisosteus injection of protein (80) or viral (26) antigen result in
the appearance of antibodies, ten to twenty days later, the immune response
163
being stronger with subcutaneous than with intraperitonal injection. The
studies were not conducted in such a way as to reveal a secondary response.
More recent work (51), devoted entirely to this latter question, shows that
a typical anamnestic response does indeed occur (table IV), with a clear
increase in the antibody titer and a reduction in response time. The antigen
dose and method of injection (intramuscular) must be carefully chosen.
After immunization by 0 or H antigen from S. typhO'sa, by red blood
cells or by bovine serum albumin, antibody activity is found in only one
protein fraction of the antiserum (6, 18, 41, 69) whose molecular weight
is between 600,000 and 645,000 and whose sedimentation coefficient is 13.9S. •
Writers disagree as to whether •the glucide content is 3.54 per cent (69)
or 4.86 per cent (41). The antibody, with a concentration of 14 mg/ml,
represents half of the total serum protein. Its sensitivity to 2-mercaptoethanol
remains at the saine level throughout the immunization period (26). Under
partial reduction, it divides into 5.3S fragments (63) and under total
reduction (41, 69) it divides into H chains (MW 70,000) and L chains (MW
22,000 - 23,000). However the remarks on method described above for
Polyodon apply here too, and it_appears that the molecular weight of the H
chains must in fact be taken as 58,100 (41, p 2035). Images obtained by
electron microscopy (6, 69) reveal that the 13.9S Ig is a tetramer (69),
making this latter molecular weight more likely:
645,000 --,: 4((2 x 58,000) + (2 x 22,000))
The 5.3S subunit would correspond to two H chains and two L chains.
With an increasing concentration of guanidine, 82 disulphide bonds are
revealed in the 13.9S Ig after one hour's incubation, and this figure does
not increase after three hours or after six hours (41). This might explain
why the antibody remains sensitive to 2-mercaptoethanol throughout the
immune response (26). The differences between the H and L chains are in the
lack of antigenic relationship (69), the peptide maps (41) and the amino
acid composition (41). Under electrophoresis, the L chains are not revealed
as heterogeneous (41). Finally, the molecule does not appear to contain
any 3 chain (37).
Two other studies of Lepisosteus should be mentioned (44, 66). It
appears that the natural and acquired agglutinins and hemagglutinins are
cold antibodies which form best at a low incubation temperature (t 2 0 ).
In some cold-blooded animais, then, antibody synthesis is not reduced at
low temperatures, but,
164
TABLE IV
The immune response sequence in the holostean Lepisosteus (51)
Legend
1
antigen
2
primary response (injection on day 1)
3
4
secondary response
-1
average titer (10 )
5
duration of immune protection
6
date of second injection
7
(time from injection)
8
diphtheria toxoid, aluminum phosphate--adsorbed
9
bovine serum albuinin, aluminum phosphate--adsorbed
•
IV
' `I'Att.rAu
1)61-ceicfmwt es la reorisc,immtmitair
e
.
..
ckez Pliolosiéell : 1.t..piso$t,eus
(5 t). •
. .......
.
i
Réporme prims.fre C
.21). •
(injcc
- tion 2U
licctiort
n ■ , , er :—......,
jour)
ter
!
..
Rep-on:7,r sccundzire @..)
,
Durée
il
cc is,. protection
Nature
i
mmunitaire
(.'••7
.
.
_....,_,„,_____
i".
: .?:j
Toxin?. diphtérique absorbe
- sur aiumin ,-
f Cr
.
••■
.
3o .T.f.
il
3oc
I.
i
Sérunr.%jumi ne ° ' °5 Enghni 2in I. '
: bovine
0 ,5 rilgirniI 21e i•
Zbsorbé , . sur
ziurni'ne •
3 Mg/I nl , il 7' 1'1;
50 riliDri 1
• .
3 2.1. f •
.
I
It
I
II
II
f
;' *-
.
I•
7e
I
I, j.n. I
f (.
:73
LI • .
date
Li
(à/.',11)1.titie
1••■■••
1 .97 1 3.
, I
.
"
5'3
! 24 " J. •
773 • ... I 11, 4 1 i. 1
.67.. 7
s.uree
•
el;: la protec:i..n
Mnk i mum
I.
•
,
23z
!
1
j
i
,
i
is)2tt
ccienlice:c- 1
1
'titre moyen -1
I
,
date
_
.
I
I
IA
1,1
'
•
4 '1
7:1
.
J.
j
.)
77.4') ,1
f 372'-'
J.
•
-- 2°
777 -
j
I.
toe I. WI ;.yi
11 0 ' i• (26 i.)I
1 8 41 i.
•
u
1
.
I
.,
I
.
I
I.54 z,
1 3 1 91 .3.
. 6:3
IJ. :
3 1 9" I.
.
84e j. il
333
.
ii4e j.
ri4'..'
66
•
e
•
.e0 •
i ^ 4 e i.
• .I.
2 4e. j• I .
z
•A 4 0
f• .233
I
.
.
.
.
. . • :.
I .
....1
. : :.
.....P.;
...:
•: -0 .
• - t?(..,
3!. 9e :.,2 :
•
/'
• .
.-
:"
• •
r
165
on the contrary, stimulated. This can be advantageous if the environment of
a species is subject to large temperature fluctuations (66). The 13.9S antibody
is also found in the mucus that covers the body surface in this species (44); its
behaviour is thus probably comparable to the secreted 10 antibodjes known to
exist in mammals.
The genus Lepisosteus displays a strange mixture of highly specialized
characteristics which make of it more the end of an evolutionary development than
a step on the road toward the teleost, unlike the genus Amia.
Work on the bowfin Amia calva (86, 106) shows that two of the serum fractions
of fish immunized against Brucella have an immunoglobulin structure (see table II).
But only the heavy fraction (13.6S) displays antibody activity; the light fraction
(6.3S) is completely inactive, even with use of the inhibition method, which makes
the fish extremely sensitive to antigen. Nevertheless the light fraction is antigenically ideniical with the heavy fraction and such a relationship also exists . with
respect to glucide content (table II), peptide map and circular dichroic spectrum.
The logical conclusion is that the light fraction is a degradation product of the
heavy fraction. Reduction with dithiothreitol divides the heavy fraction into S.SS
subunits the electrophoretic mobility of which is slightly higher than that of the
light fraction. The authors of the studies- suggest that the heavy fraction is a
tetramer. Another difference between the fractions is in the H chains, which are
eluted later in the light fraction. There is apparently a - difference of 12,000
between the H chain molecular weights of the two fractions (table II). Also the
light fraction H chain has a greater electrophoretic mobility than the heavy fraction
H chain. On the other hand, the H chains are identical with. respect to antigenicity
24,0001 are identical in all respects.
and dichroic spectrum. The L chains
The two immunoglobulins in the b.owfin appear to belong to the same class, which,
can be identified with mammal IgM. This is the third example, in the fishes
we have looked at so far, of an immunoglobblin with an H chain having a molemilar
weight much lower than 70,000. Some writers (86 ) suggest that this H chain can be
identified with a p chàin one part of which is missing. This along with what we
saw in the paddlefish (a chondrostean) and the gar provides some evidence for the
hypothesis on the origin of heavy chains according to.which they result from duplication of a precursor gene initially emding a polypeptide having about 110 amino
acids.
166
THE'TELEOSTS
1.
The immune response
The teleost fishes can be immunized against a great variety of antigens:
proteins such as bovine serum albumin, bovine gamma globulin and hemocyanin
(43, 75, 98, 100, 101, 104, 114), bacteria. (6, 43), phages (12, 117)• red blood
,
83, 89, 103).
cells from various.sources (43, 6.I_and...11aptens bound to a protein (78,
Depending on the species, one substance will have greater antigenicity thon another;
Freund's adjuvant seems to increase the immune response. After an initial injection, there is always several days' delay before antibodies appear, but they
remain present for many months (91). Reinjection definitely evokes an anamnestic
response, as has been fully demonstrated with Saline (91), Cyprinus (98) and Carassius
(75). In the goldfish Carassius initial work C12) could not reveal a definite .
secondary response, but more recent work has conclusively proved that such a. response
occurs. When the goldfish is immunized with_ bovine serum albumin, the following
results are obtained:
latent period
day of maximum
response
passive
hemagglutination
titer
Primary response
.
7th day
20th day
5
secondary response.
.
3rd day
15th day
8
This shows all the characteristics of an anamnestic response. •
Incubation temperature must be considered (12, 75). Generally speaking, up
In
to a certain point the rate of antibody formation increases with temperature.
°
C)
(12
the carp Cyprinus it has been shown (98) that a low incubation temperature
when
C
25
°
temperature
is
prevents appearance of a primary response. However if the
fish
is
immediately
the fish is immunize, a primary response appears even if the
transferred to a 12 environment. It can'be transferred before (8th day) or after
(15th day) the appearance df antibodies in'the blood. As to the secondary response,
it occurs even at a low temperature, as long as the primary response occurred before
the transfer.
In some cases at least, the antibodies- are of • an extremely specific nature (83),
Thus', antibodies in goldfish immunized with-x 174 phage have no effect on T 2 phage
(12). Similarly in carp (101), anti-bovine serum albumin antibodies do not react at
all with sheep serum albumin.
•
167
Antibody affinity for antigen changes over the response period. In goldfish (12),
the association constant goes from 10 on the fourteenth day after immunization to
200 in the third to fifth month. In the giant grouper Epinephelus, however, the
constant apparentlY tends to decrease over time (83). Another point of difference
from what is found in animals was observed in goldfish (101): the antibodies
exhibit three equivalence points during gel precipitation. The variation in immune
response from species to species may reflect the heterogeneous nature of the teleosts,
this being an artificial taxonomic grouping.
2. Immunoglobulin structure
The large number of studies and the differences between them make it difficult
to synthesize ihe results. Another reason for the difficulty, perhaps, is that
the phylogenic origins of the superorder Teleostei are numerous, as can be seen
by the taxonomy of the group which differs from writer to writer and is largely a
matter of personal opinion.
Table II gives the main results. In most of the species studied,'there is
only one type of immunoglobulin, with a fairly high molecular weight, composed of
equal numbersof light and heavy chains. Comparison of the 'moleular - weight.of
native immunoglobulin to that of its constitutent chains suggests a tetramer structure:
four subunits with two light and two heavy chains each. That this is correct has
The immunoglobulin seems to
been fully confirmed by electron microscopy (6, 48).
belong to the IgM class: the.H chains have the same characteristics (molecular weight,
electrophoretic migration properties) as mammaly chains.
Sonie results however show two different immunoglobulin forms in certain species.
In carp, for instance, several writers C12, 75) have noticed that the immunoglobulins
could be divided into two forms solely on the basis of electrophoretic migration.
However a recent study on this species (102) shows-that if the carp is immunized with
serum albumin bound to a halyten, the lowmobility antibodies react only with the
carrier protein (bovine serum albumin) while the high mobility antibodies react
specifically with the hapten.
168
The clearest results are found with the giant grouper (83, 96): two types of
antibody with sedimentation coefficients of16.13 and 6.35S, and molecular weights of
700,000 and 120,000 respectively. The 16.1S Ig consists of an equal number of heavy
(1«: 70,000) and light (blIV: 22,000) chains. A tetramer structure can be assumed,
each subunit consisting of two H and two L chains linked by disulphide bonds. It
" should be noted however that the subunits could not be separated by partial reduction.
It would appear that non-covalent, as well as covalent bonds play a large role in
the tetramer structure.
.
The amino acid composition of the two immunoglobulins is very similar. However
antigenically they have little or nothing in common. The L chains are identical
as regards both molecular weight and antigenicity. But the H chains are very
different. The molecular weights are 70,000 and 40,000. The H chain in the 6.355
Ig is very, deficient antigenically in comparison to the H chain in the Ig 16.1S.
The difference is confirmed by the peptide maps. While there are many spots in
common, thirteen are specific to the 16.15 1g heavy chain and only one is specific
to the 6.35S 1g heavy chain.
The results taken as a whole have led writers (83, 96) to the conclusion that
the 6.35S 1g may be only the F(ab) fragment of the 16.1S Ig subunit, but this is
2
unconfirmed. The 16.1S 1g chain with a certain portion deleted would be a polypeptide with a molecular weight of 30,000.
THE DIPNOAle
This group of "lung"-possessing fish is of special interest because it lies
between the fish and the amphibians. Study of the extant representatives (Neoceratodus
of Australia, Protopterus of Africa and Lepidosi:ren of South:Amerita) was , therefore
essential and because of the nature of these fishes it promised to yield important
results.
The results for the Australian lungfish are the most comprehensive (46). It was
not possible to learn whether the serum proteins having an immunoglobulin structure
displayed antibody •activity after antigen stimulation,.but the study was extremely
useful in understanding immunoglobulin phylogeny. Comparable results were obtained
for the African lungfish, which were immunized (88, 105); the authors point out
C105, p 346) that the immunoglobulins, especially those of low- molecular weight, did
display antibody activity.
169_
In the Australian lungfish (46) there are two immunoglobulins with sedimentation
coefficients of 19.45 and 5.9S and molecular weights of 950,000 and 150,000 respectively. Antigenically they are only partly related, both with rabbit antiserum
to the 19.4S Ig and with antiserum to the 5.9S Ig. • The amino acid compositions are
fairly similar, though there are appreciable differences in the histidine, arginine
and glutamic acid content. Reduction and alkylation (lin the presence of a denaturing agent) give two types of polypeptide chain. The 19.4S Ig has L chains with
a molecular weight of 23,140 and H chains with_a molecular weight of 70,280. In
electrophoresis, the latter migrate like mammal )1 chains. The 5.9S Ig H chains have
much greater mobility, slightly more than human ?J' chains; their molecular weight
is 37,950. As regards antigenicity, the L chains are very similar but there is
no relation at all between the H chains--the Situation 'found in the- higher- vertebrates.
The 19.4S Ig, according to the study, has a . pentamer structure. This, together
with its size and the nature of its H chains,puts it in the IgM class.
The 5.9S Ig, on the other hand, has two L and two H chains. -AcCording
to the writers, it is_unlikely to be a degration product of the 19.4S Ig. In chickens,
a 5.75 antibody has been found in addition to 7.85 IgG . (11); lungfish 5.9S Ig may be
comparable.
An analysis has been made (88) of the N-terminal sequences of the chains constituting the 5.95 Ig in the African lungfish and there are several points of
interest in it. First, it seems that the N-terminal residue of the H chains is almost
always pyrrolidonecarboxylic acid. Seventy per cent of the L chains have this same
acid but thirty per cent have a free amino acid, most often aspartic acid. The first
ten amino acids in the sequence have been determined.. There is some similarity to
L chains in leopard shark and to rabbit and human kappa chains (table V). A comparison can only be made, however, if one assumes a gap at positions 2 and 3 in the
African lungfish sequence:
As
- Leu - Thr Glu Asx - Ala - Ser "Val -
170
Table V
N-terminal auino acid sequence for L chains in various vertebrate species; for
the most frequent amino acid is shown. eachpositnly
Legend
1.
2
bibliographical reference
3
leopard shark
4
5
paddlefish
6
man K
7
8
9
mouse K
10
chicken
position
African lungfish
III
rabbit K
man
Asx means either Asp or Asn; Glx means either Gin or Glu, no .distinction being
possible; PCA means pyrrolidonecarboxylic acid. Note the close structural similarity
(italicized items) in positions I, 2, 3, 4, 5, 6 and 9.
CONCLUSION
The immune response of an organism is generally considered from two points of
view: the response sequence and the chemical structure of the antibodies which are
formed. Comparison of species from the first point of view only is difficdt because
experimental conditions vary from one study to another, as regards immunization
procedure, nature of antigen. method of injection and serum reaction technique. Great
care must be taken in generalizing from such results.
-
Still, all species of both jawless and jawed fishes that have been studied
have been shown to be capable of an immune response after antigen stimulation. In some
cases, hagfish for example (54), the response was obtained only after a number of
failures (5), when all necessary precautions were taken with regard to incubation
conditions. Ambiant water temperature plays an important role in triggering immune
response (Good and Pappermaster 1964);, primary response occurs only above a certain
temperature (98). There are howeverco1d,aggiutinins (44, 57, 66), a defence
•
4
7.
\
•4 • • .1.
Séquence des acides eemiai• .\--terailaaux e.,;!
de différentes es,hèces de Veriét')nis ; pour
aminé le plus fréquent.
L. de e imiaiit;ogobilliae. s
siiit;a sed fierc l'acide • .
t.
•
• ' ' s • •:
(1,1 Poon c',...::;•acicic tzt.m-,és
;Référencrs • '
îo
i
:
.
/
!
,
i
I
_ _____.........._.1..___:____I—; ...____;____1_1--;-1______/..•_.'
;
o ,
1
f ,) Triakie . .
*
,?-) Pdyndon . .i
-,1;•• Prompierns • ;
4'1 Homme Kan.;
'tzl:i) Lapin K. ., Alti
'?'„:•:i Souris K. . ;
e Homme . .-..,
.
- •* -NPOU il; .
e-..;
. . : ,
X
I, 3 11
t
■
1
it, 1 4
1
5
i
'
ï 6 1 7
I;
r,t I
Ile ■ Val! Lat1Thr 1 Gin :Pro ./ G1 y i1 .S.'c, - 1, V a/ •:
n
..,
;.A.rp! Ile 1 Val! 11c ITh r 1
i
;
.
,
.* • to
iris)) t (—): (—); Leu t 7 br ;Glu As-,t • Ala " Sei- , Val ;
68
'.4•rp ' Ile ; Val: Leu : Thr i Chi Scr Pro 1 Ser : Ser .
. •rup: Ite . t'al, Met Tbr;Glx; -,Clir: Pro ; Ser • Se.r
k
:_e‘l.rp Ile I Fa/. 'Mer. Thr IGIx'Scr ; Pro Sr ; Sec ;
l
.K.:A: Ser i Val; Leu ;Ur . Glu Pro i Pro ; Scr Val •
i
('-') . (^) : ALt ; Lai !De t GIx . Pro i •Ala ; Ala t Val i .
ni
`Arp
•
Asx indique soit Asp, soit Asn ; Glx indic:tic soit Glu, soit Glu, la dis•tinction
n'étant pas possible; PCA ...--- acide pyrolidone carboxylique. Dans les positions t,
• a, 3, .4,, 5,6 et 9, remarquer 'une communauté de structure (acides aminés en itali.
que) étroite- entre les différentes esp.èces.
•
..
•':CONC LU SVO N S
.
„L'étude de la réponse immunitaire chez un organisme e s e. considà.éé
généralement sous deux aspects : ses modalités de dé...roulement et la
structure chimique des anticorps formés. Si on ne retient que le premier
aspect, toute comparaison d'une espèce anin.yale à une autre esz dcile
car les conditions expérimentales varient d'un travail à un autre, que ce
soit pour k protocole d'immunisation, pour la nature des antigènes,
pour les voies d'injection ou pour les techniques de réactions sérologiques
eMployées. 11 faudra toujours être prudent dans 1 généralisation de ces
résultats.
Né..antnoins, toutes les espèces d'Agnathes et de Poissons étudiées
ont été capables de donner une réponse immunitaire après un c stimulation antigénique. Dans certains cas, chez EiVaireite.r par. exemple
ce résultat n'a été obtenu qu'après de- nombreux échecs (5), lorsque
toutes les précautions quant aux conditions d'élevage cies animaux
immunisés ont été prises. La température cle l'eau ambiante à laquelle
sont maintenus les animaux joue un rôle important sur le déclenchement
cle la réponse immunitaire (GooD et PAPERmAsTE-a, r.9.64); la réponse
primaire ne peut être déclenchée
„
qu'à partir d'un certain seuil de tem-
•I
I
t 1.
171
mechanism of great benefit to cold-blooded animals,
For a long time, some studies had left the impression that only viral and
cellular antigens were immunogenic. In fact, studies on various groups suggest
that there can be an immune response in fish to 'various kinds of antigen , even
in soluble or hapten form (see table II).
The primary response latent period is generally longer than in the higher
vertebrates. This is especially clear in the less evolved fishes, like the.selachians,
though in these species circulating antibodies, once formed, continue in existence
for several months.
An unambiguously anamnestic response is found only from the holosteans up
(table IV). But as we have seen, it is precisely at this evolutionary stage that
immunoglobulins appear which,though they can still be classed as IgM, have structures
not typical of this class. They are tetramers with a molecular Weight of about
650,000 and heavy chains whose molecular weight (58,000) is much lower than that of
typicalp chains (70,000). This can perhaps be understood in the light of the
situation in mammals, where iimuunocompetent cells that produce IgM are in general
incapable of an anamnestic response (b). Thus fishes like the selachians which
produce only typical IgM are incapable of an anamnestic response.
Looking at the fishes in the broadest sense of the term, we see a development
of the response sequence as regards specificity, triggering (including the appearance
of an anamnestic response at a certain state of evolution), and effectiveness
(5, 19, 26, 89). Several writers (Good et al, 1959; Papermaster et al, 1964)* have
attempted to relate this development to the evolution of the immunocompetent tissue:
the differentiation and complexity of the lymphoid tissue increases from the agnaths,
where the existence of such tissue remains debatable, to the teleosts.
But whatever may be said about evolution, work on the Myxinidae shows that the
immune function known in mammals also exists in the most primitive vertebrates.
So it seems unlikely that a zoological link displaying a primitive immune
function will be found, especially since no work to date has proved the existence
of such a function in the protochordates. All extant fishes, as we have seen, display
an immune response which in many ways resembles that found in the higher vertebrates,
172
mechanisms previously thought to exist
With advances in research
only in the most highly evolved vertebrates are being found in the most primitive
species. Thus the existence of precipitins has been demonstrated in hagfish (54),
and a hemolytic system involving complement has been recognized in numerous fish
species (though not in agnaths) (3, 24, 63).
:
Turning to immunoglobulin structure,comparisons are now much easier because
of the large number of studies that have appeared in the last four years. The
structures described for many species are similar to those that have been found in
mammals.
The results for the agnaths however do not show such a similai'ity, though .
further consideration is warranted because in one case (2) the suggested structure
seems to be a precursor of structures found in •other vertebrates, the difference
being a lack of disulphide bonds between H and L chains. In another case (107, 111),
the suggested structure is a peculiar one which in some ways calls to mind the
natural agglutinins ilot only of certain invertebrates but also of eel (130, 131).
The secondary structure - of the polypeptide chains in this case is of the a helix
type, while in other vertebrates the ei pleated sheet type is found (113).
In the cartilaginous fishes the numerous findings reveal the same situation
in different species. All antibodies are in the IgM class. There are however two
forms: 19S IgM with the structure (1,,p2 ) and 7S IgM with the structure L 9p2 . These
are very similar chemically (complete afitigenic identity) but they have different
biological properties (see table III). It is interesting that in some species
(gustelus, Negaprion, Dasgatis), 75 IgM displays no discernible antibody activity
and in other species where it does display such activity it is much less effective
than 195 IgM (compare their association constants (79)). This kind of 7S IgM has
been found in higher vertebrates, in particular in the human fetus (d). This may
be another example of ontogeny repeating phylogeny.
To sum up, in cartilaginous fishes two M immunoglobulins are found e one of
which, the 7S form, has not yet acquired an antibody function in some cases.
It is more difficult to synthesize the results for the bony fishes, because of the
dual phylogeny of the group, which is also found in the phylogeny of the immunoglobulins.
173
With the Actinopterygii, M immunoglobulins as described above are found.
However the results remain unreliable, especially as regards the molecular weight
of the heavy chains, which has caused much controversy. What can be said is thatin
the ray-finned fishes antibodies of high molecular weight (650 -;060) exist in all
. species and definitely have a tetramer structure (5, 6, 48,). There is a great
deal of information on the chondrosteans and the holsteans in particular, and it
is especially interesting in that the extant species of these two orders have
remained fairly close to the older forms that gave rise to the Actinopterygii
and the Sarcopterygii. In most cases the heavy chain of the immunoglobulin with the
high sedimentation coefficient has a molecular weight lower than that of typical )1
chains--this was shown by. the symbol p'in table II. An immunoglobulin of low
molecular weight (S °10 .,
6S) and no antibody activity has been found in paddlefish
(105), perhaps alsdin gar -(51) and definitely in bowfin (86, 106). In the latter
case it is 6.3S Ig with a typical? chain. Over the course of evolution, it would
appear, there is a trend toward differentiation of immunoglobulins into two types,
distinguished not only by their sedimentation coefficient as in the cartilaginous
fishes but also by their heavy chain structure. However, the two types remain
identical from an antigenic point of view.
. i
With the Sarcopt.erYgii, the immunoglobulins reached a new evolutionary stage,
as evidenced by the dipnoans. This group is not in the direct evolutionary line
leading to the other vertebrates (see figure 1) but a study of their characteristics
is of great use. In the extant dipnoans two types of immunoglobulin have been revealed
(.46), each with antibody activity (105). They differ in the way already described-molecular weight of the native molecule and molecular weight of the heavy chain.
But there is also a more basic difference:. there is no antigenic relation between
the heavy chains of the two immunoglobulins. The one with the high sedimentation
coefficient (19.45) has an M structure that is typical both in itS quarternary --and itspchaîn. But the other (5.951 has an frchain with a
structure--(L p
2 2
molecular weignt of 38,000. Thus the dipnoans seem to have a unique lamunoglobulin
class, though 5.75 Ig has been observed in birds (n) along with a normal IgG.
)
5
In table II we try to sum up exising data. on fish immunoglobulins.
Figure 2
174
is based on the phylogenetic hypothesis which_ is most plausible in the current
state of knowledge; it is of course subject to change with new evidence,
Fig 2 - Hypothesis on immunoglobulin phylogeny in agnath's and jawed fishes (the
Symbols iu'and.x stand for atypical iLeavy chains: see table II)
Legend
1
2
antibody activity
no antibody activity
3
or
4
antibody activity or no antibody activity
5
with IgA2 structure
6
antigenic relation between H chains
7
no antigenic relation between EL chains
8 anamnestic response
b •* .21
.
••■
étre considéré coinme l'hypothèseL. plus
.
nos connaissances; il est évident qu>ii peu:: à ;,
t'onction de résuluts acquis.
)( 1;.'4';t ).. 1'eM
.>\
1.2 1r2; ls-JG
I.
dans l'éte. actuel de
';QCnr are modifié -en
aveu,
eritir.q).Qa
entieorpt
:
f
. •
.
• é)
"(1 2k12 )4 ou
r
z)
.
P.171-7:
3 'tfort.ébré:-.4
(1, 2 122 ).4
(IWO
activeté ai t 1: cor-po
• -.., }L2ik:,')j.attze, esy.t.e.vic." aradeeri7u
«me Getivire antieorpe
.en
.
L24,2.?avee
mIte•cerpa (3)
irzTctinopt.siygill
e*
E t 2 , 21,5
K
C.11ett
ac U -.. et: te, e t: VI:G.027:3
. t2 at.ao cl. cane cz.o.,..-tre
" •'. •
'C,
tri;)
•
•
Fo I achi
crroc srriiture
ote
Cpeiyptrptidoli
K paronc4.5 c.ntig ■Iniqu.2 eritre
dei deus
.
f'4_')
L'a
( h.:in/et/ H
irn-qtato.,-1:chu 1 imee
,
,,,/
abeenee. de pc...-rent:•! cznct"9.bque antre lei(
C.igaredle0 II dee dote.: :.,:nurtog lobu 1 ine a
.
t)
i 1-,,,,,, z, pn.Frenna d'i.e:e 2•Jponee anemneatiqua
k,..,
..j
.
•
..
,
Fig. z. — 'rlypothèse proposée pour la phylogenèse des ir-ranunoglobulines des
Agnathes et des Poissons (Les symboles :J.' et x correspondent à des chaines lourdes
atypiques : volé tableau II), . .
..... .
e,
175
There are also studies which have considered immunoglobulin phylogeny in
fish from one point of view only, such as glucide composition (109, 110), heavy
chain structure (105) or circular dichroic spectrum (128).
Taken as a whole, the work that has been done not only provides a better
knowledge of the immune mechanism in fish, but also makes it possible to put forward
hypotheses on the phylogeny of the molecular structure and its function.
It is highly probable that an immune response with antibody formation appeared
very early on in the course of evolution, in primitive vertebrates that are now
extinct. This was possible only through the presence of a particular molecular
structure, that of the immunoeobulins. For all species except the.agnath Petromyzon,
where doubt remains as to the exact chemical structure of fractions having antibody
activity, immunoglobulins with a typical multichain structure have been shown to
exist. These structures must have acquired their biological effects slowly; in some
cartilaginous fishes (Mustelus, Negaprlon) and teleosts 6Imia), the 6S - 75 Ig has
no antibody . activity (as far as can be detected by the methods used).
This stability of the immunoglobulin molecule over whole geological eras is
also found in its primary. structure. Work on the N-terminal amino acid sequence of
the L chains (see table V) provides incontrovertible evidence of a remarkable
similarity between the fishes and the higher vertebrates. It is noteworthy that
the light chains of Ginglymostoma and Negaprion (17) are antigenically related though
the two species appeared about 220 million years apart (see table .1). Similarly,
the immunoglobulins of Cyprinus and Carassius, two genetically related but different
species, are similar in some ways(117).
•
These studies also make it possible to say that IgM is the most primitive
class of immunoglobulins„most fish antibodies belonging to this class (see.table II).
Only at the evolutionary stage reached by the dipnoans does a new class appear: the
heavy chain in the 5.95 Ig can no longer be related to a ji chain (46).
Certain hypotheses on the phylogeny of immunoglobulin chains. (Hill et al, 1966;
Hilschmann, 1967; Lennox et al 1967) were formulated on the basis of work on mammals.
Given new knowledge on the primary structure of mammal immunoglobulins, it
would now appear that all their chains arose from a (purely hypothetical) primordial
chain made up of 100 amino acids, having a molecular weight of 10,000 - 12,000,
‘.e
176
and a length half that of a light chain. An initial duplication of the precursor
gene controlling synthesis of this ploypeptide led to a gene controlling synthesis
of a primitive light chain; a-second duplication led to a gene controlling synthesis
of a primitive heavy chain. This highly speculative process is to some extent confirmed by the fish results. As we saw, there are some similarities between the
primary structures of fish and mammal' L chains. Also, in some species of Actinopterygii,
a heavy chain with a molecular weight of about 58,000 has been found (see table II,
chain). Comparing it to a typical» chain, the deletion is equivalent to a
polypeptide of about 12,000 MW, a figure very near that of half a light chain. A
comparable case is found in the dipnoans, where the 6.3S Ig heavy chain has a
molecular weight of about 38,000 (see table II, x chain), which is - 12,000 off the
figure for an IgG y chain. Since these immunoglobulins do have antibody activity,
it can be assumed that the deletion involves the constant rather than the variable
region of the H chain (105).
These hypothesei are of a highly speculative nature but future research on
agnaths and on jawed fishes, especially on their immunoglobulin primary structures,
is SU-re to contribute information that will be very useful in better un.derstanding
the molecular structure aspect of the immune response.