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The immune globulins.

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CURRENT COMMENT
The Immune Globulins
By EDWARD
C.FRANKLIN
I
N 1939, TISELIUS A N D KABAT clearly demonstrated that antibodies are
associated with the y globulin fraction of serum as it is defined by electro-
phoresis. With the introduction of newer technics for studying these proteins,
it has become increasingly apparent that antibodies in a variety of species are
associated with several structurally and functionally closely related proteins,
collectively referred to as the “group of gamma globulins” or the “immune
globulins.” The first of these, the 7s ( y 2 , y r 8 ) globulin fraction, and the
high molecular weight (19s 7; BZm;ylm globulins) were initially described by
Kabat, Heidelberger and Pedersen on the basis of ultracentrifugal analyses.
The application of density gradient ultracentrifugation and immunoelectrophoresis to these studies has resulted in the discovery of two other classes of
immune globulins. One of these, the Y 1 A (&) globulin fraction, carries reaginic activity and probably also some other antibodies and may be similar
to some of the 10-15s antibodies recently described by Rockey and Kunkel.
The other consists of certain low molecular weight globulins found primarily
in human urine, but also in serum. These have been reported also to possess
antibody activity and are best called y,,. The term “immune globulins,” used
widely to include each of these major groups of proteins, appears to be
particularly suitable since it emphasizes not only their common function, but
implies also that certain structural similarities exist between all these proteins.
As shown in table 1, and as will be discussed in detail later, recent physicochemical, immunologic and genetic studies have clearly demonstrated the
existence of certain structural features common to all the immune globulins
and others, unique to each class, which account for their specificity.
Much has been learned in recent years about factors influencing the type of
antibody produced in response to an antigenic stimulus, and the cell types
involved in their production. Thus the simultaneous appearance in lower
vertebrates of antibody activity and lymphocytes and plasma cells, taken in
conjunction with the localization of y globulins by fluorescent tracer technics
in plasma cells, and in certain instances also in lymphocytes and occasionally
in transitional cells resembling both types of cells, clearly implicates these
cells in the origin of the immune globulins. This is further confirmed by the
absence of plasm3 cells and occasionally also lymphocytes, as well as one or
more of the immune globulins in patients with agammaglobulinemia. There
is now abundant evidence obtained by the immunization of humans, rabbits,
horses and guinea pigs with a variety of protein, polysaccharide and particulate
antigens that 19s antibodies are formed early during the immune response
and that 7s antibodies are formed later, provided the antigenic stimulus is
sufficiently strong. Even the newborn infant and fetal sheep are able to
form 19s antibodies at birth, and these antibodies in the neonate appear to
38 1
ARTHRITISAND RHEUMATISM,VOL. 6,No. 4 (AUGUST),1983
_._
EDWARD C . FRANKLIN
t>OL
Table
1.-Properties
-____________
__
7s Y ( Y z : Y , , )
of the Four Immune Globulins
~ . ~ _ _ _ _ _ . _ _
19s Y (Ylm: / I z m )
___________________~
S Rate ( S )
Molecular weight
Common
Chain
7
160,000
19
1,000,000
YIA (/IzA)
__
7 (9, 11, 13,15)
k 160,000
Yu
1.0-3
22-45000
L (light)
L
L
L
Inv
Inv
Inv
Inv
H (heavy)
H
-
7s
19s
Gm 4*
H
YlA
Gm 3'
y , ,. myeloma
Genetic locus
Unique
Chain
Genetic locus
Paraprotein
Gm 1
y myeloma
Macroglobulin
Bence Jones
protein
*Postulated.
be similar to those seen in adults in all respects. Little is known concerning
factors influencing the formation of y1A and low molecular weight antibodies.
It appears fairly certain that the former cannot be made in significant amounts
during the first few years of life and the origin of the latter still remains in
doubt.
The physicochemical properties of antibodies is of utmost significance in
determining, to some extent, some of their biologic properties and their potential pathogenetic potential. In man, only 7s y globulins can cross the
placenta from the maternal to the fetal circulation. For this reason, it is important to determine the type of antibody in the maternal circulation in cases
of red cell, white cell and platelet maternal-fetal incompatibilities since only
7s y globulin antibodies are potentially harmful, while 19s Y and Y1A globulins remain in the maternal circulation and are therefore innocuous to the
newborn. Several screening tests are currently available to achieve this. One
consists of treatment with mercaptoethanol or other sulfhydryl reagents which
completely inactivate 19s antibodies and have little if any effect on 7s y
globulins; their effect on ylA globulins remains to be evaluated, but it is possible that these may also be inactivated since ylA reagins and the 11-15s class
of antibodies seem to be destroyed by such treatment. Another simple procedure is to determine the behavior of antibody activity recovered by elution
from DEAE cellulose columns since under appropriate conditions of fractionation, 7Sy globulins are eluted first, Y1A fractions later, while 1% y globulins are invariably the last fraction to come off the columns.
In spite of the evergrowing complexity of antibodies and their ever-increasing
heterogeneity, a remarkable state of uniformity concerning all of the immune
globulins has become apparent in recent years as a result of structural and
genetic studies performed in a number of laboratories. These have clearly
demonstrated the existence of similar structural units which are under common genetic control for all of the immune globulins, and suggest strongly
that these may be intimately related to the functional properties common
to all these proteins.
It has long been recognized that all four immune globulins are antigenically
THE IMMUNE GLOBULINS
383
related and that they cross-react when tested with appropriate antisera.
With the introduction by Porter of enzymatic technics capable of breaking
7s y globulins into two major structural units, one (slow; A and C)' having
the antibody combining sites and the other (fast; B ) * possessing many of
the other functional properties of the molecule, it became possible to evaluate
these antigenic cross reactions more closely. It was soon shown that, in the
case of human, rabbit, mouse and guinea pig y globulins, the structural similarity resided in the former and that the common antigenic determinant groups
responsible for this cross reaction resided in the part of the molecule capable
of combining with antigen. More recently, Edelman and his collaborators and
Porter have succeeded in obtaining single polypeptide chains from 7s y globulin by precise chemical technics causing the reductive cleavage of several
disulfide bonds. As a result of these elegant studies, it was clearly shown
that the common properties are due to the light ( L ) chains having molecular
weights of about 22,000 which are found only in fragment A and C and are
similar in all the immune globulins. On the other hand, the antigenic specificity which resides in fragment B is associated with the H chains having
molecular weights of about 55,000 which appear to be unique for each of
the immune globulins, Additional studies have demonstrated that the low
molecular weight urine y globulins and Bence Jones proteins are composed
only of L chains, and that the two major antigenic subclasses recently recognized as being common to all the immune globulins are due to differences in
the structures of the L chains.
Another approach which has helped to emphasize the underlying uniformity
of these proteins is the study of the genetic control of the peptide chains making up these proteins. In 1956, y globulin polymorphism was reported simultaneously in the rabbit by Oudin and in man by Grubb. Oudin's discovery resulted from the direct approach of immunizing one rabbit with the y globulin
of another and has since been extended to several other species. The discovery
of y globulin (Gm) groups in man was an outgrowth of work on rheumatoid
factors. The subject has been reviewed in these pages by Grubb and will not
be discussed in detail. Suffice it to say here that synthesis of the immune globulins in man is under the control of at least two separate and distinct genetic
loci known as Gm 1 and Inv (Gm 2 ) . The former is found only in 7s 7
globulin molecules, while Inv activity expresses itself in all four classes of
immune globulins. It seems likely that in due time, when better methods for
detecting these factors will be available, others will be discovered. The
existence of two of these, specific for Y1A and 19s y globulins, seems almost
certain, and they have been tentatively called Gm 3 and Gm 4. Their discovery
at this time is precluded by the assay system currently employed which depends upon the specific inhibition by the globulin under study, of the agglutination by a rheumatoid factor of a D+ cell coated with an incomplete
7s y globulin antibody of the same type. It is obvious that this permits the
-
*These terms refer to fractions of human y globulin: slow ( A and C) correspond to I
and I1 in the rabbit; fast ( B ) is similar to rabbit fragment 111.
384
EDWARD C. FRANKLIN
detection only of factors common to all the immune globulins or those specific
for 7 s y globulin, and that the detection of the postulated Gm 3 and Gm 4
factors specific for the other immune globulins will have to await the development of assay technics utilizing these proteins as specific reagents.
Recent structural studies on normal y globulins in a number of laboratories
have clearly shown that each of the loci regulates the synthesis of a single
type of polypeptide chain. Thus, the Inv locus, common to all the immune
globulins, expresses itself on the A and C fragments and L chains common
to all these proteins, while Gm 1 activity is limited to 7 s y globulin and
consequently expresses itself only on fragment B and the H chains. By analogy,
it seems likely that the postulated Gm 3 and Gm 4 loci will regulate the synthesis of the H chains of the other proteins.
The structural basis of these genetic factors remains obscure. Studies by
fingerprint technics in our laboratory have been hard to interpret. Of interest,
however, in this regard is our observation, subsequently confirmed by Harboe,
that Bence Jones proteins of Group B ( I ) generally are Inv+, while those of
Group A (11) are always Inv negative. This is of particular significance since
Putnam has clearly shown that these differ in their primary structure. Subsequently, we have found this to apply also to several Y1A myeloma proteins,
but in the case of 7s y myeloma proteins and pathologic macroglobulins, the
same correlation was not found and several exceptions were noted. The
reason for this discrepancy remains obscure at the present time and deserves
further study. However, the findings suggest that different L chains are under
separate genetic control and that additional alleles will soon be found.
The availability of reproducible technics of producing smaller biologically
active fragments of y globulin has permitted the precise localization of a
variety of biologic properties associated with these molecules; yet these
studies have also raised a number of very important questions which cannot
be answered at this time. It was shown initially by Porter that antibody activity
resides in fragments I and I1 ( A and C in man) which were later shown to
contain the L chains and also part of the H chains. Subsequent studies showing
differences in the L chains in purified antibodies of different specificities prepared in individual donors further supported a probable role of the L chains
in the development of antibody specificity. However, some doubt was recently
cast on this interpretation by the report of Porter that antibody combining
site resides in the H chain ( A chain),* presumably that part which remains
associated with the L chains after papain cleavage. A partial resolution to
this conflict may be found in the observation of Fran&k who showed that
specificity resides in the H chain, but that antibody activity can be potentiated
by the addition of L chains, thus suggesting that both, possibly as a result
of their relative spatial relationships, may play some role. The fundamental
question dealing with the precise structural basis of antibody specificity remains to be determined. It may well represent a difference either in folding or
in amino acid sequence.
'H chains produced by reductive cleavage in 8M urea by Edelman presumably correspond to A chains of Porter produced in the absence of urea. L chains appear similar to
Porter's B chains.
THE IMMUNE GLOBULINS
385
While the main function of antibody is to combine with antigen, there are
a number of other properties of biologic interest specifically associated with
all the immune globulins, or in several instances only with the 7s y globulin
fraction. For example, all the immune globulins can fix complement, although
not with equal efficiency. 7s y globulin, but not the others, can cross the
placenta in man and the yolk sack splanchnopleur in the rabbit, fix to the
skin of the guinea pig as measured by passive cutaneous anaphylaxis (PCA),
and combine with rheumatoid factor to form a 22s complex. In the case of
rabbit 7s y globulin, each of these properties has been recovered only in
fragment 111. In the case of human 7s y globulin, the ability to fix complement
and combine with rheumatoid factor resides in the analogous fragment B,
but the other two properties appear to be lost during the digestion. Of particular importance in the field of immunopathology is the interaction of antibody and complement and the possible consequences of this interaction in
relation to tissue damage. This interaction appears to occur with each of
the three main immune globulins, but has been best elucidated for the 7s
Y globulins. It is now generally accepted that the site necessary for combining
with complement resides in fragment 111, the fraction which does not have
the antibody combining site. This was first suggested by the observation that
pepsin treatment yields a bivalent precipitating antibody which lacks fragment I11 ( B ) and no longer fixes complement. More direct evidence has been
obtained by showing that, like 7s y globulin, fragment B or 111, but not A-C
or I and 11, can be aggregated by a number of procedures and thus made
to fix complement, although somewhat less effectively than the native protein.
While the data cited up to now suggest that the immune globulins, on the
basis of structural similarities, belong to a relatively simple and unified group
of proteins, evidence of heterogeneity is also increasing. Thus, antibodies of
different avidities to the same hapten can be isolated. Similarly, recent
studies in the rabbit, and especially the guinea pig, have clearly demonstrated
the existence of 7s y globulins with different biologic properties. For example,
in the guinea pig, Benacerraf, Ovary and Block have demonstrated the existence of antibodies of slow electrophoretic mobility which fix complement and
do not give the PCA reaction, and antibodies of the same specificity migrating
more rapidly which do not fix complement, but react in PCA. These differences
may be due to differences in the H chains, but their exact significance remains
to be determined.
In summary, observations in recent years have succeeded in uniting the
complex and biologically important group of antibody proteins into a structurally, functionally and genetically closely related group of proteins, collectively known as the “immune globulins.” On the one hand, this has resulted in many unifying concepts; however, it has also raised many additional
basic questions which may be answered in the not too distant future.
Edward C.Franklin, M.D., Associate Professor of Medicine,
Department o j Medicine, New York Unj,ersity School of
Medicine, and Senior Investiga&x, ATthritis and Rheecmatimn
Foundation, Nezu York, N . Y .
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