Suppression ofthe ImmuneResponse byMicroorganismscell which can suppress response of unstimu-lated...

BAcTmoIOOICALm REVIEWS, June 1975, p. 121-143Copyright Cc 1975 American Society for Microbiology

Vol. 39, No. 2Printed in U.S.A.

Suppression of the Immune Response by MicroorganismsJOHN H. SCHWAB

Department of Bacteriology and Immunology, School of Medicine University of North Carolina, Chapel Hill,North Carolina 27514

INTRODUCTION ................................... 121

MICROBIAL AGENTS 125

Group A Streptococcus ................................... 125Membrane-associated immunosuppressant 125

Pyrogenic exotoxin 126

Teichoic acid 127

Lymphocyte mitogens ................................... 127Corynebacterium parvum 127

Endotoxin 128

Cholera Enterotoxin 130

Gram-Negative Bacterial Cells 131

Pseudomonas aeruginosa ................................... 131

Bordetella pertussis ................................... 131Klebsiella pneumoniae ................................... 133Escherichia coli ................................... 133

Bacterial Enzymes ................................... 133L-Asparaginase 133

Ribonuclease 134

L-Glutaminase 134

Mycoplasma 134

Mycobacteria and Freunds Adjuvant 135

Mycobacterium leprae 135

Protozoan Parasites 135

Metazoan Parasites 136

CONCLUSIONS AND SPECULATIONS 136

LITERATURE CITED 138

INTRODUCTION

Man and other animals have evolved withtheir microbial flora, providing a long andintimate association between many bacterialspecies and animal tissues. Therefore, it isreasonable to expect that the microbial florainfluences the physiological systems with whichit has evolved, including the specific immunesystem. Bacteria can modulate the immunemechanism in several ways, including adjuvant(immunopotentiating) effects (108), suppres-sive effects, and through antigens cross-reactivewith tissue antigens, which may induce circum-vention of tolerance (151). Genetic regulation(98) and a self-regulating or feedback system(136, 147) are of major significance in control ofthe immune response, but in addition, mi-crobial products can participate as exogenousimmunoregulatory agents. This review is pri-marily concerned with evidence that certainbacteria, as well as protozoan and metazoanparasites, can suppress the specific immuneresponse. Immunosuppression by viruses willnot be considered here.

Presentation of bacterial cells to a host, either

as infection or vaccine, exposes the animal tomore than the immunogenic components ofbacteria. Bacteria are not inert particles carry-ing antigenic groups; they contain a variety ofpharmacologically active components. A fewillustrative examples include: activation ofthird component of complement (C3) by endo-toxin (51); the effect of peptidoglycan on phago-cytosis, cell migration, and cell growth (72); anda factor chemotactic for macrophages producedby anaerobic corynebacteria (148). In addition,both immunopotentiating and suppressing ac-tivities often can be demonstrated in the samebacterial preparation (Table 1). For this reasonit is impossible to define the mechanism bywhich bacterical cells affect immune systemswhen intact cells or crude extracts are used. It isessential to use at least partially separatedcomponents to interpret mechanisms at thecellular or molecular level. The other side of thecoin, of course, is that under natural conditionsthe animal is seldom exposed to selected com-ponents of bacteria; thus, the possibility existsthat the influence of bacteria may be quitedifferent from that obtained with their compo-nent parts.

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Table 1 lists the bacterial species or bacterialproducts reported to have immunosuppressiveactivity and summarizes some salient features.It is obvious that this represents an extremegeneralization of the data for the purpose oforganizing the discussion. The effect on anti-body response is illustrated for each agent wheninjected before, with, or after antigen. Theeffect on cell-mediated immunity (CMI) isseparated into proliferative and effector phases,which are probably mediated by two distinctsubpopulations of T cells (76). (i) Proliferativecells recognize antigenic or mitogenic structuresand are stimulated to divide and differentiate.These T cells are measured by the mixedlymphocyte reaction or by response to mitogenssuch as phytohemagglutinin (PHA). (ii) Effec-tor cells are potentiated by the proliferativesubpopulation to become the activated cyto-toxic T lymphocytes (76). The effector cellpopulation is measured in vivo by the graft-ver-sus-host reaction, delayed hypersensitivity skintests, and tumor or graft rejection. They are alsomeasured in vitro by cytotoxicity for target cells.The heading "Mitogenicity" in Table 1 indi-cates where we have information on the abilityof the microbial agents to stimulate prolifera-tion of lymphocytes through mechanisms notinvolving specific antigenic recognition. Toxic-ity reflects the lethal or debilitating effect of theagents in the range of doses needed to inducesignificant suppression in the animal.The first observation based on Table 1 is that

lists of bacterial sources of immunosuppres-sants and adjuvants are very similar. Many ofthe bacteria and bacterial products discussedhere as immunosuppressants are also known toincrease the immune response. This means thatthese agents are affecting homeostatic mech-anisms, a concept presented by White (147).Throughout this review we find that as circum-stances of dose of agent, timing of injectionsrelative to antigen, nature and dose of antigen,etc., are manipulated, the production of anti-bodies or CMI can be increased, decreased, orqualitatively changed.

Another point illustrated in Table 1 is that allpossible effects may be obtained with bacterialagents. That is, we can obtain depression ofantibody formation, or delay of antibody forma-tion followed by increased response. This canoccur with or without depression or stimulationof CMI. On the CMI side we can get depressionof proliferative T cells and/or effector T cells.Thus, it is entirely feasible that both theinduction and the expression phases of theimmune response can be selectively modified ormanipulated by these various agents.

Figure 1 is a diagram of cellular events in theimmune response. In this concept the macro-phage has a central position, reacting withantigen and interacting with T and B cells inboth affector and effector roles. All three celltypes are derived from bone marrow stem cells.Most antigens are probably "processed" bymacrophages which then present carrier andhapten-specific sites to specific recognitionstructures on T or B cells respectively (138).The macrophage itself becomes activated, asdemonstrated by increased protein synthesis,the production of factors which affect functionof T cells, and cytotoxicity for certain targetcells (137). The stimulated B cells can produceantibody cytophilic for macrophage membraneswhich affects macrophage reaction with antigen(111). The T cell may also affect macrophagefunction by releasing an immunoglobulin(IgMs)-antigen complex (or an immunoglobulin[IgT]-antigen complex, 30) which can reactwith macrophage membranes (119).T cells stimulated by antigen can apparently

differentiate into several subpopulations. Thehelper cells, which I have arbitrarily labeled T1,amplify the response of B cells to antigen. An-other subpopulation, labeled T2, is a suppressorcell which can suppress response of unstimu-lated T or B lymphocytes. This population hasbeen described by Gershon et al. (49) andothers (8). The helper and suppressor functionsmay reflect alternative activities of a regulatorcell (48). A third population, T3, comprises theeffector T cells that are stimulated to release avariety of lymphokines, which I indicate here asmediators of inflammation. Whether these aredistinct T cell populations or whether responderT cells can develop into either helper or sup-pressor cells is a moot question discussed re-cently by Kapp et al. (81).B cells stimulated by antigen differentiate

into antibody-forming cells, which may produceimmunoglobulin giving a form of feedback inhi-bition (65, 136). Basophiles and mast cells arestimulated to release mediators of immediatehypersensitivity (histamine, serotonin, etc.)when immunoglobulin E (IgE) bound to theirmembranes reacts with antigen. Polymor-phonuclear leucocytes release other mediatorsof inflammation (enzymes and cationic 'proteinsof lysosomes) after ingestion of antigen-anti-body-complement complexes. All of these cells,which are important effectors of antibody-invoked inflammation, are represented collec-tively in Fig. 1 by polymorphonuclear leuco-cytes. Another cell type can be activated tobecome a cytotoxic effector cell by virtueof a recognition site for a complex of anti-

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MEDIATORS OF INFLAMATION

FIG. 1. Outline of the development, interaction,and activities of cells involved in the immune re-

sponse. The numbers refer to possible sites at whichthe various microbial immunosuppressive agentscould function. These sites are described in Table 2and the text. The K cell function reflects several celltypes and represents the antibody-dependent CMI, inwhich cells are activated to become cytotoxic fortarget cells by binding with antibody-target cellantigen complex. The T cell subpopulations are

numbered arbitrarily. T3 represents the cytotoxiceffector, T cell subpopulation. PMN represents poly-morphonuclear leucocytes as well as mast cells andbasophiles involved in antibody-mediated allergicinflammation. PC represents plasma cell. (Repro-duced with modifications from: The Immune Systemand Infectious Diseases, E. Neter [ed.], S. KargerA.G., Basel).

body and target cell antigen (93, 94). This iscalled antibody-dependent CMI and is accom-

plished by a number of different types ofeffector cells. These have been provisionallydescribed in a recent workship (94) and includeK cells (nonadherent, nonphagocytic, no immu-noglobulin, not T) B cells, macrophages and"others."

I have indicated 10 sites in Fig. 1 where thebacterial agents we will consider could conceiv-ably function to influence either induction or

expression of immunity. Assigning such a site ofaction is highly speculative for most of thesematerials, but it serves the useful purpose ofproviding a framework on which we can attemptto organize a confusing catalog of observations.The cellular and molecular mechanisms atthese sites of action, summarized in Table 2, are

equally speculative.

Antigenic competition could be posed as anexplanation of interference of an immune re-sponse by microbial agents. Impressive evi-dence has been presented indicating that anti-genic competition is thymic dependent. Gers-hon proposes that this suppression is the resultof a nonspecific inhibiting substance releasedby stimulated T cells (48). Feldman et al. (30)propose that IgT-antigen complexes from Tcells bind to receptor sites on macrophages,preventing subsequent antigen-IgT complexesfrom reaching this critical site. Thus, the site ofantigenic competition is at the surface of macro-phages. Those microbial agents which are im-munogens, and whose immunosuppressive ef-fects may be mediated through T cells (Fig. 1,Table 2, Site 4), might function by antigeniccompetition. The problem resolves itself intothe question of whether the nonspecific suppres-sive effect can be ascribed to the immunogenicproperties of the microbial agent, or to someother biological property. In either case theessential point remains that microorganismscan produce immunosuppression.

MICROBIAL AGENTS

Group A StreptococcusA variety of immuno-active components have

been isolated from this organism. These in-clude: the adjuvant effect in rabbits of muco-peptide (peptidoglycan) isolated from the cellwall (72, 74), a mitogen for human lymphocytes(134), and three immunosuppressant agents(Table 1). It has not been determined if all ofthese are distinct molecular entities. The dem-onstration of this variety of immuno-modulat-ing agents is of interest because the group Astreptococcus is among the most common bac-teria infecting man.Membrane-associated immunosuppres-

sant. Malakian and Schwab (95, 96) demon-strated an immunosuppressant in partially pu-rified extracts of mechanically disrupted groupA streptococci. A single injection into mice 1day before sheep erthrocyte (SRBC) antigensuppressed both IgM and immunoglobulin G(IgG) plaque-forming cells (PFC) in the spleensof mice. Both primary and secondary responseswere suppressed if membrane-associated im-munosuppressant (SF) was injected before thefirst or second injection of antigen, respectively.Some suppression was obtained by injection 7days before antigen but injection 1 or 2 daysafter antigen had no effect (95). Immunologicalmemory was not impaired in spite of greaterthan 90% suppression of a primary response(95). Large doses were not toxic and repeated

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TABLE 2. Possible sites of action and cellular or molecular mechanisms of suppression by microbial agents

Sitea Reference Cellular or molecular mechanisms Examples of microbial agentsa

1 47 Bone marrow stem cell distribution or matura- Group A Streptococcus (SF)tion

2 11, 61, 137,146 Macrophage "processing" or orientation of Group A Streptococcus (SPE),c endo-antigen toxin, Mycoplasma

3 75, 110 Stimulation of macrophage inhibitor of lym- C. parvum, endotoxinphocytes

4 8, 42, 48, 50, 97 Stimulation of suppressor T cell or T cell-mac- Group A Streptococcus (SF), Group Arophage complex Streptococcus (SPE), C. parvum, T.

spiralis5 15, 39, 40, 84, 94, 142 Suppression ofT effector cells or K cells (T or K Pseudomonas, endotoxin, cholera entero-

cytotoxic lymphocytes) toxin, L-asparaginase, 7ichinella6 91 Suppression of secretion of mediators by modi- Cholera enterotoxin

fication of cAMP levels (effector lympho-cytes, granulocytes, mast cells)

7 27 C3 activation and binding to B cell receptor site Endotoxin8 2, 31, 46, 106, 152 Interfere with lymphocyte trapping, distribu- B. pertussis (LPF), L-asparaginase,

tion of lymphocytes or antigen recognition by Freunds adjuvantdirectly modifying membrane surface struc-tures

9 136 Stimulate blocking antibody10 52, 77, 88, 120, 135, 142 Lympholysis or lymphocyte depletion Endotoxin, cholera enterotoxin. M.

Ileprae, Plasmodiuma From Fig. 1.bFrom Table 1.c SPE, Pyrogenic endotoxin.

injection did not change its effectiveness (96).Finger et al. report that similar immunosup-pressive activity has been found in other strainsof group A as well as group B streptococci (37).Isolation and identification of the active compo-nent has proceeded slowly, probably because itbecomes more exposed to host degradative ac-tion as isolation proceeds. Starting with osmot-ically disrupted protoplasts, we have recentlyidentified the suppressant factor as a compo-nent of membranes. The extent of suppressionis dose related. Although there is some variationbetween batches of membranes, we have ob-tained 80% suppression of mouse spleen PFCwith a single intraperitoneal injection of 40 ,g ofprotein 1 day before SRBC. To put this in someperspective, comparable suppression by 6-mer-captopurine requires four injections of 3 to 4mg/mouse, which is very near the lethal toxicdose.

In experiments designed to define the mecha-nism of action at the cellular level, it wasobserved that some bone marrow stem cellfunctions of suppressed mice are affected (47):(i) hemopoiesis was reduced as measured by"9Fe incorporation in lethally irradiated mice re-constituted with syngeneic bone marrow cellswhich had been exposed to SF, and (ii) syner-gism of bone marrow cells from SF-injecteddonor mice with normal thymus cells was de-pressed in reconstitution of the immune re-sponse in lethally irradiated recipients. Theseresults indicate a selective suppression of bone

marrow stem cells, which corresponds to site 1in Fig. 1. We also showed that SF alone has nomitogenic effect on mouse spleen cells (47). Thiswas measured by [3H]thymidine incoroporationand by failure to induce cytotoxicity for 5 Cr-labeled target cells. In contrast, SF does stimu-late certain T cell responses: (i) [3H]thymidineincorporation in response to suboptimal doses ofPHA was increased by previous or simultaneousin vitro exposure of spleen cells to SF, and (ii) inthe mixed lymphocyte culture reaction, parentalB1O.A spleen cells from SF-injected mice re-sponded 10 times as vigorously to F1 hybridcell as normal B1O.A spleen cells. These stud-ies are still in progress, but we tentativelyconclude that a component associated withstreptococcal membrane will suppress anti-body-forming B cells while stimulating certainT cell responses. This suggests that SF mayfunction by stimulation of a suppressor T cellpopulation (49) or T cell-macrophage complex(42). This corresponds to site 4 in Fig. 1. Wehave no direct evidence, however, that the lym-phocytes stimulated by SF are a suppressor Tcell subpopulation. It is interesting that theseeffects are the opposite of those obtained withCorynebacterium parvum, discussed below,which functions as a stimulator of antibody pro-duction while inhibiting T cell-mediated immu-nity (75).Pyrogenic exotoxin. Hanna and Watson (61)

reported that pyrogenic exotoxin, a highly puri-fied extracellular protein from group A strep-

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totocci, would suppress the antibody response ofrabbits to SRBC. Rabbits given three intrave-nous (i.v.) injections with 7 tg/kg (0.002 meanlethal dose) at 3 h, 1 day, and 2 days after SRBCshowed a depressed IgM serum antibody anddirect PFC in the spleen at 4 to 5 days afterSRBC. By 10 days the animal had recoveredand the response was above control levels. Theauthors ascribe the transient suppression to theinhibition of phagocytosis and processing ofantigen by macrophages, which they had shownearlier (site 2, Fig. 1). They point out that it isnot unusual that suppression, even inducedwith purified materials, is followed by enhancedantibody production. In a subsequent paper(62) they propose a differential susceptibility,or rate of recovery, of antibody producing Bcells and T suppressor cells to explain theirfindings. This material is also a potent mitogenfor human lymphocytes (Watson, personal com-munication).Teichoic acid. Glycerol teichoic acid isolated

from group A streptococci has also been re-ported to suppress antibody formation to SRBCin mice (101). A maximum inhibition wasobtained when teichoic acid was injected 1 or 2days before antigen. Considering the relativepurity of this material, a very large dose of 5.0mg/mouse was required to achieve 50% inhibi-tion of PFC. This is more than 100-fold greaterthan the dose of the partially purified mem-brane component required for suppression. Thespecificity of the inhibition was not determined,and the question arises as to whether thisnon-immunogenic teichoic acid is cross-reactivewith a SRBC antigen and is acting as a tolero-gen.Lymphocyte mitogens. There has been con-

siderable interest in nonspecific mitogenic ef-fects of streptococcal products on lymphocytes.Taranta and co-workers demonstrated that themitogen associated with streptolysin S is sepa-rable from hemolytic activity (134). It was alsoreported (82) that hot HCl extracts of group Astreptococcal cells, cell membranes, cell walls,and cytoplasm contain a lymphocyte-trans-forming factor for human cells. This is appar-ently nonspecific since lymphocytes from cordblood can be stimulated, and a high percentageof cells are transformed. These authors statethat the HCl extract is possibly identical withtransforming activity associated with strep-tolysin S preparations. Streptococcal L-formcultures also have been reported to produce aheat-stable mitogen, not correlated with he-molysin production (21). The obvious questionsposed by these observations are the relationshipof the mitogenic activity to the streptococcal

immunosuppressants, and the in vivo signifi-cance of the mitogens. The pyrogenic exotoxinprepared by Kim and Watson is a potentmitogen (personal communication). SF studiedby Schwab and colleagues has no mitogenicactivity by itself on mouse or human lympho-cytes, which distinguishes it from these prod-ucts. Concerning in vivo significance, Quagliataand Taranta have extended their studies todemonstrate suppression of adjuvant disease inrats by crude culture filtrate injected daily for20 days before adjuvant (118). The complexityof bacterial components in the filtrates makes itimpossible to relate this effect to any of thedescribed immunosuppressants or mitogens.The same difficulty arises in interpretation ofmacrophage migration inhibition induced byadding bacterial culture filtrates to human orguinea pig cells (129). It is relevant to this lastpoint that mucopeptide from group A strep-tococcal cell walls has been shown to inhibitmacrophage migration directly without induc-tion of a detectable migration inhibition factorby lymphocytes (71).

Corynebacterium parvumThis is apparently a heterogeneous, poorly

defined collection of organisms for which theterm anaerobic coryneforms has been used(148). All of the studies with these organismsthus far reported have employed suspensions ofwhole killed bacterial cells. Thus, it is verydifficult to determine mechanisms and to de-cide if the various effects obtained are causallyrelated, or reflect distinct events initiated bydistinct bacterial components. Several reportshave shown that C. parvum is a good adjuvantwhen injected before antigen (75, 112). Injectionof this organism will also increase immunity ofmice to transplanted tumors (150) increaseresistance to infection of mice with Brucellaabortus and Bordetella pertussis (1) and in-crease protection against protozoal infection(113).

It is, therefore, most interesting that lympho-cytes from mice injected with C. parvum show adepressed responsiveness to PHA, as well asreduced mixed lymphocyte culture and graft-versus-host reactivity (126). The inability ofthese lymphocytes (probably T cells) to re-spond is reversed by removing adherent cellsfrom the spleen or peripheral blood cell sus-pensions (127). This effect of C. parvum isfurther emphasized by experiments with theT-independent antigen, type III pneumococ-cal polysaccharide (75). In this study thePFC response was increased in mice by injec-tion of C. parvum 4 days before a 2.50-gg dose of

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polysaccharide antigen. However, if the C.parvum was injected simultaneously with poly-saccharide, the immune response was de-pressed. There was also a transient suppressionof response of spleen cells transferred to irradi-ated mice which had been injected with C.parvum 4 days previously.

Recently Warr and Sljivic (141) have alsoshown that antibody responses to T-dependentSRBC and T-independent pneumococcal poly-saccharide can be either enhanced or sup-pressed in a rather unpredictable fashion. Thetime of injection of C. parvum relative toantigen and dose of antigen are importantfactors.

Further evidence for the capacity of C.parvum to depress CMI has been presented byCollins and Scott (20). They showed that miceinjected with 700 ,g of killed organisms did notdevelop characteristic delayed hypersensitivityagainst Salmonella enteritidis, although themice were more resistant to infection with thisorganism. The role of the spleen in depression ofdelayed hypersensitivity responses has beenemphasized by Scott (128).Another example of apparent suppression

rather than enhancement of immunity inducedwith C. parvum is reported by Ruitenberg andSteerenberg (121). Rats injected i.v. with 7 mgof C. parvum were challenged 3 days later withTrichinella spiralis larvae. Over the next 15days a greater number of adult worms persistedin the intestinal tract of C. parvum injectedcompared to control groups.Hovward et al. (75) concluded that the effects

of C. parvum are mediated through its effect onmacrophages. They suggested that a "factor"may be released from macrophages which canblock response of T cells to PHA or allogeneicantigens, and in higher concentrations B cellsare affected (site 3, Fig. 1). It is also possiblethat C. parvum suppresses a suppressor T cellsubpopulation (49) or a T cell-macrophagesuppressor complex (42) as described above(site 4, Fig. 1).The capacity to induce suppression of CMI

may well be a far more significant role of C.parvum than any adjuvant effect. The fact thatfactors such as timing, dose, and nature ofantigen can determine reduction or increase ofeither CMI or humoral immunity urge cautionin using agents such as this in immunotherapy.

It is possible that the affect of C. parvum on Tlymphocytes or CMI and the adjuvant effectsare distinct manifestations. Protection againsttumors and infection may result from stimu-lated activity of macrophages (20), and thesuppression of lymphocyte functions or in-

creased antibody production may be a separateactivity and reflect distinct bacterial factors. Apossibility which should be considered is thatthese suspensions of formalin-killed bacterialcells contain pharmacologically active compo-nents which could be released from the macro-phages in the process of bacterial degradation.Perhaps the simplest explanation is that morethan one active component occurs in thesebacterial cells and these may have opposingeffects. With different experimental manipula-tions, different subpopulations of T or B cells(75) or macrophages (148) are affected. Theimportance of the timing of injections of C.parvum relative to antigen or tumor cells couldreflect differential degradation and release ofthe different bacterial cell components. What-ever the explanation, these studies provide agood example of the complex interactions be-tween microorganisms and the immune system.

EndotoxinEndotoxin is a good example of a chemically

well-characterized bacterial component inwhich the capacity to enhance or suppress animmune response may be the property of asingle molecular entity, lipid A.Johnson and co-workers (77) and Franzl and

McMaster (43,100) demonstrated that Salmo-nella typhi endotoxin could function to eitherenhance or suppress antibody formation in miceimmunized with SRBC. The dose, route, andtiming of injections of both endotoxin andantigen are important determinants. If antigenis given with endotoxin or shortly thereafter, anincreased antibody response is obtained. Incontrast, injection of endotoxin 1 or 2 daysbefore antigen, and by the same route, willsuppress and often completely inhibit antibodyformation. It was suggested that these responsescould reflect different components of the endo-toxin lipopolysaccharide molecule (43). Thus,inhibitory activity may be associated withloosely bound lipid and enhancing action withthe toxic portion of the molecule. There is noevidence to support this proposal. Finger et al.(36) have also shown significant suppressioninduced in mice injected with large doses ofendotoxin given daily for 9 days before SRBC.Some information on the mechanism of sup-pression is provided by histological studies(77,100). All mice given endotoxin showed anextensive depletion of lymphocytes in thespleen and lymph nodes. This depletion per-sisted for 6 days in the suppressed mice (endo-toxin 2 days before antigen) and stimulatedlymphocytes (pyroninophilic cells) were very

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scarce. In mice giving an enhanced response(endotoxin with antigen), the lymphoid tissuerecovered cellularity after 24 h and there weremany more large pyroninophilic cells. Thus, theconclusion is that endotoxin suppression is dueto a lymphotoxic effect (site 10, Fig. 1). If this isaccompanied by antigen stimulation, rapid re-generation of the lymphoid tissue occurs to yieldincreased pyroninophilia and antibody forma-tion. How these changes are related to themitogenic effects of endotoxin measured by invitro stimulation is not clear, but this mitogenicactivity is associated with the toxic lipid Amoiety (116). We would now interpret thehistological findings of McMaster and Franzl(100) as showing a selective depletion of thymic-dependent lymphocytes from the periarteriolarsheath in the spleen and the paracortical regionof lymph nodes after injection of endotoxin.This is consistent with a description of thehistological changes induced in the thymus byendotoxin (120). Mice were injected i.v. with 100gg (200 Mg = 1 mean lethal dose) of endotoxinand histological sections were prepared fromthe thymus at intervals of 8 h to 14 days. Within8 h after endotoxin, there was obvious necrosisof lymphocytes and an increase of pyronino-philic cells in the cortex. By 3 days the changesresembled an inversion of cortex and medulla.Restoration began by the 3rd to 5th day and wascomplete by day 14. The authors suggest thatthese effects can be ascribed to release ofadrenal hormones and their subsequent lym-phocytic effect, which is primarily on T cells.

In contrast, in vitro studies show a selectivestimulation of mouse B cells by the toxic lipid Amoiety of endotoxin (116). Furthermore, otherreports indicate that endotoxin replaces the Tcell requirement of SRBC -Antigen in mice,rendering this immune response T- independent(104). This perhaps reflects a direct mitogenesisof B cells by endotoxin, thus increasing respon-siveness to antigen. According to the proposal ofDukor et al. (27), endotoxin substitutes for theT cell requirement by activation of C3 via thebypass mechanism. This is based on the con-cept that B cells require two signals: specificantigen binding and a second signal which canbe provided by helper T cells, binding of C3, ordirectly by certain T-independent antigens(117).From these in vitro and in vivo observations, I

synthesize the following hypothesis: when endo-toxin is injected there is a selective depletion ofT cells. If antigen is injected at the same time,there can also occur a synergistic effect ofantigen and endotoxin on B cells, giving anenhanced response. In this case the endotoxin

provides the second signal directly or via C3binding and renders the response T independ-ent. If antigen is delayed for 2 days, the T cellsare still depleted and the B cells have beenpartially stimulated to differentiate because ofC3 binding. This puts them out of phase in aresponse to antigen binding and results insuppressed response. According to this concept,the major effect of endotoxin is at site 7, Fig. 1.The discrepancy between in vitro and in vivostimulation of B cells by endotoxin may beanalogous to the distinctive in vitro and in vivoeffects of asparaginase (10).The earliest report of immunosuppression by

a bacterial product was the report of Bradleyand Watson (12) that endotoxin (4 mg/kg perday; Difco; E. coli) diminished actinophage-neutralizing antibody in serum of BALB/symice. These results differ from those of Franzland McMaster in three important aspects: (i)endotoxin was especially effective if given over aperiod of 6 days after antigen and the serum wascollected on day 7; (ii) the response was onlydelayed, since if endotoxin was stopped for 3days the titer of antibody was at or abovenormal controls; and (iii) endotoxin caused amore rapid loss of antibody from serum. Theseobservations suggest that the influence of endo-toxin on the immune response as measured bythese workers operates by a mechanism verydifferent from that reported by Franzl andMcMaster (43).A third mechanism by which endotoxin can

suppress the immune response to certain anti-gens was reported by Whang et al. (145).Suppression of antibody production against acommon antigen of Enterobacteriaceae in rab-bits occurred only when antigen and endotoxin(lipoid A, E. coli) were incubated togetherbefore injection. There was no effect wheninjected separately at the same time. Therefore,the effect is due to interaction of endotoxin andantigen. This might affect processing of anti-gen, corresponding to site 2 in Fig. 1. By usingendotoxin from S. dublin or lipoid A from E.coli, suppression of antibody against an arAigencommon to Staphylococcus aureus and Bacillussubtilis (probably teichoic acid) was also ob-served (146). However, suppression of responsewas not obtained against all antigens, sincethere was no effect on antibody against thepolysaccharide Vi antigen (146).

Suppression of CMI by endotoxin has alsobeen reported (40). In this report, however, onlya depression of the effector mechanisms ofdelayed hypersensitivity was demonstrated(site 5, Fig. 1). As observed with other biologicaleffects of endotoxin, poly(I) * poly(C) gave a

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comparable effect. The authors suggest thatendotoxin alters the distribution of antigen-sen-sitive lymphocytes (site 8, Fig. 1) or affects cellmembrane permeability. The latter is inferredfrom the increased toxicity of amanitin inendotoxin-treated animals (40). That inductionof CMI may be suppressed, as well as effectormanifestations, is indicated by other studies ofFloersheim and co-workers using gram-negativebacterial vaccines (39, 41). The recent report byNelson (110) that stimulated macrophages fromendotoxin-injected mice suppress lymphocyteresponses is also pertinent (site 3, Fig. 1).

Cholera EnterotoxinCholera enterotoxin (CE) has only one known

biological activity, stimulation of adenyl cy-clase, which leads to increased levels of cyclicadenosine 3',5'-monophosphate (cAMP). Otheragents including endotoxin and nucleotides canalso stimulate adenyl cyclase (149), but CE isunique among such agents in that it induces aslow, protracted elevation. Chisari et al. (17)have convincingly shown that CE can enhanceor suppress the antibody response to SRBC inmice. Both timing and dose are very important.Thus, CE is an adjuvant when 0.05 Ag isinjected with antigen and a suppressant wheninjected 12 h before or after antigen.

In vitro, CE inhibits IgE-mediated release ofhistamine from human basophiles and lysis ofallogeneic mastocytoma cells by sensitizedmouse lymphocytes (91). It was suggested thatthe common mechanism here may be inhibitionof secretion by effector cells. This correspondsto sites labeled 6, in Fig. 1. Thus, one functionof CE is to inhibit cells involved in eithernonspecific or immune-elicited inflammationby increasing intracellular levels of cAMP. Theinhibition by CE of both the antibody-mediatedand the CMI reactions can be blocked by eitherCE-specific antibody or toxoid (91). Both anti-body against CE and toxoid interfere with CEbinding to the cell by competing with themembrane site for CE. As a result CE cannotbind to the cell membrane and cannot affectintracellular levels of cAMP.The in vivo suppression of CMI described by

Henney and co-workers (67) also is primarily onthe efferent side. C57BL/6 mice were immu-nized with BDA/2 mastocytoma cells intraperi-toneally (i.p.). Cytolytic activity of spleen cellsobtained 11 days after immunization was mea-sured in vitro by 5'Cr release. One microgram ofCE injected on day of immunization (day 0) hadno effect, whereas injection of CE 4 days afterimmunization gave 70% inhibition of cytolyticeffect and injection on days 7, 8, 9, or 10 gave

100% inhibition. In contrast, no prolongation ofskin grafts across this same allogeneic barrierwas obtained with several variations of dose andtiming of CE injection. Serum antibody mea-sured by agglutination of allogeneic cellsshowed a different pattern of suppression; CEinjected with cells on day 0 resulted in a titer onday 11 of 243 compared to untreated controls of187. CE injected on day 4 gave a titer of 15 andCE on day 10 did not change the titer. Noantibody to CE was detected. Cook et al. (22)measured in vitro secondary response to keyholelimpet hemocyanin by rabbit lymph node cells.CE or dibutyryl-cAMP added to cultures en-hanced antibody synthesis, presumably relatedto increased intracellular cAMP (22). Nucleo-tides, which enhance antibody synthesis, alsoincrease adenyl cyclase in spleen cells in vitro(149). There is no obvious way to relate these invitro and in vivo studies.

In an effort to correlate their results withchanges in in vivo levels of cAMP induced byCE, Henney et al. (67) measured cAMP inspleen cells removed at intervals after CEinjection. Cyclic AMP levels returned to normalby 72 h after in vivo exposure. Thus, theinhibition of CMI is retained for a long periodafter the transitory elevation of cAMP. Theauthors conclude that the significant inhibitionof the efferent arm induced by CE injected atday 10 could be related to the increased levels ofcAMP (site 6, Fig. 1) but another mechanism isresponsible for the effect of doses given earlier,which are apparently influencing the afferent orinductive phase of the immune response.A recent report (142) confirms that CE in-

creases cAMP levels in splenic white cells ofmice, and it was further shown that this wasaccompanied by a decrease of splenic white cellcount to 21% of normal at 72 h and of peripherallymphocytes to 48% at 48 h. This indicatescaution in ascribing the effects of CE to changesin cAMP; it could be functioning through selec-tive destruction of lymphocytes (site 10, Fig. 1)or by affecting distribution (site 8, Fig. 1).Evidence on this point was presented by Chisariand Northrup (16). Using a model of delayedhypersensitivity and granuloma formation inresponse to Schistosoma mansoni eggs, Warrenet al. (142) concluded that CE produced a moreimpressive suppression (effector arm) of CMIthan antilymphocyte serum or other im-munosuppressive measures tested. Two micro-grams of CE injected 1 to 3 days before elicita-tion of a delayed hypersensitivity response(footpad swelling) was very effective. Immunegranuloma formation and migration inhibitionfactor production were also inhibited. It is

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significant that CE could also reduce somefacets of the schistosome disease in mice and incontrast to earlier reports (67) skin graft sur-vival was prolonged.The question of whether all or any of the

immunodepressive effects of CE are related tochanges in cAMP remains to be determined.The fact that nonimmune foreign body granu-loma formation was also suppressed (142) em-phasizes the point that CE can modify elicita-tion of CMI as well as induction of the immuneresponse.

Gram-Negative Bacterial CellsPseudomonas aeruginosa. Floersheim and

co-workers have investigated a series ofgram-negative bacteria for their effect on CMI.On the basis of some suggestive clinical obser-vations recorded in the literature, they initiatedstudies on Pseudomonas aeruginosa (39). Crudeextracts of sonically disrupted bacteria wereinjected i.p. into guinea pigs 30 min before and8 h after elicitation of a delayed hypersensitivityreaction with tuberculin. A dose of 0.03 mg/kgreduced the skin thickness at 24 h by 46 to 64%of controls. Pyocyanine and S. typhi endotoxindid not significantly affect the reaction. Sur-vival of skin grafts from BALB/c to C3H micewere also prolonged by large doses of 5 mg/kggiven six times a week subcutaneously begin-ning with the day of grafting. Median survivalon control mice was 11.1 i 2.2 days. The long-est graft survival achieved was 19.3 ± 1.0 days.Because of the toxicity of these large doses,there were only four surviving mice in thisgroup. Several other experimental groups, how-ever, also showed a significantly extended graftsurvival. The authors report that these resultsare the best obtained so far with bacterialreagents which include S. typhi endotoxin, S.paratyphi B, and B. pertussis. The mechanismis very uncertain. The effect may be entirely onthe effector side in the allograft response, as itcertainly is in suppression of elicitation of thetuberculin reaction (site 5, Fig. 1).These studies were extended to other immu-

nological and non-immunological inflammatoryprocesses employing fractions of the sonicallydisrupted bacteria (41). Acute microcrystal syn-ovitis in the intertarsal joint of pigeons wasinhibited by i.v. injection of bacterial fractions60 min before induction of synovitis. Anotheracute non-immunological inflammation, car-ragenin edema, was reduced 50% as measuredby edema at 3 h. Adjuvant arthritis in the ratwas reduced, as measured by foot swelling onday 16, by daily injection of 0.5 mg/kg from day0 to day 14. Skin grafts in mice across a weak,

non-H-2 barrier were somewhat prolonged byinjections of 1 mg/kg daily for 8 days. Theincidence of experimental allergic encephalo-myelitis (EAE) in rats was suppressed from 7/8control animals paralyzed to 1/6 in a groupinjected with 1.0 mg of extracts/kg i.p. twelvetimes between day 0 and day 16.These fractions of Pseudomonas were effec-

tive as anti-inflammatory agents as judged fromtheir effect on microcrystal arthritis and carage-nin-induced inflammation. This alone couldaccount for the moderation of immunologicallyinduced inflammatory models of EAE, adju-vant arthritis, tuberculin skin test, and allograftrejection. Therefore, the experiments describeddo not enable us to distinguish a suppression ofthe specific immune response.On the other hand, a significant reduction in

antibody-forming spleen cells (PFC) in miceimmunized with SRBC is also described bythese authors (41). Mice injected with bacterialfractions 1 day before SRBC showed a consider-able reduction in direct PFC measured at 4days. Although this data is incomplete, it ap-pears that P. aeruginosa does contain a cellularcomponent which suppresses the humoral im-mune response. Whether the anti-inflammatoryand humoral immunosuppressive properties re-flect activities of one bacterial component orseparate entities is unanswered.

Bordetella pertussis. The vaccine madefrom this organism has been shown to influenceimmune response in several experimentalmodels. Finger and co-workers have carefullydefined some of the variables in a series ofpapers (32-34). They confirmed the potentadjuvant effect of killed B. pertussis on anti-body response to SRBC. However, this is verydependent on timing and dose of antigen. Thus,with a suboptimal dose of 2 x 107 SRBC both19S and 7S antibody-forming cells were tem-porarily suppressed when B. pertussis was in-jected after antigen. This was followed by anenhanced response (34). The secondary re-sponse to SRBC could be suppressed if theorganisms were given 2 days before a secondinjection of 4 x 108 SRBC (32).

Floersheim (38) reported that guinea pigsinjected i.p. with very large doses of vaccine 30min before and 8 h after elicitation of thetuberculin reaction gave a significantly lessintense skin test. This hyporeactive phase wasvery transient since injection 4 days or 16 hbefore tuberculin had little effect. The hypo-reactivity may be analogous to the clinicaltuberculin anergy seen with pertussis infectionof man (38).

In other reports pertussis vaccine injected

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into mice 4 days before (height of lymphocyto-sis) or 7 days after Rauscher leukemia tumorinduced enhanced growth of tumor (73). Thiswas accompanied by a much reduced in vivoproliferation of spleen cells inducible with PHAor Freunds adjuvant.

Brent and Pinto (13) demonstrated that per-tussis vaccine injected into mice 2 days beforean allograft across an H-2 barrier greatly in-creased the capacity of either antilymphocyteserum or allotissue extract to prolong survival ofthe skin graft.Howard et al. (75) observed that pertussis

vaccine injected into mice with 5 Aig of pneumo-coccal type III capsular polysaccharide (S III)reduced spleen PFC against this antigen, mea-sured 6 days later. Pertussis vaccine given 4days before S III had little effect. There was,however, an increased response to SRBC whenthis antigen was injected with pertussis vaccine.An investigation recorded in abstract by Kong(87) showed that in a dose range of S III of 0.01to 1.0 jig the response of mice was suppressedwhen killed pertussis vaccine was injected 2days before, simultaneously with, or 2 days afterthe antigen. Response to dose of S III below orabove this range was enhanced by pertussiscells. More importantly, the switch from 19S to7S antibody occurred only when B. pertussiswas given, and some 7S and 19S antibody wasformed when only B. pertussis without S III wasinjected.

All of these studies have used suspensions ofheat-killed B. pertussis cells. Each of the aboveexperiments could be explained by the lym-phocytosis-promoting activity of B. pertussis,but with the exception of experiments with thetuberculin reaction, it is difficult to discernwhether the effect is on the affector or effectorside. The lymphocytosis induced in mice ismaximal 4 days after pertussis vaccine injectioni.v. and is accompanied by a depletion of smalllymphocytes from all lymphoid organs and thebone marrow (105). Evidence was subsequentlypresented that pertussis induces a change in thesurface of lymphocytes which causes a de-creased capacity to emigrate from the blood.Thus, lymphocytosis primarily reflects the ina-bility of lymphocytes to recirculate via postcapillary venules into the lymphoid tissue (5,106).There has been considerable debate over the

identity of the lymphocytosis-promoting, he-magglutinating, protective antigen, im-munopotentiating, and histamine-sensitizingactivities associated with B. pertussis (18).Each activity could conceivably function in

some immunoregulatory fashion. The studies ofSato et al. (123, 124) indicate that all activitiesare produced by the same molecule, a filamen-tous structure which attaches to the surface ofvarious cells including lymphocytes. This mor-phological demonstration of absorption ontocell membranes supports the concept of Morsethat a change in cell surface properties is largelyresponsible for impaired migration of lympho-cytes through postcapillary venules (site 8, Fig.1). A paper by Lehrer et al. (89) supports theconcept that histamine-sensitizing activity, leu-cocytosis-promoting activity, and adjuvant ef-fect are associated. In a recent abstract, how-ever, Morse and Morse (107) reported the sepa-ration of lymphocytosis-promoting factor (LPF)and hemagglutinin. Each was immunologicallyhomogeneous and by electron microscopy LPFwas particulate and hemagglutinin was fila-mentous. There is no information regarding theT and B cell distribution in lymphocytosis, butMorse (105) did observe a severe depletion oflymphocytes from the thymus. However, boththymic-dependent and independent areas of thespleen and lymph nodes were depopulated afterinjection of mice with culture supernatant fluid(5). Lymphocyte trapping, or the augmentedaccumulation of infused lymphocytes in lymph-oid tissue, is an early event in lymph nodesstimulated with antigens, adjuvants, or graft-versus-host reaction (46, 152). Since LPF hasthe opposite effect, we would predict that itshould be a potent immunosuppressant. Thereis one report using partially purified materialwhich indicates this factor does suppress anti-body formation in mice (3). Intravenous injec-tion of LPF 1 to 3 days before SRBC produced asmall depression in hemagglutination titers.This was accompanied by a loss in body weight,indicating that a toxic dose was necessary. Thesecondary response to SRBC and the primaryresponse to tetanus toxoid were transiently de-pressed (3). It was also shown that LPF willsuppress CMI in rats (114). Delayed hypersensi-tivity to PPD and adjuvant arthritis were signif-icantly suppressed and allogeneic skin graftsurvival prolonged by i.v. injection of 0.5 Ag ofLPF. The delayed hypersensitivity skin test wasreduced by injection of LPF 3 days beforeimmunization, but the reaction was completelysuppressed when LPF was given 3 days beforeelicitation of the skin test with PPD. Thus, bothafferent and efferent phases of the immuneresponse seem to be modified, and the degree ofsuppression seems to correlate with the amountof lymphocytosis. In summary, the most attrac-tive explanation of both affector and effector

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suppressive effect of B. pertussis is that the LPFis affecting distribution of lymphocytes by mod-ification of membrane surface properties (site 8,Fig. 1).

Klebsielia pneumoniae. In a carefully con-ducted study, Nakashima et al. (109) demon-strated that induction of immunological paraly-sis to K. pneumoniae capsular polysaccharidein mice was accompanied by a depressed anti-body response to bovine serum albumin (BSA).The BSA is serologically unrelated to K.pneumoniae polysaccharide, and these findingsare very different from the highly specific im-munological unresponsiveness obtained withDiplococcus pneumoniae capsular polysaccha-ride. As observed with other agents, appropriatemanipulation yielded either suppression or en-hancement of serum antibody against BSA.Thus, if Klebsiella polysaccharide was injectedintramuscularly 3 to 30 days before BSA incomplete Freunds adjuvant, antibody responsewas inhibited; whereas if Klebsiella polysaccha-ride was injected simultaneously with or 3 daysafter BSA there was a strong adjuvant effect.The authors believe that these effects ofKlebsiella polysaccharide are not due to con-tamination with endotoxin, but until their prep-arations have been rigorously tested for endo-toxin activities this explanation must be consid-ered. The fact that a "paralyzing" dose ofKlebsiella polysaccharide was required for in-duction of nonspecific immunosuppression andthat the duration of this suppression was com-parable to "paralysis" presents the intriguingpossibility that this bacterial product is modify-ing some basic pathways of the immune mecha-nism.Escherichia coli. The work of Kirpatovskii

and Stanislavski (85) probably also reflects theimmunosuppressive effects of endotoxin. Em-ploying cell-free extracts from E. coli with"some endotoxin present" skin allograft sur-vival in mice was significantly prolonged acrossa major H-2 histocompatibility difference. Solu-ble extracts of mechanically disrupted cells orosmotically disrupted spheroplasts (less active)given 2 weeks and 1 day before the graft anddaily after the graft increased graft survivalfrom 9.2 + 0.5 days to 22.4 + 0.8 days. Injectionof extract 1 to 3 days before SRBC or Viantigens also suppressed antibody formationagainst these antigens. No suppression wasobserved if extracts were given after antigen,and no adjuvant effect was seen. The miceremained suppressed for 2 months. Subsequentstudies (86) with Sephadex fractions of thecell-free extracts confirm these results but do

not alter our assumption that the active compo-nent is endotoxin.

Bacterial EnzymesL -Asparaginase. There have been numerous

reports of immunosuppressive effects of L-asparaginase, much of the interest derivingfrom its reported usefulness as an anticanceragent. Most of the enzyme preparations testedhave come from E. coli cultures, althoughpreparations derived from Erwinia carotovara(59) and the BCG strain of Mycobacteriumbovis (132) produce a comparable inhibition oflymphocyte stimulation in vitro. Employingvery large doses (up to 8,000 IU), Schwartz (125)showed that injection before or after SRBCsuppressed the PFC response of mice. Hersh,however, demonstrated that timing was impor-tant for this suppression since a single injectionof enzyme given 48 h after SRBC had no effect(70). Moreover, Hersh showed that the effectwas really to delay PFC response rather thansuppress (70). The recovery of antibody re-sponse in mice is ascribed to production ofasparagine synthetase by lymphoid cells (70). Inspite of this, some evidence collected from a fewcancer patients during treatment with L-asparaginase indicates induction of delayedhypersensitivity and antibody formationagainst keyhole limpet hemocyanin and syn-thetic peptide were delayed or suppressed (115).The in vivo effect of enzyme appears to correlatewith inhibition of in vitro response of lympho-cytes to PHA or specific antigens (70). Thisinhibition was reversible by washing cells, evenafter 72 h incubation with enzyme. L-Aspara-gine, but not L-glutamine, added to culturesalso reversed inhibition by L-asparaginase.Both clinical signs and histological evidence

of EAE could be prevented in rats by dailyi.v. injections of L-asparaginase derived fromE. coli (84). To achieve this inhibition, how-ever, it was necessary to inject 10,000 IU/kgdaily for 13 days beginning on the day of activeimmunization. Since the specific activity was300 IU/mg of protein, I calculate that each ratweighing 150 g received 5 mg of protein perday or a total of 65 mg of protein. There is theadditional problem brought up by the authorsthat ether anesthesia also may have contributedsome inhibition. Partial inhibition of EAE wasalso obtained by i.p. injection of 50,000 IU/kgdaily for 10 days, which is 200 mg of protein perrat. These are very large quantities of bacterialprotein, and we must question whether theeffects are due to asparaginase activity. The

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authors state that their preparation was free ofendotoxin.

Recently, Berenbaum et al. (10) showed thatL-asparaginase inhibited the immune responseof mice to SRBC more than the response tolipopolysaccharide, a T-independent antigen,indicating, therefore, a selective affect upon Tcells. However, in vitro, the response of periph-eral blood lymphocytes to PHA or lipopolysac-charide were equally inhibited by enzyme. Theauthors conclude that B cells are not inherentlyless sensitive to L-asparaginase, but rather, theapparent differential in vivo susceptibility of Tand B cells in lymph nodes reflects the ana-tomic micro-environment for B cells whichpartially protects them (10). From in vivoreconstitution experiments, Friedman (45) pro-

posed that bone marrow cells are the majortarget of L-asparaginase.The mechanism of suppression in these stud-

ies is presumed to be depletion of amino acid,but there are some reports that L-asparagine or

L-glutamine do not reverse inhibition in vitro(131), indicating that the effect is more complex.Based on reports that these enzymes can de-grade glycoproteins on cell membranes, Fidlerand Montgomery (31) have reexamined thismechanism, using rat lymphocytes. Prior treat-ment of lymphocytes with L-asparaginase de-creased binding of the 125I-labeled mitogenconcanavalin A 20%, and decreased deoxyribo-nucleic acid synthesis 95%. Treatment of lym-phocytes 90 min after mitogen had no effect on

binding. They conclude that alteration of lym-phocyte receptors could account for im-munosuppressive activity. This is supported bya report that L-asparaginase inhibits a ratmixed lymphocyte reaction through alterationof the lymphocyte surface (79). Cell membranealteration has also been presented as a mecha-nism by Han and Ohnuma (59). These reportssuggest that the site of action could be at site 8in Fig. 1 (distribution of lymphocytes or antigenrecognition).Some inhibition of histological evidence of

EAE in rats was achieved by injection of recipi-ents with 40,000 IU/kg 24 h before passivetransfer of sensitized lymphocytes (84). Thiscould be further evidence that L-asparaginaseacts directly upon the effector lymphocyte (site5, Fig. 1).Ribonuclease. Bacterial ribonuclease will

also suppress the immune response. Superna-tant medium from cultures of a bacillus resem-bling B. cereus will suppress antibody forma-tion against SRBC in mice and inhibit theresponse of lymphocytes to PHA in vitro (14).

The immunosuppressant has been purified andcorrelates with ribonuclease activity. Themechanism proposed for serum alpha-globulinribonuclease is a blockage of new ribosomalribonucleic acid in stimulated cells, and thebacterial enzyme could have the same function.L -Glutaminase. This enzyme, isolated from

E. coli, will also inhibit responses of humanlymphocytes to mitogens (69). Like L-asparagi-nase, this is not due to cytotoxicity.

MycoplasmaCopperman and Morton (23) reported that

nonviable Mycoplasma hominis could inhibitthe capacity of PHA to stimulate mitosis ofhuman lymphocytes in vitro. The inhibitionwas not the result of toxicity for lymphocytessince inhibition could be reversed by removingthe Mycoplasma organisms. Barile and Leven-thal (7) later showed that the inhibition alsowas produced by other strains of Mycoplasmawhich had in common the utilization of arginineas an energy source. They proposed that compe-tition for arginine was the mechanism of inhibi-tion. Simberkoff et al. (131) have further dem-onstrated that the arginine dihydrolase enzymeof Mycoplasma is responsible for inhibition ofresponse not only to PHA but also of sensitizedcells to tuberculin and homograft antigens.Secondary production of antibody against diph-theria toxoid by rabbit lymph node fragments invitro was also blocked. The reagent used by thisgroup was soluble freeze-thaw extracts ofMyco-plasma from which the membrane had beenremoved by centrifugation. This extract was noteffective in vivo (131). It seems unlikely thatsufficient arginine depletion could be achievedin the animal.

In contrast, studies with whole suspensions ofMycoplasma arthritidis (80) or membranes iso-lated from this organism (11) did demonstratesuppression of the immune response in vivo.When viable Mycoplasma were injected intorats concurrently with bacteriophage antigen,production of neutralizing antibody was sup-pressed (80). In addition, the response of lymphnode cells removed from Mycoplasma-injectedanimals had a greatly reduced in vitro responseto PHA. The experiments employing mem-branes were different in that antibody responsein rabbits to common bacterial antigens wassuppressed only when membranes were incu-bated with antigen before injection (11). Thismodel is similar to the suppression Whang andNeter (146) described with endotoxin or lipoid A(site 2, Fig. 1). The mechanism of immunosup-pression obtained in vivo with Mycoplasma is

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apparently distinct from in vitro inhibition oflymphocytes and is not related to argininedepletion.

Mycobacteria and Freunds AdjuvantEven the most celebrated adjuvant, complete

Freunds adjuvant (CFA), can depress ratherthan enhance an immune response in a properlycontrived experiment. Footpad injections of ratsor guinea pigs with CFA 10 days before immu-nizing with bovine gamma globulin antigen inCFA depressed the 40- and 24-h skin reactionsto antigen injected 14 days later (4). The bacte-rial component of CFA was essential for the de-pressed response. C. parvum could be substi-tuted for CFA but B. pertussis had no effect.Contact sensitivity in mice was also depressed ifthe sensitizing chemical was applied percutane-ously. Other reports show that injection of micewith incomplete Freunds adjuvant 2 or 3 daysbefore SRBC can also suppress antibody-form-ing cells (35).

In another report (2) some correlation be-tween depression of arrival of labeled lymphnode cells at recently immunized lymph nodesand depression of contact sensitivity by pre-treatment with CFA or C. parvum was shown.The authors, therefore, propose that this bac-teria-induced, depressed delayed hypersensitiv-ity is caused by a failure of T cells to arrive atlymph nodes subject to antigen stimulation(site 8, Fig. 1). The reason for inadequate celldistribution is not clear. Possibly these observa-tions are related to reports of suppression ofexperimental allergic uveitis (140) and experi-mental allergic encephalomyelitis (130) ob-tained by increasing the dose of mycobacteria inthe CFA-antigen mixture above an optimallevel. In the uveitis experiments, for example,0.01 to 0.1 mg of mycobacteria induced diseaseand delayed hypersensitivity but 0.3 to 1.0 mgof mycobacteria depressed the response to uvealantigen.

Mycobacterium lepraeThis is an example of immunosuppression

observed in certain individuals in the course of anatural bacterial infection, in contrast to theexperimental demonstration of suppressionwith other bacterial agents. Patients with lep-romatous leprosy give a negative delayed hyper-sensitivity skin test to lepromin at 72 h andalso fail to show a positive late (Mitsuda)reaction at 3 weeks after skin testing (135). Inaddition to this specific anergy to lepromin,there is a general depression of CMI against

tuberculin and dinitrochlorobenzene. Thus,70%o of lepromatous patients cannot be sensi-tized to dinitrochlorobenzene compared to 6% ofcontrols. Patients with lepromatous leprosy alsoshow delayed rejection of skin grafts (135).Antibody formation is not impaired. The re-gional draining lymph nodes are histologicallycomparable to those in animals given antilym-phocyte serum, i.e., almost complete depletionof lymphocytes from the thymic-dependent par-acortical area and replacement with histocytesis observed. The follicles are unaffected. Thissuggests an effect at site 10 in Fig. 1.

It has been proposed that the depression ofCMI in lepromatous leprosy is a host-deter-mined, genetic defect. However, evidence ofrecovery of CMI associated with successfultherapy suggests that the bacteria are in partresponsible for the nonspecific depression ofCMI. Other studies have confirmed the CMIdefect in lepromatous leprosy (52). Employing atechnique of crossed immunoelectrophoresiswith intermediate gel, an inverse relationshipappeared between depressed CMI and the num-ber of precipitating antibodies against BCG (6).The incidence of antibodies was lowest in tuber-culoid patients, who showed highest CMI,whereas lepromatous patients had highest anti-bodies and lowest CMI.

Protozoan ParasitesAutoimmune diseases are very rare in areas of

Africa with high incidence of parasitic infec-tions. From this observation, Greenwood andHerrick (55) suggested that the immunologicaleffects of repeated parasitic infection mightreduce autoimmunity. Evidence was providedby experiments on the effect of infection ofNZB/W mice with Plasmodium berghei voeliion autoimmune disease. None of the infectedmice developed proteinuria over an 11-monthperiod, compared to 100% of uninfected con-trols, and no mice died, in contrast to 100%of controls. The onset of positive Coombs testin NZB mice was delayed for at least 9 monthsin infected mice, compared to controls. Ad-juvant arthritis is also suppressed in rats in-fected with this rodent parasite (56). The ef-fect seems to be on induction of the immuneresponse rather than merely anti-inflammatorysince the depression of response persists longafter the parasitemia. Subsequent studieshave shown that during parasitemia mice havea greatly suppressed antibody response toSRBC and human gamma globulin but not tokeyhold limpet hemocyanin (57). CMI, as mea-sured by PHA response, skin graft rejection,

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and contact sensitivity to picryl chloride oroxazalone, was not affected. They concludethat the mechanism of humoral antibody sup-pression may be altered macrophage functionsince irradiated mice can be reconstitutedwith spleen cells from P. berghei-suppressedmice (site 2, Fig. 1). Others have also presentedevidence that this is a macrophage defect (92).More direct evidence, however, on the defect inlymphoid organs of malarial infected mice isprovided by Krettli and Nussenzweig (88).They showed that a large reduction in thymusweight and T cells occurs early in infection. Thenumber of both T and B cells decreases inlymph nodes later. in infection, with an increaseof a null cell population. This would correspondto site 10 in Fig. 1.Rabbits infected with Trypanosoma con-

golense showed a suppressed CMI as measuredby: delayed hypersensitivity skin tests to PPDafter immunization with CFA, peripheral bloodlymphocyte stimulation with PHA, and produc-tion of migration inhibition factor by antigen-stimulated lymph node cell cultures (97). Anactive infection was necessary, although theanimals were not debilitated. The authors ten-tatively suggested that the mechanism mayinvolve a selective depletion of regulatory Tcells (site 4, Fig. 1).Natural Trypanosoma gambiense infection in

humans will suppress elicitation of delayedhypersensitivity skin tests with PPD and Can-dida (58). Induction of CMI against dinitro-chlorobenezene was also suppressed (P < 0.001)in patients (16/38 positive, 42%) compared tocontrols (35/43 positive, 81%). Antibody re-sponse of patients to H antigens, but not 0antigens, of Salmonella typhi vaccines was alsodepressed. The authors concluded that theseresults were not due to malnutrition, but rather,the organism had some direct effect on thelymphoid system.Immunosuppression has also been associated

with leishmaniasis (19) and toxoplasmosis (68,133).

Metazoan ParasitesTrichinella spiralis infection of mice reduced

the humoral antibody response against SRBC(29). It is of interest that passive transfer of theserum from infected mice also suppressed anti-body formation in recipient mice (29). Subse-quently, evidence has been presented by Cypessand colleagues that T. spiralis-infected micehad an increased susceptibility to Japanese Bencephalitis virus (24). This was accompaniedby a 3-tube decrease in viral complement-fixingantibody titer and about one log10 decrease in

neutalizing antibody. Suppression was not ob-tained with irradiated T. spiralis or Nemato-spiroides dubius, which has no extragastroin-testinal stage. On the other hand, T. spiralisinfection increased resistence of mice to Listeriamonocytogenes infection (25).The mechanisms involved are not known, but

since viable organisms are required, effect onthe efferent phase of immune responses may beimplied (29). In more recent studies theseworkers show that infection of mice with T.spiralis either before or after sensitization withviable BCG, increases the delayed hypersensi-tivity to tuberculin (26, 103). Apparently bothinduction and expression of CMI are enhancedwhile antibody production is reduced. It ispossible that a phase of immunosuppressionallowed greater multiplication of the mycobac-teria, which led to increased antigen stimulusand delayed hypersensitivity (26). A suppres-sion of CMI is suggested by a delay in rejectionof skin allografts by mice infected with T.spiralis (15). This was correlated with the num-ber of larvae recovered from the mice. Graftrejection time also correlated with a decreasedcytotoxicity of lymphocytes for allogeneic fibro-blasts in vitro, suggesting a defect induced atsite 5 or 6 in Fig. 1.

CONCLUSIONS AND SPECULATIONSAlmost every material having adjuvant activ-

ity can also be demonstrated to suppress theimmune response with appropriate manipula-tion of timing, dose, and selection of antigen.The greater significance of these agents may beas immunosuppressants. Several of the bacte-rial agents described here have been obtained inhighly purified form and their effectiveness canbe meaningfully compared to standard im-munosuppressive drugs. Thus, 4 mg of cyclo-phosphamide injected into mice 4 days beforeSRBC produced a maximum suppression, mea-sured 5 days after antigen, of 75% of direct PFCand 84% of indirect PFC (28). As noted above,6-mercaptopurine requires even larger dosage toachieve this level of suppression. In contrast,cholera enterotoxin, endotoxin, and the strep-tococcal products are effective in microgramdoses.One of the problems which concerns us is the

evidence that bacterial immunoregulatoryagents are produced and function in nature. It islogical to question whether these agents areproduced in the course of infection and natu-ral habitation of tissue, or if these activities areconfined to contrived experiments. There aresome observations in germfree animals whichcould be interpreted to support the concept that

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the microbial flora does influence immune ca-pacity. Lev and Battisto (90) report that germ-free guinea pigs did not develop delayed hyper-sensitivity to picryl chloride or bovine alpha-globulin. However, other workers found contactsensitivity and tolerance to dinitrochloroben-zene in germfree guinea pigs to be very similarto responses in conventional animals (44).Other studies have shown that germfree micehad a lower incidence of plasma cell tumorsthan conventional mice when injected withmineral oil (99). Similarly, fewer carcinogen-induced hepatomas developed in germfree com-pared to conventional mice (53). Another reporthas shown that primary antibody response per-sisted in germfree chickens without showing thecharacteristic switching to secondary antibodyresponse (143). In a study on the effect ofmicrobial flora on macrophage function, it wasshown that macrophages from conventionalmice digested ferritin and S. marcescens anti-gens more rapidly than those from germfreemice, in which antibody formation was delayed,although eventually the response was moreprolonged and sometimes greater than in con-ventional animals (9). Finally, the distributionof Salmonella flagellar antigens in lymph nodesof germfree mice differs from that in the conven-tional animal (102). No coherent pictureemerges from these studies, but they indicatethat microbial factors affecting immunity areproduced and do function in vivo.The next question is the possible significance

of the finding that bacterial agents influence theimmune response. Situations in which theseagents could be participating in the host'simmune capacity are outlined as follows.

(i) Effect on infection by the organism fromwhich the immunosuppressive agent was de-rived. There is no evidence that this occurs. Inthe case of some agents this apparent lack ofinfluence could reflect the timing of exposure ofthe lymphoid tissue to microbial antigen rela-tive to effective levels of the suppressant fac-tor. In the case of the group A Streptococcus,for example, the membrane-associated im-munosuppressant is only effective experimen-tally when given before antigen (95).

(ii) Effect on sequelae to infection such asrheumatic fever, acute glomerulonephritis, andpossibly rheumatoid arthritis. There is no directevidence for an effect in these diseases, butthese are diseases associated with immunologi-cal abnormalities. It is an intriguing hypothesisthat the microorganism responsible for initiat-ing the disease process contributes to the immu-nological abnormality, whether this can bemanifested as autoimmunity or immune defi-ciency.

(iii) Effect on infection by other microorga-nisms. Some examples which may reflect aninfluence through depression of the specificimmune response include the effect of C. par-vum on infection of mice with Salmonellaenteriditis (20) or Trichinella spiralis (121). T.spiralis infection is reported to greatly enhanceinfection of mice with Japanese B encephalitis(24). Trypanosoma brucei infection of rats re-duced IgG and IgE antibody against Nippost-rongylus brasiliensis and immune expulsion ofadult worms did not occur (139). Mice infectedwith Toxoplasma gondii suffered a more severemalarial parasitemia when subsequently in-fected with Plasmodium berghei yoelii (124).Patients infected with Trypanosoma gambiensehave an increased occurrence of lobar pneumo-nia and other secondary infections (58). Beforewe draw any general conclusions from theseexamples, we must recall that quite oppositeeffects have also been recorded. Thus, T. spi-ralis infection increased resistence of mice toListeria monocytogenes (25), and C. parvumcan also enhance resistance to infection (1, 113).It is certainly premature to ascribe any of theseeffects directly to modification of specific im-mune mechanisms. A related problem pre-sented by Greenwood and co-workers is theeffectiveness of vaccines in areas where malariaand other agents of immunosuppression areendemic (54). They have shown that childrenwith acute malaria have a reduced antibodyresponse to tetanus toxoid and Salmonellatyphi 0 antigens.

(iv) Effect on response to tumor cells. Thereare reports accumulating on the effect of BCG,C. parvum (150) and streptococci (66) in experi-mental and human cancer. Other reports haveappeared showing that nematodes and proto-zoan parasites can significantly influence tumorgrowth in experimental animals (78, 83, 122,144). One of the most interesting observationson this point is the report that an anaerobicCorynebacterium liquefaciens was isolated fromthe sternal bone marrow of 80% of normal youngadults, while it was obtained in much lowerincidence from patients with cancer (63). Cul-tures of this organism were subsequentlyshown to have antitumor activity in mice (64).The problems presented by host microflora andinfections on the investigations of susceptibilityto cancer have recently been considered byHanna et al. (60).

This is an incomplete list of situations inwhich microorganisms are demonstrated toeffect the hosts' susceptibility to other infec-tions or disease processes. In none of these ex-amples can it be claimed that the mechanism isthrough modification of immunoregulatory sys-

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tems. These observations provide, however,fascinating speculation on our inescapable in-volvement with our microbial environment.The importance of environmental factors

which influence immunoregulation is probablythrough subtle and transient effects whichcould shift the balance between host and in-vader. For example, the effectiveness of theimmune surveillance system could be compro-mised at a time coincident with a mutagenicevent, allowing establishment of malignantcells. "Spontaneous" relapses and exacerba-tions in human cancers or in chronic viralinfections could reflect encounters with mi-crobial agents affecting the immune capacity.Other environmental factors could also functionin this way but certainly bacteria are the mostpervasive source of such exogenous immuno-regulatory agents.

ACKNOWLEDGMENTS

The author's studies included in this review weresupported by a grant-in-aid from the American HeartAssociation.

Parts of this review were presented at the IVInternational Conference on Immunology, 3 to 6 June1974, Buffalo, N.Y., and will be published under thetitle, The Immune System and Infectious Diseases(ed. E. Neter), published by S. Karger A.G., Basel.

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