Antibody-Mediated Immunity against Intracellular Pathogens: Two-Dimensional Thinking Comes Full Circle

The view that antibody-mediated immunity against many prokaryoticand eukaryotic intracellular pathogens is not important waspopular until recently (6). The concept of a division of laborwhereby antibody-mediated immunity protected against extracellularpathogens and cell-mediated immunity protected against intracellularpathogens may have had its intellectual origins in the greatdebate between the advocates of humoral and cellular immunityat the turn of the 20th century. The humoralists, championedby Paul Ehrlich, viewed immunity as being conferred by solublesubstances in the blood and the generation of an effective antibodyresponse, with phagocytic cells functioning primarily to cleanup microbial debris (42). The cellularists, championed by ElieMetchnikoff, viewed immunity as being conferred by macrophagesand other phagocytic cells, with the role of humoral factorsbeing to provide opsonins (42). This debate was fueled by thesuccess and difficulties associated with demonstrating antibody-mediatedprotection against certain pathogens in passive immunizationstudies. Administration of immune serum protected against toxin-mediateddiseases such as tetanus and diphtheria and a certain subsetof bacterial pathogens exemplified by the organisms now knownas Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilusinfluenzae. However, passive immunization provided little orno protection against other microbes such as Mycobacterium tuberculosis(reviewed in reference 19).

By the 1960s, classical studies with facultative intracellularpathogens such as Listeria monocytogenes had shown that effectivecontrol of infection depended on cellular immunity, as manifestedby granuloma formation and participation of T lymphocytes (28).The microbes for which passive antibody was not protective andcell-mediated immunity appeared to be paramount for host defensewere often facultative intracellular pathogens. This associationgave credence to the concept of an immunological division oflabor whereby humoral and cellular immunity provided effectivecontrol for extracellular and intracellular pathogens, respectively(3, 8, 28). Furthermore, this division of labor was conceptuallyconsistent with a large body of experimental observations thatindicated an inverse and mutually antagonistic relationshipbetween humoral and cellular immunity (35). In recent years,the view that antibody-mediated immunity protects against extracellularpathogens and cell-mediated immunity protects against intracellularpathogens has been modified and extended by the Th1/Th2 paradigm,which posits a division of labor at the level of T-cell differentiation.According to this view, Th1-polarized responses result in granulomatousinflammation that effectively controls intracellular pathogens,whereas Th2-polarized responses result in the production ofantibodies that control extracellular pathogens and parasites.

The fact that a microbe inside a cell is separated from serumantibody has contributed to the belief that serum antibody cannotbe effective against an intracellular pathogen. However, thetwo-dimensional separation and categorization of microbes aseither intracellular and extracellular pathogens was never absolute,since tissue examination often revealed that pathogens classifiedas intracellular could be found in the extracellular space andvice versa. Furthermore, at some point in the infectious cycle,most intracellular pathogens reside in the extracellular space,where they are vulnerable to antibody action, and Fc receptorcross-linking can have profound effects in the intracellularmilieu through signal transduction.

In this issue of Infection and Immunity, we have an exampleof how the investigation of mechanisms by which passive antibodyprotects against the obligate intracellular pathogen Ehrlichiachaffensis led to the discovery of an extracellular phase thatmay include replication (27). Hence, the wheel has turned fullcircle, since an investigation to explain how antibody protectsagainst an obligate intracellular pathogen has revealed thatit may not always reside in the intracellular space and thuscould become accessible to serum antibody.

DECONSTRUCTING A PARADIGM

Top

Deconstructing a paradigm

The notion of an immunological duality whereby immunity to intracellularpathogens is conferred by cell-mediated mechanisms and immunityto extracellular pathogens is conferred by antibody-mediatedmechanisms was a reigning paradigm in the closing decades ofthe 20th century and still has wide credence. However, thisview is problematic because it is not universally applicableto all pathogens and because the induction of antibody mediated-immunityis sufficient to prevent infection with some intracellular pathogens.For example, the major childhood viral diseases and smallpoxwere drastically reduced in incidence or eradicated by vaccinesthat elicited antibody-mediated immunity despite the fact thatall viruses are obligate intracellular pathogens. For some intracellularbacterial pathogens, such as Salmonella enterica serovar Typhimurium,it was clear that antibody responses were protective in certainhosts (13). The concept of an immunological division of laborbased on whether or not a microbe assumed intracellular residencedefied the common-sense view that the most effective immuneresponse was one that combined both humoral and cellular components.

Perhaps the most important advance in suggesting a resolutionto the cellular versus humoral controversy was the applicationof hybridoma technology to investigate the potential of antibody-mediatedimmunity against certain pathogens for which immune serum didnot manifest efficacy. In contrast to immune serum, which variedgreatly in the composition, isotype, and specificity of microbe-bindingantibodies, monoclonal antibodies provided a homogenous preparationor defined reagents with which to investigate the variablesthat contributed to antibody-mediated protection. Studies withmonoclonal antibodies have now demonstrated passive protectionfor several microbes where experiments with immune serum hadprovided negative or inconsistent results, including Candidaalbicans (20), Cryptococcus neoformans (9, 17, 32, 40), Listeriamonocytogenes (11), Leishmania mexicana (1), Mycobacterium tuberculosis(45), and Histoplasma capsulatum (J. D. Nosanchuk, A. Casadevall,and G. Deepe, Abstr. Annu. Meet. Am. Soc. Microbiol., 2001,abstr. F-143). For these pathogens, the identification of protectivemonoclonal antibodies established the precedent that antibodycould be effective and dispelled the notion that humoral immunitywas ineffective due to an inherent limitation in the activityof this arm of the immune system. The list of intracellularpathogens for which antibody has been shown to modify the courseof infection to the benefit of the host is extensive (Table1).

View this table:
[in this window]
[in a new window]
TABLE 1. Prokaryotic and eukaryotic intracellular pathogens for which antibody has been shown to modify the course of infection to the benefit of the host^a

INTERPRETATION OF NEGATIVE RESULTS

Top

Interpretation of negative...

A central argument for the concept that antibody lacked efficacyagainst certain intracellular microbes was the observation thattransfer of immune serum was not protective in animal modelsof infection. In fact, Mackaness proposed six criteria for establishingthe importance of cellular immunity, of which the first onestated that "there should be no evidence that protection canbe conferred by passive transfer of antibody alone" (29). However,the absence of demonstrable protection in passive antibody experimentsdoes not mean that antibody has no role in protection, sincethis conclusion cannot be made from a negative experimentalresult. In recent years, studies with monoclonal antibodiesto Cryptococcus neoformans and other pathogens have providedseveral insights as to why passive antibody experiments canproduce negative results even when protective antibodies existand protective antibody responses are possible.

A dramatic example of the limitations of passive antibody transferexperiments is provided by the observation that transfer ofeither too little or too much antibody can result in no protection.In 1987, Dromer et al. generated a protective immunoglobulinG1 (IgG1) monoclonal antibody to Cryptococcus neoformans anddemonstrated that a certain amount of immunoglobulin was necessaryto observe protection in a murine model of cryptococcosis (9).This observation suggested that the inability to protect withimmune serum may have been a consequence of inadequate amountsof protective antibody. Similarly, it was noted that a monoclonalantibody to listeriolysin O was protective against Listeriamonocytogenes if administered in large doses but that antibodieswith that specificity were not common in immune serum (11).More recently, my group has shown prozone-like effects withprotective IgM and IgG, such that the administration of largeamounts of immunoglobulin can result in reduced or abolishedprotective effects (43, 44). Consequently, too much or too littleantibody can yield a negative result in a passive protectionexperiment despite the fact that antibody can be protectiveagainst the relevant pathogen.

Apart from antibody amount, immunoglobulin-related variablessuch as antibody specificity (31), isotype (49), and idiotype(39) can have profound effects on antibody protective efficacy.However, host-related variables can also determine the outcomeof passive protection experiments. For example, the protectiveefficacy of passive antibody to Salmonella enterica serovarTyphimurium is dependent on the mouse strain used (13). Forsome pathogens, the efficacy of passive antibody is dependenton the presence of intact cellular immunity (48). Adding tothe uncertainty associated with negative results in passivetransfer experiments is the observation that antibody efficacycan depend on the microbial strain used despite the presenceof the target antigen (33).

Clearly, negative results in passive protection experimentsdo not exclude the existence of protective antibodies. Conversely,the discovery that it is possible to make protective monoclonalantibodies against several intracellular pathogens does notnecessarily imply that antibody immunity plays a major rolein natural resistance, since the antibodies that mediate protectionmay be absent or rare in the immune response to natural infection.Experimental variables that can lead to a negative result inpassive protection experiments are listed in Table 2.

View this table:
[in this window]
[in a new window]
TABLE 2. Outcome of antibody protection experiments is dependent on multiple independent variables

LESSON FROM ERHLICHIA CHAFFEENSIS

Top

Lesson from erhlichia...

The obligate intracellular bacterium Ehrlichia chaffensis isthe causative agent of human monocytic ehrlichiosis. Accordingto the immunological division of labor discussed above, hostprotection against E. chaffensis would have been expected tobe conferred exclusively by cell-mediated immune mechanisms.However, there was evidence that specific antibody could mediateprotection against Erhlichia spp. (23), possibly by blockingcellular entry or promoting the expression of proinflammatorycytokines (25, 30). Studies by Winslow and colleagues subsequentlyestablished that specific antibody could protect against E.chaffensis in both normal and SCID mice (47). That result wassurprising because it might have been anticipated that cell-mediatedimmunity would play a major role in promoting antibody efficacyagainst an intracellular pathogen, as was shown for Cryptococcusneoformans (48).

The efficacy of passive antibody against E. chaffensis in SCIDmice suggests that antibody-mediated protection was independentof T cells and implied that other mechanisms must be operative.In pursuit of that question, Li and Winslow now describe anextracellular phase for E. chaffensis during which the bacteriaare potentially susceptible to serum antibody (27). Althoughit has not been proven that antibody-mediated protection againstE. chaffensis occurs in the extracellular phase, this observationsuggests a mechanism that is fundamentally different from thatreported for Listeria monocytogenes (12), where antibody isactive intracellularly. Ironically, the finding that E. chaffensishas an extracellular phase that is presumably susceptible toserum antibody is consistent with the older view that antibodiesare active only against extracellular microbes. Nonetheless,antibody may be effective against E. chaffensis when a thresholdportion of the microbial pool is extracellular and accessibleto antibody. This discovery suggests that other obligate intracellularpathogens may also have extracellular phases during which theyare susceptible to humoral immunity. This elegant study illustratesthe connectivity of scientific thought in that pursuing an explanationfor an observation that defied one paradigm led to findingsthat undermined another and, in so doing, provided new insightsinto microbial pathogenesis and immunology.

FOOTNOTES

* Mailing address: Dept. of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-4259. Fax: (718) 430-4259. E-mail: casadeva{at}aecom.yu.edu.

The views expressed in this Commentary do not necessarily reflectthe views of the journal or of ASM. Editor: T. R. Kozel

REFERENCES

Top