Carrier-Specific Induction of Hapten-Specific Suppression Leonore A

Home , Hapten

40 immunology today, March 1981 ) Carrier-specific induction of hapten-specific suppression Leonore A. Herzenberg, Takeshi Tokuhisa and Leonard A. Herzenberg

Department of Genetics, Stanford University School of Medicine, Stanford, California, 94305, USA

Two functionally opposing, regulatory T cell popula- regulation of hapten-carrier responses. Therefore, our tions are found in carrier-primed mice. Both popula- data throw a substantially new perspective on the tions are raised by essentially the same immunization regulatory interactions involved in the response to protocol, both control antibody production to deter- common antigens. minants on typical carrier proteins, both are 'carrier- specific' in that they control responses to haptens The mechanism of carrier-specific suppression is added to the initial priming (carrier), and both are indeterminate commonly studied as regulators of such anti-hapten Carrier-specific suppressor populations were generally responses in adoptive or in-vitro assays. However, one presumed to regulate antibody production by control- population helps the antibody response while the ling the supply of carrier-specific help because they other substantially suppresses it. suppress in-vitro or adoptive in-vivo responses to 2, 4- These two populations have captured the limelight as regulators of heterogeneous antibody responses to T-dependent antigens. The helper T cells, because 150~ they are required for a successful response to these antigens, are generally thought to stimulate and thereby to directly control the expression of anti- hapten memory B cells. In contrast, the suppressor cells are thought to regulate B-cell expression indirectly by reducing the size or activity of the avail- I O0 able helper population. In addition, helper cells are DNP-KLH DNP-KLH thought to regulate suppressor activity through 1 O0 ug 1 ug homeostatic 'feedback loops' that can prevent exces- IgG2a on alum aqueous sive suppression. Thus, over the last few years, a anti complex but fairly consistent picture of response DNP regulation has evolved which places carrier-specific regulation at the center of the immunological uni- 5(2~ verse 1. Nevertheless, as we shall show, this picture is based on data which can (and probably should) be interpreted quite differently. Our recent studies 2 on KLH first the regulation of it~-situ responses to hapten-carrier conjugates reveal assumptions that mar previous conclusions about the role of the carrier-specific -1 0 1 2 3 4 5 6 7 8 suppressor population and suggest a novel alternative Weeks for the contribution these cells make towards regulating antibody production. Furthermore, the charac- Figure l. Carrier-priming prevents hapten-carrier stimulation of anti-hapten antibody responses. teristics of response regulation revealed by our studies (BALB/c x SJL) FI responses shown are representative of suggest that mechanisms akin to idiotype-specific and responses in BALB/c, BAB/14 and G3H. SW mice. Protocol and allotype-specific regulation may be central to "the assay details are described in the legend to Fig. 2. Elsevier / Non h-Ilolland Biomedical Press 1981 immunology today, March 1981 41

dinitrophenyl (DNP) on the priming carrier but are Anti-DNP responses are suppressed in ineffective in controlling antibody production by the KLH-primed mice exposed to DNP-KLH same B-cell population to DNP on an unrelated carrier. This interpretation of the suppression specifi- K ~ I 3 city data is consistent with the demonstration that allotype suppressor T cells remove allotype-specific K,DK -I f,o.31 i help and is reinforced by evidence that idiotype K,DK,DK 00"3) I suppressor T cells remove idiotype-specific help. These data have been taken as evidence that carrier- DK --i (5.) specific suppressor T cells interfere with the carrier- DK,DK l(too? i specific help interactions necessary for the production i I I I I I of antibody to determinants on the carrier molecule. 50 100 150 100 200 300 We suggest, however, that the logic underlying this Igh-la Anti DNP Igh-la Anti KLH analysis is flawed. The suppression of adoptive and in- (ug/ml) (units) vilro responses to DNP on the priming carrier, but not *K = KLH; DK = DNP-KLH on an unrelated carrier molecule, clearly demonstrates Fig. 2. Hapten-specific suppression selectively reduces the that the suppressor population is carrier-specific magnitude and affinity of anti-hapten responses. (since its function depends on being confronted with Animals were exposed to 100pg of the indicated antigens (on hapten on the appropriate carrier). But this specificity alum) at 6 week intervals. Responses were measured two weeks is consistent with a variety of mechanisms and does after the last indicated exposure. Units of ariti-carrier antibody are measured in radioimmunoassays (RIA) and calculated relative to not necessarily indicate that the suppressor cells a standard adoptive secondary response serum (to DNP-KLH). interfere with carrier-specific help for antibody Anti-DNP responses were measured in an RIA modified to responses to determinants on the carrier molecule. measure magnitude and affinitylL Data in parentheses after anti- Theoretically the suppressor population could DNP response 'bars' show the average K a M -~ x 106 for that establish a carrier-specific induction mechanism using response. the hapten-carrier conjugate to induce a second indicated, that carrier-primed animals produce a (hapten-specific) mechanism to specifically suppress minimal anti-hapten response when stimulated with the production of anti-hapten antibodies. This hapten on the priming carrier (Figs. 1 and 2). By the hapten-specific mechanism could operate ana- time we discovered the original papers which sug- logously to idiotype-suppression mechanisms by gested that the minimal responses were due to inter- specifically preventing the production of antibodies ference with anti-hapten memory development 5, we that have combining sites for the hapten and could had shown that anti-DNP memory development therefore suppress anti-hapten responses without occurs normally in keyhole limpet hemocyanin interfering with carrier-specific help or with the (KLH)-primed animals exposed to DNP-KLH (Table response to other determinants on the hapten-carrier 1) and that the anti-hapten responses fail because ex- conjugate. Consequently, carrier-specific help could posure to the hapten-carrier conjugate induces a be present and functioning normally in the presence of specific and persistent suppression of antibody p'ro- the suppressor population even though anti-hapten duction to the (new) DNP hapten on the carrier mole- antibody production is suppressed. cule (Fig. 3). This theory seems complex when compared with the relative simplicity of the hypothesis that carrier- The suppression-effector mechanism is hapten- specific help is depleted but the immune system, being specific more concerned with function than with appearance, The specificity of this suppression for responses to the may have a different idea of what constitutes a work- DNP hapten was established conclusively by the able set of regulatory interactions. In any event, as we observation that anti-DNP responses were suppressed shall show, a hapten-specific mechanism such as the in KLH-primed mice exposed first to DNP-KLH and one we have just described regulates antibody produc- then to DNP on an unrelated carrier molecule (i.e., to tion to hapten-carrier conjugates; and a carrier- DNP-CGG (chicken gamma globulin) ). These specific mechanism suppressing induction such as the animals produce normal primary responses to the one described operates in carrier-primed animals to CGG determinants on the DNP-CGG molecule and induce the hapten-specific system to suppress the thus have normal carrier-specific helper populations. response to DNP on the priming carrier. Furthermore, as indicated above, they have normal Our studies are rooted in a set of observations well anti-DNP memory populations. Therefore, if their known in the early 1970s but largely forgotten since anti-DNP responses were not specifically suppressed, then. We inadvertently repeated some in-situ studies they should have produced secondary level anti-DNP reported in the first papers on hapten-carrier help responses comparable with those in the control mice interactions 3,4 and found, as these papers had exposed to DNP-KLH and then to DNP-CGG. 42 immunology today, March 1981

TABLE I. Hapten-carrier stimulation of anti-hapten memory B minants on these antigens suggests that the hapten- cells is normal in carrier-primed mice~ specific mechanism normally operates to selectively control responses to each of the determinants on an immunizing molecule, regardless of whether the Memory B cell donors' form Adoptive IgG (haptenic)determinants are added chemically or are of antigenic stimulation anti-DNP response defined by the natural structure of the molecule. However, understanding this mechanism of action in Mean K a 'ordinary' immunizations (to haptens on the priming Group KLH DNP-KLH Units (x 10 6) carrier) first requires consideration of how it suppresses responses and makes animals persistently l * Alum 73 10 responsive or non-responsive to individual deter- II Alum Alum 73 8 minants on an antigen. III * Aqueous 18 0.7 The induction of hapten-specific suppression by the IV Alum Aqueous 50 8 KLH/DNP-KLH immunization sequence does not appear to be due to anything unique in the KLH carrier molecule or DNP hapten. Exposure to a GGG/DNP-CGG sequence induces similar suppres- Donors (BALB/c x SJL) were killed 3 weeks after the first indicated stimulation with DNP-KLH. Splenic B cells were prepared by sion for anti-DNP responses 6, and exposure to a eytotoxic depletionof T cells usingmonoclonal anti-Thy-1.2 (30-H12, KLH/PC-KLH sequence induces suppression of anti- J Ledbetter). Cells obtained from 107 spleen cells were co-transferred PC (phosphoryl choline) responses (J. Kenny, with 2 x 106 nylon-passedsyngeneic T cells from KLH-primed donors personal communication). Therefore, suppression (100 gg on alum plus 109 Bordetella pertussis at least 6 weeks before induction must represent the animal's response to ex- transfer). Recipients (650 rad irradiated BALB/c) were stimulated with 1 lag aqueous DNP-KLH (intravenous)at time of transfer and posure to the carrier/hapten-carrier sequence. That bled 1 week later for assay. Assay details are as described in Fig. 2 is, it must be due to mechanism(s) within the immune legend. Igh-lb (IgG2) response data are sllown here. Igh-la (IgG2~) system that become functional some time after anti- and IgG~ response~data were similar. Responses in mice receiving genic exposure and then induce suppression of the nylon T (no donor B) were three units of very low affinityantibody. response to determinants subsequently 'seen' in The proportion of IgD+memory cells (also an index of memory maturity~) was similarin groups I, 1I and IV and higher in group III, physical association with the priming antigen. indicating less maturation (to IgD-) in mice primed with aqueous antigen. Anti-KLH memory was similar in groups I, II and IV and The suppression-induction mechanism is lower in group 1II. carrier-specific The operation of this suppression-induction * No KLH injection mechanism is carrier-dependent since DNP-CGG induces suppression for anti-DNP responses in CGG- ~Adapted from Table 2, Herzenberg, Tokuhisa and Herzenberg, (1980) Nature 285, 664-667. primed animals but not in KLH-primed animals and vice versa. Therefore, it must have at least one carrier- specific component that recognizes determinants on the carrier molecule and 'presents' the hapten in such Nevertheless, as data in Fig. 3 show, they produce a way as to induce a specific suppression for anti- only minimal low-affinity anti-DNP responses that are hapten responses. Anti-carrier antibodies present in substantially below primary level. the carrier-primed animal could, in principle, serve These data effectively rule out non-specific or this purpose; however, we have shown (1) that the carrier-specific mechanisms as being responsible for induction of suppression is not correlated with levels of suppression since such mechanisms should affect endogenously produced or exogenously introduced, primary responses to CGG determinants on the DNP- anti-carrier or anti-hapten antibody; and (2) that the CGG molecule equally or more severely than they suppression-induction mechanism acts rapidly and affect secondary responses to the DNP hapten on this persists for at least twelve weeks after initial stimula- molecule. Thus, our studies define a 'hapten-specific' tion with carrier ('. Therefore, the carrier-specificity is mechanism capable of controlling individual antibody mediated by one or more cell populations generated responses mounted to different determinants present by carrier-priming. on a complex antigen. This function could be provided by carrier-specific This apparently novel regulatory mechanism, helper T cells or by an unknown carrier-specific cell which has gone unrecognized until now, emerges in population. But, as indicated above, the defined more familiar surroundings when considered in the properties of the carrier-specific suppressor T cell pop- context of the production of antibodies to complex ulation are consistent with the idea that these cells antigens such as those used for immunogenetic constitute the mechanism responsible for the induc- studies. The tendency for genetically identical animals tion of hapten-specific suppression in carrier-primed to produce antibodies to different subsets of deter- animals. In addition, we have shown that the immunology today, March 1981 43 immunization conditions used to generate carrier- stimulation with any molecule capable of serving as a specific suppressor T cells generate an efficient induc- carrier for the determinants associated with it, is tion mechanism for hapten-specific suppression 6. clearly present by this time and is probably functional Thus, we return to our earlier assertion that sup- to some extent 3 - 4 days after priming. pressor T cells play a different role in regulating res- The precise time at which the suppression- ponses than that currently perceived. induction mechanism becomes fully functional is The accuracy of this assertion is not crucial to the difficult to determine because antigenic-competition arguments raised here concerning the mechanism of mechanisms that interfere with primary antibody induction of hapten-specific suppression, since a responses to new antigens or determinants are opera- carrier-specific population of some sort must be tive 3 - 7 days after stimulation with an initial priming involved in this process. However, the likelihood that antigen. Preliminary observations in a related system carrier-specific suppressor T cells serve this purpose indicate that these competitive mechanisms may introduces ambiguities that must be resolved. At represent a special case of induction of hapten-specific present, it is impossible to state, with any certainty, suppression where the second antigen becomes that carrier-specific T-cell populations, which regulate physically associated with the first on antigen- responses by controlling the supply of carrier-specific processing or antigen-presenting cells and conse- help, actually exist. Furthermore, assuming that such quently all of its determinants are treated as new populations do exist, the current experimental haptens on the first antigen 6. If this proves correct, evidence does not permit distinction between the then the presence of a fully active suppression- suppressive effects these cells have on responses and induction mechanism three days after priming has the effects attributable to the carrier-specific cells that already been well documented. induce hapten-specific suppression. We hope that the This means that the suppression-induction application of diagnostics used here (differeritial mechanism begins to operate several days after the effects on anti-hapten and anti-carrier responses) can initial priming antigen has had an opportunity to help to clarify these complex issues which presently induce antibody production but while this antigen is cloud our interpretation of the existing data. still present and capable of inducing hapten-specific suppression for determinants associated with it. Even The suppression-lnduction mechanism in its if the antigenic competition mechanisms are distinct normal habitat from those inducing hapten-specific suppression, the The rapid appearance of the suppression-induction same arguments hold, since our studies show that the mechanism after primary exposure to antigen provides suppression-induction mechanism is functional at the key to its normal role in the regulation of antibody some level a few days after priming. Thus, in responses to individual determinants present on principle, suppression could be induced for any or all antigenic molecules. Exposure of KLH-primed mice determinants on the initial priming antigen by the to DNP-KLH as early as seven days after KLH- same mechanism that induces suppression for new priming induces a typical hapten-specific suppression determinants (e.g. DNP) introduced subsequently on for anti-DNP responses 6. Thus, the induction that antigen. mechanism, which can be expected to appear after Priming. usually leads to antibody production to at least some determinants on the priming antigen The carrier/hapten-carrier exposure sequence (especially DNP). Therefore, the overall regulatory induces hapten-specific suppression system apparently has the paradoxical ability to permit response to take precedence over suppression- K*,DK,DC -Igo.3) I induction when the determinants are initially presented on a carrier molecule, even though the induc- DK, DC l(100) I tion of suppression takes precedence over response when 'new' determinants are presented on a carrier to ,1(2) ] K,DC which the animal has already been primed. This DC "--q (2) I schizophrenic regulatory potential, revealed here by

2~5 , i i I carrier-related manipulations of immunization 50 10 20 30 protocols, traces to the surprising but basic Igh-la Anti DNP Igh-la Anti CGG (ug/ml) (units) capabilities of the hapten-specific system. *K = KLH; DK = DNP-KLH; DC = DNP-CGG The suppression-effector mechanism can establish Fig. 3. Anti-hapten responses to the hapten on an unrelated individual response patterns to the different carrier are specifically suppressed once hapten-specific determinants on a typical antigen suppression has been induced. Protocol and assay details are described in the legend for Fig. 2. The effector mechanism responsible for hapten- Data in parentheses after anti-DNP response 'bars' show the specific suppression appears to be similar to a con- average K a M -~ x l0 b for that response. certed set of idiotype-suppression mechanisms (for 44 immunology today, March 1981 examples, see Refs 7-9), each of which prevents the animals immunized 6. However, when these variables expression of memory B cells committed to produce are held constant, the deciding element is whether the immunoglobulins with a certain variable region carrier-specific mechanism inducing hapten-specific structure (anti-hapten combining site). However, two suppression becomes functional before or after the additional properties distinguish hapten-specific from animal has established antibody production to a idiotype-specific suppression. First, the hapten- determinant on the relevant carrier. Therefore, in a specific system regulates responses according to both given animal responding to a given antigen, the rate at constant region (allotype/isotype) and variable-region which a determinant first presented on the antigen determinants on the IgH chain whilst idiotype-specific drives the hapten-specific mechanism into a respon- mechanisms do not make isotype or allotype distinc- sive configuration is critical. tions. Secondly, the hapten-specific system shows an That is, Since the suppression-induction mechanism unprecedented bistable regulatory pattern in which matures several days after antigenic stimulation the the response first established (suppression or antibody determinants on a newly introduced carrier molecule production) tends to prevent a shift to the alternative (antigen) have a brief period during which to establish response 6. antibody production and impair the subsequent For example, the hapten-specific suppression in- induction of suppression. For each determinant that duced by exposure to the KLH/DNP-KLH sequence succeeds, antibody production will ensue; for each lasts during one to three subsequent DNP-KLH or determinant that fails, a stable suppression is estab- DNP-CGG stimulations but is eventually reversible lished that can be reversed (occasionally) by intensive (more easily for IgG1 antibody responses than for exposure to antigen. Thus, determinants like DNP, responses in other IgG isotypes). Similarly the anti- which induce broadly heterogeneous antibody DNP responses established by initial exposure to responses when presented initially on an appropriate DNP-KLH or DNP-CGG are reversible by exposure carrier, are capable of generating a sufficiently rapid to a carrier/hapten-carrier sequence where the hapten response to prevent the induction of suppression, is on an unrelated carrier, but the suppression induced whilst determinants on antigens commonly studied in under these conditions is less marked (IgG1 responses immunogenetic analyses (for example) appear to be are not suppressed and other IgG isotype responses inherently less able in this way and consequently tend become suppressed in about half the animals). These to fall prey to the suppression-induction mechanism response characteristics, partly reported in the early more frequently. 1970s but interpreted differently at that time 5, demonstrate that regardless of whether antibody production or suppression is established initially, the response established will tend to be maintained Regulatory circuits can account for hapten-specific against substantial pressure towards establishment of regulation the opposite response. Devising a set of plausible cellular interactions to This bistable regulation constitutes a novel form of account for bistable immunoregulation could be quite immunological memory whose existence accounts for difficult a priori; however, the theoretical 'regulatory the difference between expectation and actuality in the circuits' we recently proposed for quite different response of animals to antigenic stimulation. Once reasons 1° actually form a ready-made model that this type of 'configurational' memory is considered serves to predict most of the properties of the hapten- along with the response to individual determinants on specific system, including its bistable capabilities. The complex antigens, an overall picture develops in which design of these circuits grew from an attempt to the probability of antibody being produced to an develop an integrated picture of the regulation of individual determinant is fixed by a complex set of heterogeneous antibody response in which idiotype- parameters that influence whether the hapten-specific specific regulatory processes were placed centrally and system will be established in a responsive or suppres- considered to result from a closed circuit rather than sive configuration, and in which the responses to the an open-ended network of T-cell and B-cell interac- different determinants on the stimulating antigen tions. Following this premise to its logical conclusion, made by individual animals reflect those probabilities. we arrived at a 'Core' regulatory circuit that could be Therefore, most antigens can be expected to induce induced to help or suppress production of individual varied but relatively stable response patterns to the antibodies according to conditions in the immuno- array of determinants on the antigen. genic environment. Our studies have shown that the parameters which In this model, 'Core' circuit helper and suppressor influence whether antibody production or suppression T cells use a complementary receptor recognition becomes established include the characteristics of the system based on IgH constant region and variable determinant and carrier molecule, the physical form region determinants to recognize each other and the B in which the molecule is presented, the adjuvants that cells that the circuit regulates. In addition, these T accompany it and the genetic constitution of the cells are organized to maintain the circuit in one of immunology today, March 1981 45

mechanism that ultimately determines whether an individual B cell will be permitted to expand and differentiate to an antibody-producing cell population. Th(9 = Tso~"~Tho p B id- id + id- id + These properties, defined before we were aware of the existence of the hapten-specific regulatory system, TRIADS TS O ~,-*,~Th (~ =Tso provided considerable comfort and guidance as the id ÷ id- id + surprising results of our current studies began to unfold. Thus, although the circuit model as such Tho ~Ts @---::.-;Th O id- id + id- would have to be expanded and modified to accom- modate some findings, and although other models could presumably be devised to account for these findings, we feel for the moment that the kinds of CHAIN •.. Tho----*TsE)*,~*Tho----~Tso~ThO-----~ B theoretical cell interactions suggested offer a quite id- id + id- id + id- id + reasonable approach to exploring the actual cell i ...... i interactions involved in hapten-specific regulation. We must admit to some surprise at finding that our attempts to explain the curious and unexpected inability of carrier-primed animals to produce anti- CIRCUIT TS 0 ~--~----'-~--~ T h 0 ~ B hapten responses when stimulated with DNP on the id + id- priming carrier resulted in the description of a CRC regulatory system matching the properties of the model which had previously occupied our attention. Similarly we did not expect that these studies would

Tho ~-----:----:.-::. TS ~) lead us to question contemporary views on the id- id + mechanisms involved in carrier-specific suppression or to suggest a drastically different alternative to such views. Although we specifically excluded consideration of this suppression mechanism in drafting the Fig. 4. Theoretical core regulatory circuit. circuit model, because the information available The B cell carrying id + V H Ig surface receptors is helped by a T cell, Th C), with complementary (id~ receptors. This Th, which seemed inadequate to define a consistent circuit, we is analogous to an idiotype-specifichelper T cell, is depleted by a never thought to question the basic concepts of suppressor T cell, Ts(D, analogous to an idiotype suppression T carrier-specificity. Nevertheless, while searching the cell. It carries id ~ receptors similar to the B cell and therefore literature for the roots of our current findings, we could bind the same ha~enic determinant. Th (~) and Ts (~) are realized that most of our ideas about how the immune distinguished from Th (.!) and Ts C) by non-VH related surface determinants. The configuration of this circuit is such that it will system works were derived from conclusions predating tend to stabilize either with Th C) and Ts (~ dominant or Th (~) modern descriptions of the complex interactions and Ts(~ dominant. Thus it will tend to maintain itself (stabilize) possible within this system. In addition, we found that either in a help or suppression configuration depending on the bringing current data to bear on the results from these initial conditions of antigenic stimulation. older studies cast a new light on a number of (This figure appeared in Herzenberg, L. A., S.J. Black and L. A. Herzenberg (1980) Eur. J. Immunol. 10, pp. 1-11) unexplained findings that had more or less drifted into oblivion during the intervening years. Thus, our ex- perience confirmed anew the existentialist view of his- two stable configurations (suppression or help) that is tory which, paraphrased slightly, states that while the maintained unless the conditions of antigenic stimula- results of experiments do not change, the perspectives tion change dramatically (Fig. 4). Thus, the model on their meaning undergo substantial alteration with defines a series of circuits that individually regulate each new relevant discovery. the production of a given anti-hapten antibody molecule according to its combining-site and IgH The authors wish to express their thanks to Dr. Kyoko allotype or isotype and also tend to individually Hayakawa, Mr. F. T. Gadus and Ms. Jean Anderson for stabilize according to the conditions under which the their help in preparing this manuscript. animal first 'sees' the relevant hapten, remaining in This research was supported, in part, by grants from the this configuration until stimulatory conditions dictate National Institute of Health (HD-01287). change. Carrier-specific interactions are placed in auxiliary regulatory circuits in this model. These contribute References (along with other types of regulatory interaction) to ] Cantor, H., Hugenberger, J., McVay-Boudreau, L., Eardley, the configuration of the 'Core' circuit. The 'Core' D. D., Shen, F. W. and Gershon, R. K. (]978) J. Exp. Med. circuit is therefore viewed as a central integrative 148, 87]-877 46 immunology today, March 1981

2 Herzenberg, L. A., Tokuhisa, T. and Herzenberg, L. A. (1980) 7 Eichmann, K. and Rajewsky, K. (1975) Eur. J. Immunol. 5, Nature (London) 285, 664-666 661-666 3 Rajewsky, K., Schirrmacher, V., Nase, S. and Jerne, N. K. 8 Hetzelberger, D. and Eichmann, K. (1978) Eur. J. Irnmunol. 8, (1969)J. Exp. Med. 129, 1131-1143 839-846 4 Mitchison, N. A. (1969) Eur. J. Irnmunol. i, 10-17 9 Woodland, F. and Cantor, H. (1.978) Eur. J. lmmunol. 8, 5 Katz, D. H., Paul, W. E., Goidl, E. A. and Benacerraf, B. 600-606 (1970) J. Exp. Med. 132, 261-282 10 Herzenberg, L. A., Black, S. J. and Herzenberg, L. A. (1980) 6 Tokuhisa, T., Herzenberg, L. A. and Herzenberg, L. A. (~981) Eur. J. Immunol. 10, 1-11 in Herzenberg, Leonore a., Ph.D. Thesis, 202 pp. University Paris 11 Herzenberg, L. A., Blac,, S. J., Tokuhisa, T. and Herzenberg, VI, France L. A. (i980)J. Exp. Med. 151, 1071-1087

(,°.,°,, ) New directions in research

Eosinophil effector functions

Some new and interesting properties of eosinophils remains uncertain. More significant, in the context of have come to light as a direct result of studies on the parasite-killing mechanisms, was the discovery that interaction of eosinophits with schistosomula. This neutrophils attached to the surface of antibody-coated work was stimulated by the finding that eosinophils, schistosomula, showing membrane fusion, and then but not neutrophils, killed IgG-antibody-coated migrated away, leaving behind 'footprints' of inverted schistosomula in vitro ~. This effect has been confirmed membrane on the parasites (which seemed remark- in vivo and has raised many questions about its sig- ably unaffected by this encounter). On the other hand nificance in parasitic diseases in general z. eosinophils attached themselves firmly to the parasite, An initial surprise was that although eosinophils without such membrane fusion, and the schisto- develop the capacity to bind IgG (measured as IgG- somula died as a consequence. The spaces between coated erythrocytes) as they mature in the marrow, eosinophils and parasite surfaces sometimes contained eosinophils in the blood of normal individuals have a electron-dense material: peroxidase and the eosino- very low binding capacity compared with other phil major basic protein (MBP) 9. MBP itself was phagocytic cells 3. However, it was then found that in found to kill schistosomula ~°. many diseases, including filariasis and schisto- Two stages in the binding of eosinophils to somiasis, the binding capacity could be greatly antibody-coated schistosomula were defined I~. The increased 4. This appeared to be the result of interac- first was IgG-dependent, and could be reversed. It was tion between secreted lysosomal enzymes and eosino- mimicked by binding through concanavalin A. The phil cell membranes. The removal of sialic-acid resi- second occurred later, apparently involved MBP and dues from membrane constituents induced additional was irreversible. Comparable effects occurred when binding capacity s. Under these conditions eosinophils the calcium ionophore A23187 acted on con A-bound bound complexed IgG as effectively as neutrophils. eosinophils. Because a principal action ofA23187 is to This mechanism has been recently confirmed in beige induce the secretion of lysosomal enzymes, it was (Chediak-Higashi) mice which have defective cyto- thought that the secretion products themselves, in- skeletal control of lysosomal granule fusion and cluding MBP, were responsible for the irreversible release. In these mice eosinophils had reduced IgG attachment. This could be another property of lysoso- binding capacity but were responsive to lysosomal mal enzymes and one which also induced an addi- enzymes secreted from normal cells 6. tional binding capacity for IgG-coated erythrocytes. Biochemical studies defined several important A further step forward in understanding the pro- structural and functional differences between eosino- perties of eosinophils has come with the discovery that phils and neutrophils. ~2SI-labelling techniques for the number of blood eosinophils in patients with defining individual cell-membrane constituents schistosomiasis or other diseases correlated with their showed that eosinophils had some membrane com- capacity to kill schistosomula in vitro ~2. Although ponents which were not present in neutrophils 7 and direct measurements of the eosinophil binding these altered after stimulation with complexed IgG 8. capacity for IgG-coated erythrocytes were not made, it Although it is suspected that these constituents were was probable that it increased in parallel with eosino- receptors for IgG or chemotactic factors, their n~tture phil counts. These results suggested that eosinophils in