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 .