[Advances in Enzymology - and Related Areas of Molecular Biology] Advances in Enzymology and Related...

THE PHYSIOLOGICAL ROLE OF ../-GLOBULIN

By VICTOR A. NAJJAR, Boston, Massachusetts

C O N T E N T S

I. Introduction 11. Molecules and Organs of Defense

111. Mechanism of Antibody-Antigen Interaction A. Conformational Change Following Interaction B. Complementarity of the Reactive Sites C. Isohemagglutinins

A. Binding of Erythrophilic y-Globulin to Erythrocytes in Vitro B. Binding of Erythrophilic 7-Globulin to Erythrocytes in Viuo

A. Reduction of Erythrocyte Viability Following Splenectomy B. Concomitant Reduction of Erythrophilic y-Globulin and Erythrocyte

IV. Association of Specific Cytophilic y-Globulin with Autologous Erythrocytes

V. Physiological Function of Erythrophilic y-Globulin

Viability Following Splenectomy VI. Association of Specific Cytophilic y-Globulin with Autologous Leukocytes

A. Binding of Leukophilic y-Globulin to Leukocytes in Vitro B. Binding of Leukophilic y-Globulin to Leukocytes in Vzuo

A. Stimulation of the Phagocytic Activity of Polymorphonuclear Neutro-

B. Reduction of Leucokinin Activity Following Splenectomy VIII. The Unique Properties of Leucokinin Stimulation of the Phagocytic Activity

of Polymorphonuclear Neutrophils IX. Isolation of the Active Polypeptide Fragment of Leucokinin, Thr-Lys-Pro-

Arg (Tuftsin) X. Chemical Properties of the Tetrapeptide in the Native Leucokinin Molecule

A . Possibility of a Free Carboxyl-Terminal Arginine B. Possibility of the Existence of an Ester Linkage at the Amino-Terminal

XI. Increased Rate of Migration and Prolongation of the Viability of Polymor-

VII. Physiological Function of Leukophilic y-Globulin

phils

Threonine

130 130 132 132 133 137 138 138 145 147 147

147 150 150 152 153

154 155

156

158 161 162

163

phonuclear Leukocytes by Tuftsin 164 164

XIII. Chemiral Synthesis of Tuftsin and Its Analogs 168 XIV. Human Tuftsin-Deficiency Syndrome 169

Function 170

XII. The Tetrapeptide Tuftsin as a Biological Entity with a Specific Function

XV. Human Tuftsin Deficiency Following Removal or Curtailment of Splenic

129

Advances in Enzymology and Related Areas ofMolecular Biology, Volume 41 Edited by Alton MeisteI

Cowright © 1974 bv John Wilev & Sons. Inc.

130 VICTOR A. NAJJAR

XVI. Association of Cytophilic y-Globulin with Autologous Blood Thrombocytes A. Binding of Thrombophilic y-Globulin to Blood Platelets tn Vitro B. Binding of Thrombophilic y-Globulin to Blood Platelets in Viuo

170 171 171 172 173

XIX. Summary 173 References 175

XVII. Association of Cytophilic y-Globulin with Arterial Endothelium XVIII. Effects of Normal Mouse Serum on Neonatally Thymectomized Mice

I. Introduction

Since the discovery of the major components of serum proteins, a-, p-, y-globulin and albumin, numerous lesser components have been found, and in some instances their function has been clearly delineated. However, of the major serum components, the y-globulin fraction was the first to be assigned a definite biological role. It was the only serum protein that possessed antibody activity (1). So important is this antibody function to the defensive mechanism of the animal that it overshadowed any other consideration. In fact, antibody activity has been found to be astonishingly versatile in combating all varieties of bacterial and viral agents and their toxins as well as a multitude of foreign substances that the investigator can conjure or concoct and introduce into the animal. Thus the receptive mind acquiesced and concurred, with satisfying comfort, that one function, particularly so vital a function, suffices for one molecule, the y-globulin molecule.

In contrast to other protein molecules, such as enzymes, which the antibody molecule resembles in its high degree of specificity, the y-globulin molecule has an incalculable variety of primary structure with a similar overall molecular design. It would not be surprising, therefore, to find a variety of possible functional patterns attributable to y-globulin.

11. Molecules and Organs of Defense

The question as to the manner by which the y-globulin molecule was conceived to perform one single and simple reaction with a defensive function was never raised until recently. Can nature generate a molecule with the sole purpose of combining with and neutralizing an intruder molecule? Can nature anticipate events to come and create the necessary corrective measures? Clearly the answer is in the negative.

Molecular reactions with an obvious defensive posture (not function) are known. Various acetylation reactions neutralize toxic amines much as the antibody neutralizes diphtheria and tetanus toxins. Similarly

PHYSIOLOGICAL ROLE OF y-GLOBULIN 131

methylation, sulfation, glucuronidation, hydroxylation, and phos- phorylation, to name a few, all neutralize possible toxic effects of foreign molecules that gain admittance into the animal body. However, these reactions, though admittedly helpful in detoxification, possess primarily well-defined and well-studied biological roles of vital importance to the metabolism of various compounds involving synthetic, catabolic, and ex- cretory pathways.

One can go further to a higher level of organization. A number of organs are, in variable measure, defensive in character: the horns of the bovine, the antlers of the cervine, the hoofs of the equine, the teeth of the canine, the claws of the feline, the tusks of the porcine, the horn of the rhinoceros, and the high voltage battery of the electric eel. Again these organs are either adaptations of an existing physiologically functioning entity or organs that perform both biological and defensive functions.

It is possible to ascertain, from these simple but well-founded exam- ples, that nature may allow the modification of a part of an existing biologically useful organ and in so doing generates a structure that for the most part can be utilized for defense against agents external to the animal. Alternatively nature may also allow a dual function for the same organ, physiological and defensive. In the latter case the defensive aspect is of minor and temporary character. In all cases, however, the defensive deployment of an organ is realized in response to selective evolutionary pressures. Animals that used their teeth only to chew with, their hoofs and claws to cushion their walk, their hair for warmth, and their skeleton only for support could not stand their ground in the struggle for survival.

How does this all apply to the problem of y-globulin as the possessor of the sole function-antibody activity? It does in the sense that it is more intellectually useful and perhaps more in keepng with biological principles to assume that the immunological function of y-globulin arose at some time during the evolutionary process as a valuable outgrowth of an ongoing and a necessary physiological function. Clearly these offer attractive arguments in favor of the notion that y-globulin might indeed still play a physiological role and that antibody formation arose from a modification of this role that made the new function well suited to combating intruding molecules from external or perchance internal sources. This thought seemed to elude many of us in this field. This argument has been of necessity lengthy and presses the issue squarely. Its object is to condition the reader no less than to strengthen the conviction of the


author. The highly useful function of antibody dominated all thought on the subject of y-globulin. The insight into the possible physiological role of y-globulin did not arise from this type of argument. Had this idea emanated from such teleological deductions, the initiation of a successful experimental approach to the role of y-globulin would have been quite difficult if not impossible. If not by such logical deductions, how then did the notion arise that y-globulin might have a physiological role? This has already been documented. However, it is imperative to the under- standing of the role of y-globulin that a short narrative be presented of the thinking and results that led to this notion whose ramifications were altogether unexpected. Some very important and interesting results appeared during a study of the antibody-antigen interaction, as a model of protein-protein interaction. The results repeatedly asserted themselves and could not be explained away.

111. Mechanism of Antibody-Antigen Interaction

A. CONFORMATIONAL CHANGE FOLLOWING INTERACTION

Several years ago (1955), we began a study of the antibody-antigen interaction as a typical and attractive model for an investigation into the consequences of macromolecular interaction (2). At the time, crystalline yeast alcohol dehydrogenase was used as the antigen. The use of an enzyme as antigen offered an obviously singular advantage for such a study. The results were unexpected and did not fit into the pattern prevailing at the time. An adequate explanation of the results could be made on the assumption that both antibody and antigen could undergo conformational change on interaction. Consequently we proposed a theory incorporating this suggestion (2-5). As all theories do, this one included certain formulations and predictions. In olden times, theories were regarded as prophecies, and any prophecy that ran counter to prevailing beliefs brought serious indictment to the prophet, who was swiftly af- fixed to the cross or mercifully stoned to death. The early 1950s were the early days of protein chemistry, and such a change in the conformation of the protein molecule was neither expected nor accepted. However, this has since been adequately documented immunochemically in several laboratories (5) and by other approaches: polarimetry (6), depolarization of fluorescence (7), electron microscopy (8), and the antibody-enzyme- substrate interaction (5,9-13).


B. COMPLEMENTARITY OF THE REACTIVE SITES

The question was further raised as to the possible cause of the conformational change and as to the type of reactive surfaces between antibody and antigen that would be conducive to such profound alteration of the secondary and tertiary structures of the two interacting molecules. It was reasoned that a complementary lock-and-key fit would not inflict any serious strain on the molecules other than a minimal adjustment of participating amino acid residues within the range of a few angstroms. In this manner the interacting molecules would be relatively easily dissociable, falling within the dissociation range obtained between haptens and antibody surfaces; witness the case of dissociaton between a lock and its complementary key. However, if the interacting surfaces were short of complete complementarity (subcomplementary), then the forces of at- traction between such slightly discordant sites, as determined primarily by ionic, hydrophobic, and hydrogen bonds, would compel major adjust- ments in the orientation of the apposed surfaces, which in turn would engender secondary interactions and subsequent unfolding of random areas on the molecules. This type of interaction is reminiscent of that between a warped key and a defective lock. This type of antibody-antigen combination is not readily dissociable largely because of the occurrence of secondary interactions and the consistent increase in the entropy of the system.

It must be emphasized here that the lymphoid system cannot make antibodies with the right degree of subcomplementarity. It can only make complementary antibodies to the antigenic surface that is presented to the antibody-forming cell. In this connection, it is well known that antigens break down in the body, and various fragments with varying geometry of a particular haptenic site are available for antibody induction. Since a given antigenic area present on fragments of various sizes differs in conformation from that on the undegraded antigen, it follows that most of the antibodies generated would indeed be subcomplementary to that haptenic area on the intact antigen. The intact native antigen is invariably used for most in uitro studies of the antibody-antigen interaction. Such subcomplementary antibodies would react in the fa- miliar manner, form precipitins and bind complement. However, it is quite conceivable that some of the antibodies would be formed against the particular haptenic area of the undegraded antigen if the antigen ar- rives intact at the site of antibody synthesis. Alternately, some fragments

134 VICTOR A. NAJJAK

of the antigen may well possess an unaltered haptenic site. In both instances complementary antibodies would be formed that by definition would not be strained nor alter conformation nor bind complement on interaction with antigen. Experiments to explore these possibilities showed this to be the case.

7. Immunization with Covalently Linked Haptens

The experimental approach to this problem entailed immunization of rabbits with antigens of inherently varying degrees of antigenicity, each covalently bonded to arsanilic acid through the diazo reaction (7,13). Thus p-azobenzene arsonate (paba) served as the common hapten for the various antigens-insulin, gelatin, casein, bovine-serum albumin, and horse globulin.

Because of the differing chemical nature of the antigens, the haptenic group was expected to assume various orientations toward its adjoining amino acid residues, depending on the protein. The antisera, generated by such immunization of individual animals, were first absorbed out by the underivatized protein with which the animal was immunized. In this manner only antibodies to the hapten paba remained. If indeed both types of antibodies, complementary (cryptic) and subcomplementary (overt) antibodies, are formed, it should be possible to demonstrate their existence quite readily. For this purpose antisera derived from one serum sample of a single animal were first treated successively with single amounts of the derivatized paba-immunizing antigen. This was intended to remove all overt antibodies. This is one of the usual procedures com- monly used to assess the antibody content of an antiserum. Cryptic or covert subcomplementary antibodies, if any, remain in the antiserum sample since they are presumed to be readily dissociable. These can be brought down if they are allowed to react with the paba groups that have a different orientation from that existing on the immunizing antigen. This is done by reacting the antiserum, which has been denuded of antibodies to the immunizing paba-antigen, with a paba-protein to which the animal had never been exposed. Indeed, in every case, antibodies to paba were demonstrable in relatively large amounts on several hapten carrier proteins after exhaustive absorption by each separately.

A typical example is shown in Figure 1 (7). Here antiserum to paba- gelatin was exhaustively absorbed by paba-gelatin to remove all the antibodies to this immunizing antigen. Subsequently the absorbed serum was successively and exhaustively absorbed out with paba-casein, paba-

PHYSIOLOGICAL ROLE OF ?-GLOBULIN 135

albumin, paba-insulin, and paba-globulin in varying orders of absorption. In each case, after all antigelatin antibodies were removed, there remained antibodies that reacted with the other paba-proteins. This type of result was also obtained in antisera where the immunizing antigen was paba-insulin and paba-albumin (7,9,14,15). The classical explanation that all antibody-antigen reactions are a consequence of a complementary lock-and-key fit cannot be sustained by these results. Were this true, one would have to accept the notion that the animal can produce antibodies with a complementary fit for any orientation paba might possess and for an adjoining amino acid residue to which the antibody-forming system of the animal had never been exposed. This clearly cannot be the case. These cryptic antibodies become typically overt, precipitate, and bind complement, when they react with an unfamiliar paba-carrier protein. They can only be considered subcomplementary by

ANTI- GELATIN . D a b SERUM e55.14

0.41

ABSORPTIONS - DAYS

Fig. 1. Successive 24-hr absorptions of the immune sera, anti+-azobenzene arsonate (paba) conjugates of gelatin. The first absorption was done with the immunizing antigen conjugate, followed by a variable order of absorptions with the hapten carrier proteins as shown. Each graph represents four such variations according to the key shown for each antiserum. Where no values were recorded, no precipitate was formed. It is clear that, after exhaustive absorption with the particular immunizing antigen, a good deal of antibody remained in the supernatant that precipitated with one or another hapten carrier protein

(7).


default since they would otherwise be complementary, which is logically as unacceptable as it is inconceivable. Thus by the same token the overt antibodies to paba-gelatin must therefore be subcomplementary to the immunizing antigen paba-gelatin. It should be emphasized that the immunizing antigens do indeed show a strong association to the antihap- tenic site of the antibody since in excess amounts they are capable of in- hibiting the precipitating action of the overt subcomplementary antibodies in the manner that excess hapten does (7,14).

These experiments were devised and executed to evaluate the hypothesis outlined here, and so they did. However novel the idea was, it was gratifying to find that similar results had already appeared in a limited manner in early immunological literature and had long been ignored (16,17). These were deservingly detailed in an earlier review (5). Antisera to gelatin-azobenzene arsonate failed to react with the antigen, but reacted with ovalbumin-azobenzene arsonate (5,16). Antibodies generated by immunizing rabbits with an 0-P-glucosidotyrosyl- and 0- 0-glucosido-N-carbobenzyloxytyrosyl-protein, such as gelatin and insulin, did not form precipitins to the immunizing antigen, but did so with glucosido derivatives of horse globulins (17). Many similar reports have been reviewed previously (5). Chief among these are the results of immunization with synthetic polypeptides (18,19). One such antiserum to a copolymer of glutamic acid and lysine lost only 46% of its antibody content by absorption with the copolymer. The remainder reacted with other copolymers containing tyrosine or phenylalanine (1 9).

2. Immunization with Erythrocytes of Various Blood Groups The indications so far favor the concept that a reaction with the usual

antibody in a subcomplementary manner does not always require identical hapten modified only by its orientation to different amino acid residues in its determinant environment. The same can obtain if the hapten itself is chemically modified to a minor degree. This type of hapten is best exemplified by human blood-group determinants. Type A and type B determinants are quite similar. They differ only in one ace- tamido group in the outermost galactose residue (20,21).

If this is true, then immunization with A-cells should produce the usual subcomplementary antibodies, which would be anti-A. It might also produce complementary ones, which would not agglutinate A-cells. They would necessarily be less than perfect fits (subcomplementary) to the B-determinant and consequently would agglutinate B-cells.

PHYSIOLOGICAL ROLE OF -(-GLOBULIN 137

Such results were in fact reported in a limited manner over 50 years ago (22) and later (23,24). These, however, were relegated to the realm of the odd and unusual as befits the times. Without prior knowledge of this, these important results were reproduced in an extensive manner. Rabbits and chickens were immunized with human cell types A, B, 0, and AB. After absorption of the serum with the immunizing cell obtained from the same original donor, the serum nevertheless retained antibodies to other cell types. Antisera generated by immunization with A-cells had anti-B agglutinins. Those that were induced by B-cells had anti-A agglutinins, and those induced by 0-cells developed both anti-A and anti-B agglutinins. However, animals immunized with cells of the AB type after absorption with the immunizing AB-cells as above, unlike the results obtained with A-, B-, and 0-cells, and indeed true to form, did not agglutinate any of the other cell types (9,24).

C. ISOHEMAGGLUTININS

The isohemagglutinins are naturally occurring antibodies that form the basis of blood typing of the individual’s red blood cells. Individuals who are type A have type A cells and isohemagglutinins that react with type B cells of another individual. Similarly type B individuals have B cells and anti-A isohemagglutinins. Both interactions typify antibody-antigen interactions and are identical in every way with agglutinins induced by immunization.

In the light of the preceding discussion, the behavior of the isohemagglutinins parallels closely the results obtained by immunization with paba-antigen and blood-group determinants.

A significant part of antibodies induced by type A cells react with the reciprocal B-cell and are presumed to be patterned in a complementary manner after the immunizing type A cell. The same holds for some of the antibodies that were induced by B-cells and agglutinate type A cells. What then, one may ask, are the anti-B isohemagglutinins of human type A blood complementary to? The answer is that they are patterned in a complementary manner after type A cells, the very cells that circu- late in type A individuals. The same type of reasoning dictates that the anti-A isohemagglutinins of type B individuals are complementary to the B-cells of his own blood. Thus the natural antibodies, the isohemagglutinins of individuals of the various blood groups, are synthesized with a complementary fit to specific areas of the autologous blood-cell membrane.


It is proper then to inquire whether the isohemagglutinins and other nonantibody y-globulins that might also be complementary to the autologous circulating erythrocyte can manifest any binding capacity to specific cell receptor groups. The answer to this question holds the clue to the proposed induction and physiological purpose of the isohemagglu- tinin in particular and nonantibody y-globulin in general.

There have been several theories regarding the origin of the isohemag- glutinin (25-29); they have been reviewed before (5) and will not be detailed here. The genetic theory proposes that the isohemagglutinins are dictated by linked gene pairs representing the determinant and its antibody (25). Suffice it to say that the other idea (26) and its subsequent variations (27,28) required that the individual be regularly immunized with outside antigens that produce antibodies to all types of cell determinants. These may number in the dozens. Those antibodies that react with the individual determinants either are not induced or crossreact with the cell determinants to the detriment of the absorbing cell. The remaining antibodies become the so-called isohemagglutinins, which react with the determinants not represented in the host. Although this idea has some measure of plausibility, it would necessarily mean that through this type of immunization there may well follow cell destruction through the induced crossreacting antibodies. This would not be limited to erythrocytes since the specific determinants are distributed throughout the various organ cells. Were this to take place, no tissue could be shielded from this crossreaction.

IV. Association of Specific Cytophilic y-Globulin with Autologous Erythrocytes

We have so far assumed that the isohemagglutinins are synthesized with their reactive sites designed to bind in a complementary manner to the cell determinants of the host, yet react in the manner of the antibody with the determinants of the related cell of another host. O n this basis the isohemagglutinins must then be capable of association with the cells of the host.

A. BINDING OF ERYTHROPHILIC ?-GLOBULIN TO ERYTHROCYTES

IN VITRO

In an aqueous milieu like the intracellular fluid, the dominant type of molecular interraction is expected to be electrostatic in nature. Con-

Fig. 2. Reduction of y-globulin after adsorption on autologous naked erythrocytes. This is apparent both in the pattern on paper electrophoresis and the corresponding tracings by the Analytrol. None of the other major components of serum appears to have been diminished. Human erythrocytes (O+) (15 ml) were washed five times each with three volumes of 0.15 M NaCl in 5 + lo-$ M sodium phosphate, p H 7.4. This was followed by washing five times, each with three volumes of the isotonic buffered sucrose solution (see text). Autologous serum was dialyzed against the sucrose solution for 2 hr. The dialyzed serum (0.5 ml) in 5 ml of sucrose solution was then mixed with 10 ml of the packed cells at 4-6OC for 10 min. The supernatant (0.06 and 0.08 ml, before and after adsorption, respectively) was applied to the paper. The difference in the volume used takes into account the dilution resulting from the added erythrocytes. Coated erythrocytes are washed with the sucrose medium and therefore retain their membrane boundy-globulin coat. Naked erythrocytes are washed with 0.1 5 M NaCI, which elutes bound y-globulin. From Fidalgo et al.

(31).

139


sequently such interactions become most pronounced in a low-ionic- strength environment that minimizes ionic competition with the electrostatic forces between macromolecules. For binding studies an isotonic low-ionic-strength medium was devised; it consisted of 0.27 Msucrose, 5 x lo-' M phosphate buffer, and 1 x M CaC12, p H 7.4. T o remove any y-globulin bound to erythrocytes, 0.15 M NaCl or 0.1 M phosphate buffer, pH 7.4, was used.

Human or dog erythrocytes were thoroughly washed with isotonic electrolyte solution and mixed with a suitable concentration of autologous serum in isotonic sucrose solution for a few minutes. The serum sample before and after erythrocyte addition differed only in one respect. The serum sample taken after erythrocyte addition showed a dramatic reduction in y-globulin. No other serum component was affected (Fig. 2) (7,29-31). The supernatant serum became devoid of all hemagglutinin activity since it was all bound to the erythrocytes. This activity could be recovered almost completely by elution from the sedimented erythrocytes with 0.15 M NaCl (7,29). These results were subsequently confirmed using exactly the same technique and showing in addition that some complement activity was also bound to the erythrocytes in the sucrose medium (32). Further support of the cytophilic activity of y-globulin came from studies of experimental encephalitis. It was found that y-globulin was bound to the cell membranes of the human central nervous system. It was noted that fluorescein-labeled antihuman y-globulin stained normal brain sections. This was used as the control in a study of possible immunological damage to brain tissue in encephalitis. In particular, myelin sheaths and glial cells were found to stain with the specific antibody. The affinity of y-globulin for brain tissue appears to be specific to 7s y-globulin (33).

1. Characteristics of Erythr@hilic y-Globulin

a. Immunochemical Characteristics. It was possible to show, by means of immunochemical studies, that erythrophilic y-globulin consisted predominantly of y-G-globulin and a minor component of 7-M-globulin. However, not all y-G-globulin could bind to the red cell. This suggested a good measure of specificity. Another immunochernical method was used to pinpoint the binding of erythrophilic y-globulin to the surface of the cell, using fluorescein-labeled antibody to human y- globulin. ThL cells were coated with autologous y-globulin in the sucrose medium, and the labeled antiserum was added. As expected, the cells ag-

PHYSIOLOGICAL ROLE OF r-GLOBULIN 141

glutinated and showed peripheral fluorescence (31). This is shown in Figure 3.

b. Chromatographic Characteristics. Further characterization was needed; consequently fractionation of y-globulin on phosphocellulose (PC) columns was undertaken using batch elution with 0.05 M acetate buffer at increasing pH values (4.8-5.4) and salt concentration (0.15-0.2N NaCl). Serum y-globulin exhibited four fractions, PC I through PC IV (34). The first fraction contained yG- and yA-globulins,

A

B

Fig. 3. ( A ) Agglutination of coated red cells carried out in sucrose solution with anti- human y-globulin as compared 10 the lack of agglutination with the naked cells under identical conditions. (B) The presence of fluorescence with the coated erythrocytes and the virtual absence with the naked cells. Fluorescein-labeled anti-human y-globulin was used. The reaction was also carried out in sucrose solution. For details, see Figure 2. From Fidalgo et al. (31).

142

2 3 h m

r

C .- I-’

C

UI

.- I-’

VICTOR A. NAJJAR

Before aosorption on H H C After adsorption on R B C

RBC Eluate

Fig. 4. Diminution of fractions I I I and IV after adsorbing whole y-globulin with naked autologous erythrocytes. y-Globulin was dialyzed against sucrose solution overnight. Packed erythrocytes (25 ml) were mixed with 100 mg of y-globulin in 50 ml of sucrose solution. After incubation at 0°C for 10 min, the sample was centrifuged and the supernatant 7-globulin was prepared by precipitation of 0.33 ammonium sulfate saturation followed by dialysis against 0.05 M sodium acetate buffer, pH 4.8. This was then chromatographed. A 15-mg quantity was chromatographed on a 1.5 + 12-cm phosphocellulose column (34). The cells used for adsorption were then washed three times with three volumes each of sucrose solution and finally eluted two times each with three volumes of 0.15 M NaCI. The combined eluates were evaporated in thin cellophane bags in the cold room, and the 7-globulin precipitated a t 0.33 ammonium sulfate saturation. The y-globulin was prepared for chromatography as hefore, and 15 mg applied to the column. For comparison, unadsorbed whole y-globulin is included in the figure (upper left). Other details as in Figure 2. From Fidalgo et al. (31).

the second y G- and yM-globulins. The third and fourth fractions showed only yG. This fractionation procedure did in fact separate yG into four fractions, which would help in the characterization of the erythrophilic component. The latter was obtained by washing freshly isolated cells with the sucrose solution followed by elution with 0.1 M phosphate buffer. It was apparent that the major components of the erythrophilic y-globulin were fractions 111 and IV. A very minor amount of fraction I1 containing yM was obtained (30,31,35).

A more informative demonstration is shown in Figure 4. Here prepared y-globulin from a human subject was fractionated before and after absorption with the autologous cells previously washed with 0.15 M


NaCI. The reaction was carried out in the buffered sucrose solution. It is clear that the only two fractions that showed a definite reduction were fractions I11 and IV. Furthermore, when the sedimented erythrocytes were eluted with the isotonic salt solution, the two major fractions 111 and IV were recovered with a minor fraction I1 (30,31).

It was clear from the beginning that only a portion of serum y-globulin fractions 111 and IV could bind to the erythrocyte. These have a remarkable degree of cytophilic specificity. Both fractions could be eluted and made to rebind completely onto the same cell (29). These same fractions do not bind any other cellular component of the blood (29-31).

c. Chemical Characteristics. Amino acid analysis of erythrophilic fractions 111 and IV showed marked differences in certain amino acids in

TABLE I

Amino Acid Composition of Erythrophilic Fractions I11 and IV Compared with the Corresponding Fractions of y-Globulin'

~

Amino Erythrophilic Serum Erythrophilic Serum

acid Ill 111 IV IV y-globulin y-globulin y-globulin y-globulin

~~ ~

LYS 88 71 77 72 His 56 23 45 24 Arg 38 32 35 31 Asx 92 84 85 87 Ser 111 128 96 129 Pro 83 118 67 92 Ala 66 56 56 53 CYS 28 26 36 28 Met 4 4 12 6 Ile 23 23 32 23

*The number of selected amino acid residues per mole (150,OOO daltons) of y-

globulin that show a clear difference between erythrophilic fractions I l l and IV and the corresponding fractions in whole pooled y-globulin (Cohn 11). A 1.0-mg quantity of each was hydrolyzed and assayed in a Spinco-Beckrnan 121 automatic amino acid analyzer (35). Among several outstanding differences the most notable is that erythrophilic y-globulin IV has double the number of methionine residues of the corresponding fraction of whole y-globulin.

144 VICTOR A NAJJAR

comparison with the corresponding fractions of serum y-globulin. Table I (35) contrasts the values of selected amino acid residues of the erythrophilic fraction with the corresponding fraction obtained from whole-serum y-globulin (Cohn 11). The values are given as residues per mole of 150,000 daltons. There is obviously a marked difference in most of the residues listed. Other residues are either the same or less dissimilar.

2. The Effect Erythrophilic y-Globulin on Hypotonic Lysis of Red Cells

Erythrocytes or prepared erythrocyte ghosts (36) coated with erythrophilic y-globulin showed considerably increased resistance to shearing forces. Coated intact cells showed a definite retardation of the movement of intracellular electrolytes toward an extracellular hypotonic environment. Consequently coated cells lysed more readily and at a lesser degree of hypotonicity of the extracellular fluid than did the un- coated cells. The latter were capable of more rapid passage of ions down the gradient and did not swell and lyse as early as the coated cells (7). The movement of [ ‘“c] aminoisobutyric acid in the opposite direction, from the external to the intracellular fluid of the cell, was retarded by the presence of a y-globulin coat as compared to its absence.

3. The Effect of Erythrophilic y-Globulin on the Shape of the Erythrocyte

It has been known for some time that erythrocytes washed with isotonic saline solution became crenated, star-shaped, or burr cells (37, 38). The addition of autologous serum causes an immediate reversal to the normal biconcave-disk shape. Studies of this reversal with the various phosphocellulose fractions and albumin showed that the erythrophilic fractions 111 and IV were most potent in causing the reversal. Only a few micrograms per milliliter suffice for a v/v 4% cell suspension. This is shown in Figure 5. The other fractions (i.e., fractions I , 11, and albumin) were much less effective (39). It will be shown later that, when the blood levels of the erythrophilic fractions are reduced in vzvo, this reduction is followed in time by a striking appearance of a large number of burr-shaped or crenated cells. Of the two fragments of erythrophilic y- globulin, the Fc-fragment was shown not to bind the erythrocyte and did not affect its shape. However, the Fab-fragment binds strongly and is as


effective as the parent molecule in causing a correction of the burr shape to the normal biconcave disk.

B. BINDING OF ERYTHROPHILIC y-GLOBULIN TO ERYTHROCYTES

IN VIVO

It was thought possible, but not probable, that the use of the unphysiological sucrose solution might affect the cell membrane in such a manner as to cause nonspecific binding of certain types of y-globulin. Consequently an experiment was designed to verify the existence of y- globulin bound on the erythrocyte as it exists in freshly drawn blood with citrate or heparin as anti-coagulant without any resort to the use of isotonic sucrose medium.

Essentially, the blood sample was gently centrifuged at 600s for 10 min, the plasma removed, and the y-globulin of the supernatant plasma fractionated on a phosphocellulose column as before (34). The eryth-

Fig. 5. Top: the star shape of the naked erythrocyte; bottom: the same cell preparation with added erythrophilic y-globulin. The identical change can be effected by the individual erythrophilic fractions 11, 111, or IV. Of the various fragments of erythrophilic y-globulin, only Fab-A and F(ab),, at concentrations equimolar with that of the parent molecule, also casued an identical reversal to the normal biconcavedisk shape. Naked erythrocytes are washed with 0.15 M NaCl and consequently lose their bound y-globulin coat. From La- hiri et al. (39).


c 0 Serum Y- globulin - - ._

W

y - g l o b u l i n from RBC woshings

Fig. 6. The considerable augmentation of fractions 111 and IV in NaCl washings of erythrocytes packed in situ. There is also some relative increase in fraction 11. This indi- cates that these fractions represent y-globulin bound on erythrocytes under the conditions existing in blood. (100 ml) containing citrate as anticoagulant was centrifuged, and the clear plasma and leukocyte layer were removed. The sedimented cells were then resuspended in their plasma and recentrifuged. This was repeated three more times to minimize leukocyte contamination. Finally, the supernatant plasma was separated, and the cells were washed twice with 2.5 volumes of 0.15 M NaCl each. The combined eluates (200 ml) were concentrated to a volume of 15 ml in cellophane bags by evaporation in the cold room. Solid ammonium sulfate was added to 0.33 saturation and allowed to stand for 8 hr in the cold. The precipitate was dissolved in 2 ml and dialyzed against 0.05 M sodium acetate buffer (pH 4.8) for 6 hr. It was then chromatographed on phosphocellulose columns. For details, see text. From Fidalgo et al. (31).

rocyte sediment was suspended in 0.15 M NaCl to recover the contaminating plasma, which would show the same fractionation pattern as the supernatant plasma. However, if the erythrocytes were indeed coated with fractions I11 and IV, as found by in uitro studies, then the relative quantities of fractions 111 and IV, in relation to those of fractions I and 11, would be altered. There would be a relative increase in erythrophilic fractions 111 and IV. Such was indeed the case, as seen in Figure 6. Fractions I11 and IV, usually representing 15-2070 of the combined fractions, are now over 50%. It is clear therefore that fresh blood as obtained with no exposure to sucrose or any other unphysiological manipulation contains erythrocytes with a typically erythrophilic 7-globulin coat.


The degree of association between erythrophilic y-globulin, as any association between two interacting moieties, depends on the concentration of the two reactants. The amount of erythrophilic y-globulin bound to erythrocytes is about 250 mg/l of packed cells. The concentration of erythrophilic y-globulin in plasma is about 3000 mg/l. Thus, despite the presence of 150 m M ionic concentration in plasma, the concentration of erythrophilic y-globulin along with a relatively tight binding is sufficient for near maximum association. This also explains why for complete elution of the bound y-globulin it was necessary to repeat the process two to three times with 10 volumes of isotonic salt solution (29).

V. Physiological Function of Erythrophilic 7-Globulin

A. REDUCTION OF ERYTHROCYTE VIABILITY FOLLOWING

SPLENECTOMY

In the preceding section it was shown that the binding in vitro of erythrophilic y-globulin to the erythrocyte membrane had a salutary effect on the cell. One can assume that such a beneficial result may hold in uiuo. It has been known for some time that removal of the spleen in some animals resulted in a significant, though temporary, reduction in the red- cell count, while others showed no such response (40). Furthermore, it was shown that the half-life of the red cell was diminished due to early lysis, as evinced by increased excretion of bile products (41). It has also been suggested that the rate of red-cell synthesis is reduced (42). However, others found no untoward effect on the mature red cell (42, 43). In all stances of splenectomy, it is well known that the animals, whether mice or dogs, are subject to severe Bartonella infection, much as human are to pneumococcus and meningococcus infections. This will be dealt with in detail in a subsequent section. It is possible that undetected Bartonella infection may have affected the results of some of the workers in this field. It is also possible that the discrepancy in the results stem- med from the variation in the interval between such studies and the time of splenectomy. This point is of singular importance, as we shall see.

B. CONCOMITANT REDUCTION OF ERYTHROPHILIC y-GLOBULIN

AND ERYTHROCYTE VIABILITY FOLLOWING SPLENECTOMY

In view of the in vitro effects of the binding of erythrophilic y-globulin to the erythrocytes, it seemed profitable to inquire whether splenectomy

148 VICTOK A NA,J,JAR

Dog # 5 BEFORE SPLENECTOMY

55 DAYS AFTER SPLENECTOMY

Fig. 7. Fractionation pattern of dog (MII) serum y-globulin before and after splenectomy, showing marked reduction of fractions 111 and IV and no detectable diminution of fractions I and 11. In each casc 2(1 rng of serum y-globulin was chromatographed on phosphocellulose columns (10 + 1.5 cm) and the pattern of elution was recorded. All steps in the preparation, dialysis, and chromatography were done under identical conditions. Details in text. From Fidalgo ct al. (30).

might possibly alter the quantity of these fractions. In that event, the effect on the permeability, tensile strensth, and shape of the cell might explain the decrease in the viability of the cell. For these experiments several provisions were deemed essential:

1. Infection should be prcvcntccl, whenever possible, by antibiotic preventive therapy.

2. Studies on the red cell should be done at a time when the regular turnover of y-globulin ensures that little presplenectomy y-globulin remains.

3. Assessment of the relative ratios of the four fractions should be done concomitantly with blood studies.

4. Sham-operated animals would servc as controls. 5. A curative effect of administered rrythrophilic fractions should be

demonstrable in animals that show a rcduvtion in these fractions along with a reduction in the survival of the erythrocyte.

PHYSIOLOGICAL ROLE OF -/-GLOBULIN 149

A total of 15 splenectomized dogs were used (30). In every instance infection was prevented. All but one of the 13 splenectomized nontreated dogs showed marked reduction in fraction 111, amounting to an average of about 50% of the level found in the presplenectomy period. There was some reduction in fraction IV in half the animals. In a few of those a marked reduction of both fractions occurred simultaneously. One of those is shown in Figure 7 (30) and occurred 45 days after splenectomy, at which time about 3% of the presplenectomy y-globulin was still present in the animal. The half-life of y-globulin in the dog was found to be approximately 9 days (44). Sham-operated animals did not show either a reduction in any of the erythrophilic fractions or the half-life of the red cells. Two animals that were splenectomized were treated by

TABLE I1

The Parallel Reduction of Erythrophilic r-Glohulin Fraction 111 and the Half-Life of the Erythrocyte in Splenectomized Dogs"

Fraction HIb Erythrocyte half-life"

Quantity of Reduction Reduction y-globulin after Days after

Type of before operation operation before operation operation (mg/100 mg) (70) operation (76)

Shamd Shamd Splenectomy Splenectom y Splenectomy Splenectomy Splenectomy Splenectomy Treated Treated

15.2 13 .O 14.7 11.2 11 .2 11.6 13.7 14.0 10.6 13.5

3 0

65 45 38 40 41 21 20 19

23 22 21 23 21 23 22 23 23 23

5 0

34 39 48 44 50 26 13 13

* From Fidalgo et al. (30).

of the y-globulin fractions (34).

operation.

cept that their spleen was not removed.

Serum was withdrawn before operation and 45 days after operation for the assay

c The half-life of 51Cr-tagged cells was determined before and 45-50 days after

d Sham-operated controls were treated exactly like the splenectomized animals ex-

150 VICTOR A. NAJ,JAR

weekly intramuscular injections of 250 mg of fractions 111 and IV com- mencing 8 days after splenectomy. These animals showed minimal reduction in the fractions and cell half-life. A sample of these results is shown in Table I1 (30).

It was possible to confirm certain reports (40,41) that dogs do show a low red-cell count, particularly if splenectomy is performed on poorly fed newly acquired animals (35).

VI. Association of Specific Cytophilic y-Globulin w i t h Autologous Leukocytes

Preparations of blood leukocytes containing a high proportion of polymorphonuclear (PMN) cells were made from freshly drawn blood by sedimentation in dextran (45). Alternatively, highly purified PMN neutrophils were prepared by chromatography on glass-bead columns (46).

Both preparations showed the same binding characteristics to specific y-globulin under the conditions used. Consequently most of the work was done on purified buffy coats. The phagocytosis reaction, which served as a measure of the biological effects of leukophilic y-globulin, was carried out on unpurified buffy coat freshly prepared from the same host. This is a long-established method of measuring phagocytosis (47-49). Results obtained in the dog (47) and in man (48) were identical in all details. The fractionation of 7-globulin was carried out as described in Section IV.

A. BINDING OF LEUKOPHILIC ?-GLOBULIN TO LEUKOCYTES

IN VITRO

White blood cells were repeatedly purified (45) while in constant contact with plasma to the point where a maximum of 3% contamination with erythrocytes was obtained. These cells were washed with low-ionic- strength isotonic buffered sucrose solution then eluted with 0.15 M NaCl. The eluates, after dialysis in 0.05 M acetate buffer, p H 4.8, were chromatographed on phosphocellulose columns (34). One predominant fraction, IV, was obtained along with a small amount of fraction I. Simi- larly, when leukocytes were stripped of their y-globulin coat and binding determined in an appropriate solution of autologous 7-globulin in isotonic sucrose solution (47,48), the fractionation pattern of the supernatant y-globulin solution differed from the pattern before absorption by showing a relative diminution of fraction IV. Again, when these absorbing cells were eluted with the salt solution, a small fraction I


and a much larger fraction IV were the only fractions present in more than trace amounts (Fig. 8). In both man and dog the relative amounts of fractions I and IV, as the only bound fractions, were quite similar. 'The leukophilic y-globulin could be eluted and rebound to the cells with no detectable change in the amount and ratio of bound y-globulin. It was found that 1 ml of packed purified leukocytes bound about 2 mg of specific y-globulin, which contrasts with about one-tenth of this amount per milliliter of erythrocytes. This is most likely due to the presence of a much larger number of receptor sites per unit area of membrane surface. The rate of binding of leucokinin, in isotonic sucrose medium, was found to be very fast. As with erythrophilic y-globulin, the association of the

i Before odsorption on leukocytes E

+

c .- c b $ After odsorption on leukocytes X w

Eluote from cooted leukocytes

Fig. 8. Evidence that naked leukocytes bind mainly y-globulin IV from serum y-globulin, causing a reduction i n the fraction. Serum y-globulin was first dialzyed against sucrose solution. A 5-ml volume containing 15 mg was then mixed with 2 ml of packed naked leucocytes and incubated for 10 min. The cells were then sedimented, and the supernatant was dialyzed against 0.05 M sodium acetate buffer, p H . 4.8. The leukocytes were then washed three times each with 6 ml of the sucrose solution and finally eluted with 6 ml of 0.15 M NaCl two times. The combined eluates were concentrated by evaporation and precipitated in ammonium sulfate at 0.6 saturation. This was followed by dialysis against the acetate buffer. Aliquots containing 4 mg ot y-globulin derived from the sample before adsorption (top graph), after adsorption (middle graph), and that derived from the eluate of the leukocytes (bottom graph) were chromatographed on a 12 + 0.5-cm phosphocellulose column at room temperature. All other manipulations were performed at 0-4'C. From Fidalgo and Najjar (48).


i E 8 N

c 0

C 0

0 C t Y

.- t

.- W

Fig. 9. The presence of leukophilic y-globulin bound 10 leukocytes in situ. The pattern of the eluate (lower graph) from saline-washed leukocytes shows augmentation of fraction IV as compared with the pattern of whole y-globulin (upper graph). Heparinized blood (500 ml) was centrifuged at 1000 g for 5 min. The plasma and bury coat were removed separately. The leukocyte layer (3.6 ml) was resuspended in 15 ml of plasma and centrifuged. Again the leukocyte layer was separated from the contaminating red cells. This process was repeated five more times. The packed cells (1.8 ml) along with the contaminating plasma were then washed twice each with 4 ml of saline. T h e pooled washings were then precipitated with solid ammonium sulfate at 0.33 saturation, and the y-globulin recovered was dialyzed against 0.05 M acetate buffer ( p H 4.8) and chromatographed. From Fidalgo and Naijar (48).

cytophilic y-globulin with the respective cell reached completion within 1 min.

We have stressed that the main component of leukophilic y-globulin is fraction IV. Fraction I , though diminutive, is nevertheless quite prominent. However, in every fractionation, in both man and dog, trace amounts of fractions I1 and 111 were invariably obtained. It is quite likely that these are indeed minor leukophilic fractions rather than simple contaminants. These two fractions have not been studied further. Leukophilic fractions I and IV are yG, as identified by immunodiffusion and immunoelectrophoresis (48). Fraction I is the least basic and fraction IV the most basic of the four fractions.

As was shown for erythrophilic y-globulin, only the Fab-fragment binds to the white cells. Not even traces of bound Fc-fragment were detectable.

B. BINDING OF LEUKOPHILIC y-GLOBULIN TO LEUKOCYTES IN VIVO

The indication that the circulating leukocyte possesses a specific y-globulin coat was obtained through the same in situ experiment as that outlined for erythrocytes.

PHYSIOLOGICAL ROLE OF *-GLOBULIN 153

Freshly drawn blood, containing citrate as anticoagulant, was used for purifying the leukocyte fraction. The red cells were separated, and the buffy coat was purified by fractional sedimentation in its own plasma without the addition of sucrose or dextran. The process was repeated until a highly purified mixed leukocyte preparation was obtained with little or no contamination with red cells or platelets. After centrifugation at 600 g in the cold, the supernatant plasma was removed. The sediment, containing leukocytes and contaminating plasma, was then eluted with isotonic salt solution, and the fractionation pattern of the eluted y-globulin on phosphocellulose was compared with that of the y- globulin pattern obtained in the supernatant. Had there been no y-globulin bound onto the leukocytes, the pattern of the contaminating plasma of the sedimented cells would have shown the same relative pro- portions of the four fractions as that of the supernatant. O n the contrary, the pattern in the cell eluates showed a definite augmentation of fraction IV (47,48). Fraction I is too small to affect the relative ratio in a discer- nible way. This is clearly demonstrated in Figure 9.

VII. Physiological Function of Leukophilic 7-Globulin

Nature had provided, in the white corpuscles as you call them. . . in the phagocytes as we call them. . . a natural means of devouring and de- stroying all disease germs. There is at bottom only one genuinely sci- entific treatment for all diseases, and that is to stimulate the phagocytes. Stimulate the phagocytes. Drugs are a delusion.

The Doctor’s Dilemma,

GEORGE BERNARD SHAW

One of the advantages of the P M N neutrophilic leukocyte over the erythrocyte is that its functional activity involves motility and engulfing cell debris and particles. Consequently the effect of leukophilic y-globulin on these parameters can be evaluated. However, the P M N cell has a distinct disadvantage in that it does not lend itself to reliable evaluation of its viability in uzuo since it does not remain in the circulatory system but spends a good deal of its life in the extravascular tissue spaces. The P M N neutrophil is the preponderent P M N leukocyte and is mainly responsible for phagocytosis. All these studies are therefore confined to the activity of the neutrophil. The eosinophil and basophil P M N cells


are few in number and do not participate as actively in phagocytosis. Consequently they were not studied.

A. STIMULATION OF THE PHAGOCYTIC ACTIVITY OF

POLYMORPHONUCLEAR NEUTROPHILS

A study of the effect of leukophilic y-globulin on the PMN neutrophil showed that it increases the phagocytic activity of the cell to a considerable degree. Furthermore, in the presence of this protein, the PMN cell is capable of phagocytosis for a much longer period (47,48). Because of these effects on the PMN cell and in the interest of brevity, leukophilic y-globulin was termed leucokinin (48). Of the two fractions of leucokinin, fraction IV has the capacity to effect a maximal stimulation of phagocytosis with a high specific activity, Fraction I is capable of a definite stimulation, but at a much lower specific activity. It must be recalled here that fraction I of whole y-globulin contains all the ?A-globulin of the serum. This can amount to about 50-75% of that fraction. It is entirely possible that, if fraction I of the leukocyte eluate were assayed for its phagocytic effect, it might have a high specific activity. This has not been done because of the prohibitive effort that is required to prepare pure leukophilic fraction I for such detailed studies. A comparison of the stimulatory effect of fraction IV of whole y-globulin with that of serum showed that almost all the stimulatory activity of serum resided in its content of fraction IV. For this comparison serum complement activity was destroyed by heating at 56°C for 30 min. For maximal stimulation 0.03 ml of inactivated serum and 90 pg of fraction IV were required. The content of fraction IV in that amount of serum is approximately 60-80 pg. Figure 10 shows the comparison between serum and fraction IV stimulation in sucrose medium as contrasted with Hank’s physiological medium. The relative amount of fraction IV required to attain the same maximal stimulation of phagocytosis in Hank’s medium and in the buffered isotonic sucrose medium is 5 : 1 (48). On the reasonable assumption that the extent of phagocytosis is directly related to the amount of bound y-globulin, these results suggest that in isotonic salt solution approximately five times the concentration of fraction IV is required to bind the same amount of this fraction as in the low-ionic- strength sucrose medium. Based on this, it would be expected that even in the face of the normal ionic strength of the serum, the binding sites on the PMN cells in the circulating blood are likely to be completely saturated with leucokinin. This conclusion stems from the


fact that blood contains 75-100 mg of leukophilic y-globulin per 100 ml, of which approximately 0.8-1.4 mg is bound to the leukocytes (48).

B . REDUCTION OF LEUCOKININ ACTIVITY FOLLOWING

SPLENECTOMY

It was shown earlier that after splenectomy in the dog there was a drop in at least one of the erythrophilic fractions and a parallel reduction in the half-life of the erythrocyte. On the basis that the spleen may be the

50 -

8 40-

.SERUM 0.03ml

10 20 30 40 50 60

MINUTES

- SUCROSE MEDIUM 0---o HANK‘S MEDIUM

10 20 30 40 50 60

MINUTES

Fig. 10. Relative stimulatory effect on phagocytosis of isolated y-globulin fraction IV as compared to serum containing the same amount of the fraction. The advantages of the sucrose medium over Hank’s medium are obvious. In order to attain a level of phagocytosis in Hank’s medium comparable to that obtained in the sucrose medium, five times the amount of fraction IV was needed. The reaction was carried out as described in the text. The or- dinate represents the number of polyrnorphonuclear leukocytes containing one or more staphylococci per 100 cells. From Fidalgo and Naijar (48).

156 VICTOR A NAJJAR

site of synthesis of cytophilic y-globulin for the cellular elements of the blood, a similar study was conducted for the leukophilic fractions (49).

Six dogs were splenectomized, of which two were supplemented with weekly injections of 250 mg of fractions 111 and IV. Two were sham- operated. Of the four splenectomized animals, all showed a marked reduction of fraction 111, as before (30), 55 days after operation. Only one showed about 40% reduction of fraction IV as compared with the pre- splenertomy value. All four dogs showed complete absence of phagocytic stimulation in either their serum or corresponding amounts of fraction IV. Their autologous P M N cells served as the test cell in all cases. However, presplenectomy serum was as effective in stimulating the autologous P M N leukocytes as it was with cells obtained before splenectomy. This indicated that the deficiency did not lie in the P M N cell but in the activity of fraction IV. Essentially no leucokinin was being processed at that time. T h e level of leucokinin activity was measured pe- riodically for 8 months after the operation. As expected from the previous experience (30), all dogs regained their leucokinin activity 4-8 months later, and again splenic tissue was found in the peritoneal cavity. The half-life of the erythrocyte was assayed on these dogs with "Cr (42), and the level of fraction 111 as determined by phosphocellulose chromatography (34). Again there was a remarkable correspondence between the level of reduction in both and a similar correspondence during the recovery period (49). Before complete regain of leucokinin activity, such animals would be susceptible to severe infections. This had been noted earlier as a common occurrence in splenectomized animals.

As we shall discuss later, the curtailment of phagocytosis in splenectomized dogs has its counterpart in splectomized humans, who, unlike dogs, lose all leucokinin activity permanently. They are also prone to severe and overwhelming infections.

VIII. The Unique Properties of Leucokinin Stimulation of the Phagocytic Activity of Polymorphonuclear Neutrophils

In order to understand the properties of the phagocytic reaction, a series of kinetic studies were performed (50,51). For this, fraction IV, as isolated from autologous y-globulin, was utilized. About one-fourth of this fraction is leucokinin. A similar quantity is erythrophilic, and approximately the same amount is thrombophilic. It was not practical or necessary to prepare pure leucokinin samples for this study since the


nonleukophilic portion of fraction IV does not bind to P M N cells nor in- terfere.

Inasmuch as erythrophilic y-globulin is capable of adequate association with the red cell in uitro as well as in uzuo, it was assumed that in this capacity it was able to prolong the life span of the red cell. It was therefore a justified expectation that the association of leucokinin with the specific receptor on the neutrophil imparted to the membrane the proper stimulus for the initiation of the engulfment process. However, kinetic studies revealed otherwise:

1. No stimulation of phagocytosis was observed if leucokinin was preincubated with the cell for 20-30 min before the addition of the staphyloccoccus particle.

2. The addition of fresh leucokinin to this reaction mixture gave the usual twofold to threefold stimulation. This indicated that the P M N cell was unaffected by the preincubation.

30 I 20

10

0

0 10 20 30 MINUTES

Fig. 11. Loss of phagocytic stimulation by leucokinin after preincubation with thoroughly washed intact leukocytes. Under these conditions, intact erythrocytes show no effect on leucokinin activity. 0, 100 fig of fraction IV was preincubated with a human buffy coat preparation of polymorphonuclear cells for 20 min in Krebs-Ringer buffer, p H 7.4, at 37%, and only bacteria were added at zero time; 0, a similar preparation was made, except that bacteria with another 100 fig of fraction IV were added together at zero time; X,

100 pg of fraction IV, cells, and bacteria were added rapidly within 30 sec, without preincubation. As indicated, samples were taken at 10, 20, and 30 min, and the phagocytic index was determined by visual counting. For details, see the text. From Nishioka et at.

(51).


z - 10

Q I a 0

I 1 I 1

0 10 20 30 40

MINUTES

Fig. 12. Short-lived stimulation of phagocytic activity by Ieucokinin. The reaction mix- tures consisted of (0) 50 pg of fraction IV, polymorphonuclear cells, and bacteria; and (.) a similar reaction mixture, except that at 20 rnin another SO pg of fraction IV was added. At the indicated times, samples were taken, and the phagocytic index was determined by visual counting. From Nishioka et al. (51).

3. When leucokinin that had been preincubated with PMN cells was recovered and assayed with fresh PMN cells, no stimulation of phagocytosis was elicited (Fig. 11). Clearly under these circumstances the leucokinin molecule had become inactive. However, its physical properties, solubility and electrophoretic mobility, immunochemical reactivity, and molecular weight were not detectably altered (51). Thus the effectiveness of the leucokinin molecule is short-lived. In harmony with this idea were the peculiar kinetics observed. When half the maximal stimulatory dose of leucokinin was added to the phagocytic reaction, phagocytosis did not proceed at a constant and diminished rate during the 30-40 min of the reaction. O n the contrary, after about 10-15 min of stimulation, the rate fell off to control levels. When another aliquot of leucokinin was added, stimulation was resumed, only to fall off again after a similar lapse of time (Fig. 12).

IX. Isolation of the Active Polypeptide Fragment of Leucokinin, Thr-Lys-Pro-Arg (Tuftsin)

These observations gave rise to the postulate that the leucokinin molecule suffered the loss of an active fragment small enough not to af-

PHYSIOLOGICAL ROLE OF ?.-GLOBULIN 159

fect the molecular weight and other properties. The loss of the fragment was presumed to occur through cleavage by a membrane enzyme located on the outer surface of the PMN cell in the vicinity of the binding site of the leucokinin molecule. This postulate proved correct in all details. Membrane preparations were indeed found to contain an enzyme, leucokininase, which cleaves off an active peptide from leucokinin. The peptide, termed tuftsin (52), was extractable in 76% ethanol. O n a molar basis, it was responsible for the full activity of the parent molecule. The enyzme is tightly bound to the membrane. The purified membrane preparation (51) showed highly active marker adenylate cyclase activity (52). The enzyme requires no cofactors, has a p H optimum at 6.9, and is remarkably active. A 4-pg quantity of membrane protein per milligram of fraction IV yields over half the available tuftsin in less than 30 min. It releases tufstin activity only from fraction IV and to a much lesser extent from fraction I . Fractions I1 and 111 did not yield any detectable activity. Thus the peptide tuftsin is small and is covalently bonded to the parent molecule since it is not dialyzable in 0.05 M acetate buffer, p H 4.8, 0.15 M NaCl, 8 M urea, and 6 M guanidine HCl. It becomes readily dialyzable after treatment with leucokininase. Tuftsin is destroyed by leucine aminopeptidase, carboxypeptide B, pronase, and subtilisin. It is not affected by trypsin, chymotrypsin, clostripain, pepsin, papain, al- kaline phosphatase, deoxytribonuclease, and ribonuclease nor by aeration in the presence or absence of cysteine.

Tuftsin is also cleaved by controlled short treatment with trypsin for 1 hr in 0.1 M phosphate buffer, p H 8.1. The properties of the trypsin product are identical with those of leucokininase in specific phagocytic stimulation as well as in dialyzability and susceptibility to the enzymes cited above. Both have identical elution volumes in Sephadex G-10 or Aminex AG 50 W-X4. Whether underivatized or dansylated, they have identical R , values on silica gel. Both yield measurable tuftsin only from fraction IV, and only from the heavy chain. Once tufstin is removed by trypsin, further treatment with leucokininase does not yield any more tuftsin activity. Digestion of leucokinin-rich fraction IV with either trypsin or leucokininase yields only one peptide fraction that stimulates phagocytosis. No other peptide fraction shows any stimulation. Over 56 such fractions were assayed, and only the tuftsin peak showed activity. Products of autodigestion of trypsin and leucokininase were inactive. Furthermore, both enzymes fail to produce tuftsin activity from the y- globulin of patients with the tuftsin-deficiency syndrome (53,54). Simi-


larly, neither of the two enzymes releases an active peptide from fraction IV of patients with loss of splenic function, be it through surgical removal or through infarction as occurs in sickle-cell anemia or through leukemic infiltration (54,55).

Large-scale preparations of tuftsin using trypsin digestion yielded a pure product with unit residues of lysine, arginine, threonine, and proline. T h e sequence was determined as Thr-Lys-Pro-Arg. This peptide proved to have the identical properties that were determined from the leucokininase digest before its structure was known (50,512). At that time, tuftsin activity was located in the heavy chain. No activity was detected in the light chain following leucokininase treatment. The tetrapeptide is definitely localized in the carboxyl-terminal Fc-fragment of leucokinin (56). The stimulatory activity of the Fab-fragment (52,57) was due to the formation of a monomer Fab-Fc molecule or to contamination with intact leucokinin that was not detected with the particular antiserum. The failure of the Fc-fragment, as such, to show any stimulation of phagocytosis is due to its inability to bind to cell receptors, a property unique to the Fab fragment.

Subsequent studies with trypsin digestion of highly purified Fab and Fc fragments showed that all the activity resided in the Fc portion of the molecule (58). Furthermore, only leukophilic fraction IV possessed with an exposed carboxyl-terminal which can be cleaved by leucokininase and made available to the cell. The isolation of the tetrapeptide from y-globulin and its fragments is performed as detailed in several previous publications (50-52). Following performic acid oxidation of 65-70 mg of the fraction, complete digestion of the oxidized protein was done with trypsin. The products were chromatographed on Sephadex G-10, and the eluate having tuftsin activity was chromatographed on Aminex AG 50 W-X4 in a pyridine acetate gradient (51). The localization of the tetrapeptide tuftsin on the Aminex column shown in Figure 13 is highly re- producible. Fractions I and IV are the only two fractions that contain leukophilic y-globulin; fractions I1 and I11 have only trace amounts.

The same tetrapeptide has been identified in yG, myeloma H-chain residues 280-2532 (59). Not all yG, possess a leucokininase-cleavable tetrapeptide. Fractions I1 and 111 are immunochemically predominantly 6, (53,54), yet the two fractions, as we have already noted, show no tuftsin activity following leucokininase digestion but yield normal amounts by chemical isolation. It is clear that tetrapeptide that is present in the H-chain of such molecules cannot be cleaved by leucokininase. It


~ 2.0 E -

is proposed that in these instances the tetrapeptide does not have a free arginine residue which is essential for leucokininas reactivity (58).

Arninax AG 5 0 W - X 4 -

X. Chemical Properties of the Tetrapeptide in the Native Leucokinin Molecule

When a n active fragment was first discovered, a n attempt to characterize it was made by studying its susceptibility to various enzymes (see above). Two of these were leucine aminopeptidase and

I- I80

P

I A 200

- 220

ELUANT ml

Fig. 13. A 750-mg quantity of pooled comercial y-globulin (Cohn 11) was digested with trypsin, 5 mg/100 mg in 150 ml of 0.1 M tris-HCI buffer, pH 8.1, and 0.01 M CaCI, for 1 hr at 37OC. The TCA supernatant was extracted with ether, and the aqueous layer was lyophilized and dissolved in 0.1 M acetic acid. The material was then chromatographed on a Sephadex G-10 column, 1.5 x 90 cm, using 0.1 M acetic acid saturated with chloro- form, with a flow of 6 ml/hr. The active fraction was rechromatographed on the same column under the same conditions. One active fraction was recovered between elution volume 146-1 56 ml. This was lyophilized and taken up in 1 .O ml of pyridine-acetate buffer, p H 4.0, having a pyridine concentration of 1.2 M . The sample was then applied to an Aminex AG 50W-X4 column, 0.6 X 56 cm equilibrated with the same pyridine-acetate buffer at 60OC. The peptides were fractionated in a linear gradient, using the equilibrating buffer as the starting solution and pyridine-acetate bufffer, pH 6.0, containing 2.5 M pyridine as the limit buffer. Flow rate was 8-10 ml/hr, and 0.6 ml was used for the ninhydrin reaction. The only fraction showing phagocytosis-stimulating activity was the vary last fraction, which yielded ratios of Lys. 1 .O, Arg 0.92, T h r 1 .O, and Pro 1.05.

162 VICTOR A NAJJAR

TABLE I11

Effect of Treating Fraction IV with Either Leucine Aminopeptidase (LAP) or Carboxypeptidase B (CP-B) on the Phagocytic Activity of

P M N Leukocytes8

y-Globulin treated y-Globulin treated with CP-B with LAD

Experiment Cohn I1 Fresh Cohn I1 Fresh

1 1 5 18 36 40 2 19 18 36 38 3 17 17 34 35 4 18 18 35 36 5 20 21 38 35 6 18 17 38 38

After digestion of 1.0 mg of fraction IV with 20 fig of LAP or CP-B per milliliter of reaction mixture, a t the appropriate pH and temperature (51, 56) for 1 hr, the bound tuftsin was cleaved with 4 pg of Ieuco- kininase membrane preparation (51 , 52). The liberated tuftsin or its breakdown products were extracted with 76% ethanol and assayed for phagocytic activity. Phagocytosis was carried out in Krebs-Ringer phosphate buffer, p H 7.4, in a total volume of 0.4 ml. Dog P M N leukocytes were obtained from fresh dog blood and Staphy2ylococcus aureus from an 18-hr culture. The ratio of bacteria to neutrophils was approximately 1:2 (2 X lo6 P M N leukocytes and 4 X lo6 bacteria were used in the reaction mixture). The reaction was started by adding bacteria immediately after tuftsin extract. Reagent control for all experiments was 18 f 2.

carboxypeptidase B, which completely destroyed its activity. Con- sequently these enzymes were used to probe the chemical linkage of the tetrapeptide within the H-chain of the leucokinin molecule.

A. POSSIBILITY OF A FREE CARBOXYL-TERMINAL ARGININE

The tetrapeptide as it exists in fraction IV can be inactivated by treating fraction IV with carboxypeptidase B, but not with leucine aminopeptidase. This was discovered long before the isolation of the peptide. In these experiments (56) native fraction IV was treated either with carboxypeptidase B or leucine aminopeptidase. The reaction was


stopped by heating, and each sample was treated with leucokininase to release tuftsin, which was then assayed in the usual manner. Table 111 shows the results of six such experiments. In every case, carboxypeptidase treatment yielded an inactive product. This finding is consonant with the fact that this enzyme destroys the activity of free natural tuftsin released either by leucokininase or trypsin. On the other hand, leucine aminopeptidase treatment of fraction IV failed to inactivate the tetrapeptide. This indicated clearly that the tetrapeptide as it exists in the parent molecule has a free carboxyl-terminal arginine residue. This is further strengthened by the fact that free arginine was released following carboxypeptidase B treatment of leucokinin-rich fraction IV. Inacti- vation of tuftsin by the enzyme occurred readily whether fraction IV was prepared from freshly drawn blood or from commercial y-globulin (Cohn 11). It is therefore unlikely that the free carboxyl-terminal arginine resulted from a chemical cleavage through storage. The threonine residue in both samples remains covalently linked to the H-chain (56).

B. POSSIBILITY OF THE EXISTENCE OF AN ESTER LINKAGE AT THE

AMINO-TERMINAL THREONINE

The linkage at the amino-terminal threonine in the intact leucokinin molecule is at least partly, if not totally, an ester linkage, presumably at the threonine hydroxyl group. Treatment with hydroxylamine yielded the tetrapeptide with the same sequence. Hydroxylamine cleavage was equally effective whether it is done with or without prior dansylation of the parent molecule. In the latter case dansylated tuftsin was obtained. The yield in each case was low, presumably because of the high pH needed for the reaction and the expected 0 + N shift. Further support for the existence of tuftsin in an ester linkage (56) is in the observation that trypsin readily releases tuftsin in the presence of 2-chloroethanol. Under these conditions it was shown that only the esterase activity of trypsin was demonstrable (60).

0 shifts in peptide chemistry are well recognized. It is difficult therefore to ascertain whether the ester bond proposed here is the true bond in the native molecule or whether it occurred as a result of handling or aging of the preparation. The inability of leucine aminopeptidase to destroy bound tuftsin suggests that the threonine residue may be in a peptide linkage. On the other hand, an ester linkage with a free threonine amino group may afford steric hindrance for the

The N

164 VICTOR A . NAJJAR

enzyme. Another point worth noting is that in myeloma protein yG, the threonyl residue of the tetrapeptide is preceded by lysine. It is not known that a lysyl-threonyl bond is susceptible to hydroxylamine cleavage. This remains to be determined.

XI. Increased Rate of Migration and Prolongation of the Viability of Polymorphonuclear Leukocytes by Tuftsin

The migration of P M N leukocytes vertically inside a capillary tube (61) was studied in the presence or absence of tuftsin. Equimolar quantities of the constituent amino acids were added in control tubes. Figures 14 and 15 are illustrative of the results obtained (62). Other than the stimulation of migration, which is quite apparent in Figure 15, it was observed in the middle three tubes that the cells were still motile after 16 hr in the presence of 12.5 Fg of the tetrapeptide per ml. In other tubes, after 16 hr of incubation, the cell that had migrated a much smaller distance fell off and lysed (62).

Tuftsin analogs such as Thr-Lys-Pro-Pro-Arg, Thr-Lys-Pro, and Lys-Pro-Arg did not stimulate migration or phagocytosis. In fact, the Lys-Pro-Pro-Arg proved to be a very effective inhibitor of tuftsin activity both for migration and phagocytosis (Figs. 14 and 15).

XII. The Tetrapeptide Tuftsin as a Biological Entity with a Specific Function

With each identification of a biologically active polypeptide two questions arise:

1 . Does the isolated and identified peptide have the same properties that were described for the original active crude material: the stimulation of phagocytosis by leukokininase or trypsin digest?

2. Is the isolated peptide a true physiological compound that performs a meaningful function on a specific organ or cell?

A few highly active peptides do not fulfill these criteria. The answer to

First, all the properties that were described for the biologically active both questions is in the affirmative with regard to this tetrapeptide.

crude fragment apply for the tetrapeptide:

1. Both the biological activity and the tetrapeptide are restricted to the heavy chain and are not found in the light chain of leucokinin-rich fraction IV (52,57).

PHYSIOLOGICAL ROLE OF ?GLOBULIN 165

2. Both are destroyed by carboxypeptidase B, leucine aminopeptidase, pronase, and subtilisin.

3. Both are not destroyed by trypsin, chymotrypsin, pepsin, clostripain, papain, pepsin, ribonuclease, and desoxyribonuclease, nor by air oxidation with and without cysteine (50-52).

0.8

0.6

E E 0.4

0.2

HOURS

Fig. 14. Freshly drawn dog blood was treated with heparin at a final concentration of 0.1 mg/ml and washed three times, each with 4 volumes of Hank’s solution containing 0.1% glucose. A 0.1-mI volume of packed cells was mixed with 0.1 ml of Hank’s solution containing tuftsin to yield a final concentration of 5 nmoles (0) and 25 nmoles (8) per milliliter. Eight nanomles of the pentaopeptide Thr-Lys-Pro-Pro-Arg was mixed with 25 nmoles of tuftsin per milliliter of reaction mixture (0). The amino acid control (A) contained 25 nmoles each of threonine, lysine, proline, and argine per milliliter. At all times, this showed a migration rate equal to the reagent control. From each sample, six si- liconized microhematocrit tubes were filled by capillary action, heat-sealed at one end, and centrifuged. The buffy coat containing polymorphonuclear granulocytes is at the cell-fluid interface. These were then mounted vertically in a 37°C chamber, and the migration of cells from the buffy coat into the solution was measured microscopically with an ocular mm scale. Note the increased migration with synthetic tuftsin at both concentrations, the lack of increase above the control when all of the constituent amino acids were present, and the strong inhibition of tuftsin stimulation by the pentapeptide Thr-Lys-Pro-Pro-Arg. From Nishioka et al. (62).

Fig. 15. The details are exactly as in Figure 14, except that the observation period was extended to 16 hr. The first three tubes from the left contained 25 nmoles/ml of the constituent amino acids threonine, lysine, praline, and arginine. These served as controls. The middle three tubes contained 25 nmoles/rnl of synthetic tuftsin, and the three tubes on the right contained 5 nmoles/ml of the same tetrapeptide, tuftsin. From Nishioka et al.

(62).

166

PHYSIOLOGICAL ROLE OF ./-GLOBULIN 167

4. By activity assay, tuftsin was found principally in fraction IV and to some extent in fraction I , but not in fractions I1 and 111 (47-49).

5. The specific biological activity of tuftsin calculated on a molar basis in fraction IV of the leukophilic fraction was remarkably close to its content of the tetrapeptide (50,51,62).

6. Tuftsin activity is released completely from the parent molecule by the membrane enzyme leucokininase and becomes ethanol extractable. The tetrapeptide was also released by the same membrane enzyme and was isolated and identified in the ethanol extract following Sephadex and ion-exchange chromatography (50,51,62).

7. Tuftsin is responsible for the full biological activity of the carrier molecule. In the same vein, the isolated tetrapeptide is the only peptide that showed biological activity, among all fractions of trypsin and leucokininase digests of whole y-globulin or fraction IV, as well as all fractions from the autolytic digestion of trypsin and leucokininase controls (50-52).

8. Finally, the specific activities of natural tuftsin released by leucokininase and that of the synthetic tetrapeptide are identical (62).

Second, several characteristics indicate that the tetrapeptide tuftsin is a true biological entity with a specific physiological activity:

1. It is present on a distinct carrier leukophilic molecule. 2. This molecule binds specifically to P M N granulocytes on which

the tetrapeptide exerts its effects in stimulating phagocytosis, migration, and viability of the P M N cell.

3. It is not released by leucokininase or short trypsin digestion from the y-globulin of splenectomized subjects. This correlates well with the high rate and severity of infection in these patients. Presumably the spleen is involved in the availability of tuftsin in some manner (54,55).

4. It is defective in the tuftsin-deficiency syndrome, an inherited fami- lial disorder (53,54). These subjects also suffer severe infections that respond to the administration of normal y-globulin, presumably due to their content of the tetrapeptide. The presence of such human mutants is the most compelling argument, since the loss of a functional activity through a mutation is an affirmation of the presence of its counterpart, a normally functioning gene with a defined phenotypic expression.

5. Tuftsin is active in uitro at very low, hormone-like, concentrations of 0.05-0.1 pg/ml of reaction mixture, as shown in Figure 16 (50,51, 63).

168

0 5

VICTOR A . NAJJAR

:fi +

2o 1

TUFTSIN,yg per ml

Fig. 16. The stimulatory effect of various concentrations of tuftsin on dog-blood P M N leukcocytes (open circles) and mouse peritoneal macrophages (solid circles). Phagocytic stimulation is the phagocytic index obtained with added tuftsin minus that obtained in the controls lacking tuftsin (A index). From Constantopoulos and Naijar (63).

6. The tetrapeptide is equally active on phagocytic cells other than blood PMN granulocytes. These are rabbit peritoneal granulocytes, peritoneal and lung macrophages (63). Its receptor sites on the PMN cells require the presence of a full complement of sialic acid.

7. The tetrapeptide appears to have specific membrane binding sites on the PMN cell. The presence of sialic acid on the membrane is an absolute requirement for its activity (64).

XIII. Chemical Synthesis of Tuftsin and Its Analogs

The tetrapeptide was synthesized by the insoluble-resin-support technique of Merrifield (65,66). All manipulations were carried out under strictly ahydrous conditions and at room temperature except for the este- rification of arginine to the resin. The starting material, Na-BOC-NG- nitro- L-arginine, was esterified to the chloromethyl function of the resin in ethanol and triethylamine for 48 hr at 8OOC. The BOC-group was cleaved off the nitroarginine with 50% trifluoroacetic acid in methylene chloride. Then Na-BOC proline was coupled with N,N'-dicyclohexyl-


carbodiimide, also in methylene chloride. The BOC-group was then removed as before with trifluoroacetic acid. Successively, Na-BOC-Nt- carbobenzoxy- L-lysine and Na-BOC-0-benzyl-L-threonine were then coupled using the same deprotection and coupling steps. Finally, the tetrapeptide was cleaved off the resin, and the remaining protecting group was removed by treatment with hydrogen bromide in trifluoroacetic acid. The reductive cleavage of the nitro group was accom- plished by hydrogenation at 40 Ib/in.' hydrogen pressure, with palla- dium as catalyst. The cleaved tetrapeptide was then purified on the same Aminex column used to isolate tuftsin. The purified product was identical in physical and biological properties with the natural compound. The analogs Thr-Lys-Pro-Pro-Arg, Thr-Lys-Pro, and Lys-Pro- Arg were synthesized and purified in the same manner.

XIV. Human Tuftsin-Deficiency Syndrome

The possibility that tuftsin deficiency exists as an inherited syndrome was first considered when it was noted that patients with a history of repeated intractable infections responded rather dramatically to y-globulin administration.

So far four families have been reported (53,54,67). Staphylococcus, pneumococcus, and meningococcus a re the predominant infecting organisms. However, the severity of infection varies considerably. It can be manifested as a mild and persistent skin infection or an upper respira- tory tract infection. It can also be an infection of the overwhelming type, with infected eczematous-looking skin, pneumonia, meningitis, and septicemia. Such severe manifestations are almost indistinguishable from the well-known granulomatous disease (68) or deficiency in complement components C3 (69) and C5 (70). T h e latter two complement components are essential for the opsonization of the bacterium for effective capture by the phagocyte. In every instance of tuftsin deficiency one of the parents exhibited the defect as well as one or more children in the family (53,67).

Further study of the biochemical aspect of tuftsin deficiency in these patients showed that the peptide that is cleaved by leucokininase or trypsin proved to be strongly inhibitory to the biological activity of either synthetic tuftsin or natural tuftsin obtained from the enzymatic digestion of y-globulin of individuals with normal tuftsin activity (52,53,67). The


inhibition is of the same order of magnitude as that obtained by the only inhibitory peptide so far synthesized, Thr-Lys-Pro-Pro-Arg (62). However, so far no attempts have been made to identify the inhibitory peptide from these patients.

XV. Human Tuftsin Deficiency Following Removal or Curtailment of Splenic Function

Because of the leucokinin deficiency noted in splenectomized dogs, a survey of tuftsin activity in splenectomized humans was undertaken. This is of particular importance since it has been known for some time that a high rate of infection of great severity occurs in splectomized children (71). Furthermore, splenectomized animals in general suffer fatal infections (72).

Other patients in whom splenic function is curtailed also exhibit an abnormal susceptibility to infections. The damage to splenic function may result from vascular involvement, as in sickle-cell anemia (73), or from the infiltration of splenic tissue, as in acute myelogenous leukemia (74,75). All these cases show no measurable tuftsin activity (54,55,75).

An interesting contrast to tuftsin syndrome is the finding that in these cases involving splenic function, no inhibition of tuftsin was obtained with limited trypsin or prolonged leucokininase digests of their y- globulin (54,55,75). Chemical analyses of the 7-globulin of splenectomized subjects show normal values of tuftsin. However, leucokoninase is incapable of releasing the tetrapeptide. Since an exposed carboxyl- terminal is essential for such a release, it is proposed that the spleen contains an enzyme that cleaves tuftsin at the arginine residue (58).

XVI. Association of Cytophilic 7-Globulin with Autologous Blood Thrombocytes

Thrombocytes (platelets) are a major formed element of the blood. Numbering about 1-3 x lo5 per cubic millimeter, they occupy an inter- mediate numerical position between erythrocytes and leukocytes (4-5 x lo6 and 5-7 x lo3 per cubic millimeter, respectively). They play a vital role in blood coagulation. It was expected that, as in the case of the erythrocytes and leukocytes, platelets would also be coated with y-globulin. For quite some time platelets have been suspected to have several blood protein components, including albumin, either adhering to their surfaces or within the cytoplasm (76).


A. BINDING OF THROMBOPHILIC y-GLOBULIN TO BLOOD

PLATELETS IN VITRO

Platelet-rich plasma was collected from human donors. The platelets were purified (77) and washed with low-ionic-strength isotonic sucrose as before. The eluate obtained with 0.15 M NaCl was subjected to paper electrophoresis at p H 8.6 (29). Only y-globulin was found. Im- munochemically the y-globulin was predominantly yG, but yM was definitely present, as was the case with erythrocytes; no yA was detectable. O n phosphocellulose chromatography, the y-globulin showed predominantly fractions I and IV (Fig. 17). However, fractions I1 and 111 were found in more than trace amounts and should be considered minor fractions that actually participate in the binding (78). Further demonstration of y-globulin binding to thrombocytes was done through fluorescein-tagged anti-human y-globulin. This is depicted in Figure 18.

B. BINDING OF THROMBOPHILIC ?-GLOBULIN TO BLOOD PLATELETS

IN VIVO

Tha t the circulating platelet has bound y-globulin on its cell membrane was shown by using the same simple approach as in

I

I

Fig. 17. The pattern of fractionation of donor human y-globulin (top graph) and that of the eluate of autologous platelets (bottom graph). The platelets were washed with buffered low-ionic-strength isotonic sucrose. A 0.5-ml volume of washed thrombocytes was eluted with 0.15 A4 NaCI, and 3 mg of protein was obtained. This was chromatographed on a phosphocellulose column, 10 X 0.5 crn, as described in the text. The relative quantities of

each fraction were monitored automatically by absorption in the ultraviolet (LKB Uvicord).


Fig. 18. Shows immunofluorescent staining of y-globulin bound to the thrombocyte plasma membrane. The right frame shows the minimal fluorescence in intact platelets that were washed with 0.15 M NaCI. Note also the absence of agglutination. T h e left frame shows strong fluorescent staining of thrombocytes washed with low-ionic-strength buffered isotonic sucrose solution. Fluorescein-labeled goat anti-human y-globulin (yG, yM, and yA) was used for reaction with human y-globulin bound to prepared platelets. Platelet suspension in the sucrose medium (0.1 ml) was mixed with 0.1 ml of 1 : l O dilution of the fluorescent antiserum and incubated for 30 min at room temperature.

demonstrating the in situ presence of erythrophilic and leukophilic bound 7-globulin. Phosphocellulose chromatography of the eluates of the platelets sedimented in their plasma showed essentially the same pattern except that fractions I and IV were clearly augmented as compared with the pattern of the supernatant plasma y-globulin (78). It has not yet been determined whether thrombophilic y-globulin binds only to the thrombocyte. This is assumed to be the case since the erythrophilic and leukophilic y-globulins were shown to be highly specific

It has not been possible so far to investigate the physiological role of thrombophilic y-globulin. Suffice it to say that the spleen appears to play a role involving the life span of the platelets. After splenectomy, there is an increased rate of platelet production resulting in a twofold to sixfold rise in the platelet blood level (79). Furthermore, there does not appear to be a change in the life span of platelets after splenectomy (79, 80).

(47-49).

XVII. Association of Cytophilic y-Globulin wi th Arterial Endo t he l ium

The binding of 7-globulin to the endothelial lining of the large arteries of cattle was not studied extensively. The y-globulin has not

PHYSIOLOGICAL ROLE OF 7-GLOBULIN 173

been characterized as was the case in the preceding sections. The limita- tions imposed on this investigation reflect those inherent in the limited characterization of cattle y-globulin.

Fresh vessels, aortic, carotid, subclavian, and ilial, were thoroughly washed in the low-ionic-strength buffered isotonic sucrose solution, p H 7.4. This was followed by one wash with isotonic NaCI. The saline wash was concentrated, and the protein precipitated at 0.70 saturation of ammonium sulfate and dialyzed against isotonic salt solution. The material was then subjected to electrophoresis at p H 8.6 for 16 hr. Only y-globulin with traces of albumin was detected. (81)

XVIII. Effects of Normal Mouse Serum on Neonatally Thymectomized Mice

The beneficial physiological effect of y-globulin on the survival of the erythrocyte is not limited to this cell. It was shown that the polymorphonuclear leukocyte is similarly influenced in its function and survival. Further support for this is afforded by results obtained on the whole animal (7). Thymectomized mice have a deficiency of the lymphatic system and consequently of y-globulin synthesis. They seem to have the normal complement of other serum proteins. Groups of these animals were subcutaneously given either serum from the same inbred strain, or an equal amount of serum from another strain, or an equal volume of 0.15 M NaCl. The first group grew considerably faster than the se.cond, which in turn grew faster than the NaCl control. The first group survived about twice as long as the other two, which showed equal survival time. The 50% survival time for the first group receiving serum from the same strain was 55 days, whereas the 50% survival times of the other two groups was markedly less, 28 and 30 days, respectively. It was assumed that the supplementation by serum from the same strain provided unaltered quasi-autologous y-globulin since these animals are defective only in y-globulin. An attempt to prepare y-globulin from the serum of the experimental mice yielded an unacceptable amount of dena- tured y-globulin, which might be harmful to thymectomized newborn.

XIX. Summary

Through a study of the molecular mechanism of the antibody-antigen interaction it was shown that two general types of antibody may be generated by immunization. This led to the postulate that the naturally


occurring isohemagglutinins are patterned after the receptor sites of the autologous cells. This in turn was followed by an investigation of the binding of y-globulin to erythrocytes and the physiological role of bound y-globulin. As a result of this study, autologous y-globulin was shown in uitro to bind specifically to the erythrocytes and polymorphonuclear leukocytes of man and dog. The bound y-globulin appears to be highly specific for the particular cell type. Human thrombocytes have also been shown to bind autologous y-globulin. In all these instances it was also shown, by in situ studies of freshly obtained blood, that ail three cellular elements have membrane-bound y-globulin of the same chromatographic characteristics as those obtained in uitro. It is reasonable to assume that the particular circulating cell in uiuo is coated with its specific y-globulin. A limited study of bovine large arteries showed y-globulin to be bound to the inner endothelial lining of the vessels. There is excellent in situ evidence of y-globulin binding to the myelin sheath and cellular elements of the human brain.

The physiological role of bound y-globulin has been demonstrated for the erythrocyte and the polymorphonuclear leukocyte. It was shown that erythrophilic y-globulin is essential for the normal configuration of the cell as well as the tensile strength and the permeability of the erythrocyte membrane. Following splenectomy in the dog, there is a parallel drop in the major component of erythrophilic y-globulin and in the half-life of the erythrocyte. Leukophilic y-globulin, leucokinin, is essential for the expression of the maximal phagocytic activity of the polymorphonuclear leukocyte. Following splenectomy in the dog there is a disappearance of the capacity of leukophilic y-globulin to stimulte the polymorphonuclear cell.

An enzyme, leucokininase, normally present in the cell membrane of polymorphonuclear cells cleaves tuftsin, a Thr-Lys-Pro-Arg tetrapeptide, from leucokinin. The tetrapeptide is fully responsible for all the stimulatory activity of the leucokinin molecule. Splenectomized humans show a complete absence of tuftsin activity. Two other types of patients that have splenic involvement also show absence of tuftsin activity. These are patients with sickle-cell anemia and patients with myelogenous leu- kemias. Patients with the tuftsin-deficiency syndrome, which is manifested by repeated and sometimes severe infections, show absence of tuftsin activity. Leucokininase or trypsin digests of their y-globulin yield a strong inhibitor to natural or synthetic tuftsin. The possible physiological role of thrombophilic y-globulin has not been inves- tigated.

PHYSIOLOGICAL ROLE OF ./-GLOBULIN 175

Acknowledgments

The work described in this chapter was supported by the National Institutes of Health, Bethesda, Maryland (Grant Al-O9116), and the National Science Foundation, Washington, D.C. (Grant GB-31535). The author is an American Cancer Society Professor of Molecular Biology (Massachusetts Division).

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Addendum

Edward J. Goetzl (personal communication) has recently studied the effects of tuftsin of human leukocytes in uitro random and chemotactic migration utilizing both modified Boyden micropore filter chambers and a radiochemotactatic assay. Preincubation of purified neutrophils or eosinophils with tuftsin at 1-100 ng/ml, which is known to enhance phagocytosis, produced a dose-related inhibition of random migration of up to 50%, and of chemotaxis of up to 90%, without any influence on mononuclear leukocyte mobility. The inhibitory action of tuftsin on leukocyte mobility is in contrast to the stimulatory action on mobility seen in studies utilizing glass capillary tubes and whole buffy coat cells. He suggested that this may be due to possible alteration of leukocyte surface adherence by tuftsin.

178 VICTOR A NAJJAR

Fractionation of bovine 7-globulin on phosphocellulose columns (34) was found by Elton Erp (personal communication) to be essentially similar to that found in humans and dogs (30,31). The percent values obtained were (kSD): fraction I , 52 & 8; fraction 11, 28 f 7; fraction 111, 10 f 5 ; and fraction IV, 10 & 3 . Bovine red blood cells bound a certain amount of all fractions but predominantly fraction IV. In isotonic NaCl and isotonic buffered low ionic strength sucrose solution (29-31) the uptake per milliliter of packed erythrocytes was 1.3 and 3.3 mg, respectively. Bovine white blood cells showed phagocytic stimulation of latex particles only with fraction IV. The tetrapeptide tuftsin also showed considerable stimulation.

It appears that tuftsin stimulation of phagocytic cells is a general phenomenon including cells from mouse, guinea pig, rabbit, dog, cow, and man.

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