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|ES OF PATIENTS WN'ITH TRACHOMA ,LLPOX VACCINATION / f __,' IN SEMISYN'THETIC MEDIA THIOACETAZONE SIDE EF""'f' CHOLERA VACCINE DISSECTION OF DRY AND PRE' ! A1-i-NAL LIBFRARY OF MNEDICINE SEASONAL CHANGES IN POPULATf _______ MEMORANDA: VIRUS-ASSOCIATED -__
193 MARG2 1
WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTI
Virus-associated immunopathology: animal models and implications for human disease*
1. Effects of viruses on the immune system, immune-complex diseases, and antibody-mediated immunologic injury
The tissue damage caused by virus infection has been traditionally explained by the ability of viruses to multiply in cells and thereby injure or destroy them. Recent evidence suggests, however, that lesions may also be caused by the host's immune response to viral antigens and that the immune system itself may be perturbed by some viruses. This memorandum reviews recent developments in viral immunopathology, with special reference to animal model systems, and indicates the possible relevance of the new concepts and techniques for certain diseases of man. Certain viruses, notably the leukaemia viruses and some of those causing persistent infections, depress the host's ability to mount an antibody response to antigens, while other viruses may enhance the antibody response. Cellmediated immunity may also be depressed. Another immunopathological manifestation of virus infection is immune-complex disease. When viruses or their antigens persist in the circulation they combine with specific antibody, and the resulting complexes lodge in various sites, especially the kidney. Further combination with complement leads to the release of tissue-damaging substances. A third condition associated with virus infection is antibodymediated immunologic injury. Both oncogenic and non-oncogenic viruses frequently induce new antigens on the surface of the cells they invade. When antibody attaches to these antigens in the presence of complement, the cells are destroyed.
The lesions associated with virus infections have traditionally been explained by the ability of viruses to replicate in cells and hence cause cell injury and even death. However, recent studies indicate that virus-associated tissue damage may be due in part to the immune response of the host to viral antigens. The properties of viruses are seemingly ideal for producing immunopathological damage. Viruses are foreign antigens and, being self-replicating, can continue to produce antigen for long periods of time. Certain viruses are also known to be able to induce new antigens on the surface of cells they infect. The host's immune system can respond to these antigens. In view of these properties, immunopathological changes may be initiated by a number of different mechanisms in the course of virus infection: (1) Certain viruses can infect the cells of the immune system and cause direct immunologic
* This memorandum was prepared by the signatories listed on page 262.
derangements. Many processes and parameters of immune function may be thus affected, including graft rejection, the induction of immunologic tolerance, antibody production, graft-versus-host reactions, lymphocyte transformation, immunoglobulin levels, phagocytosis, and delayed-type skin reactions. (2) The host's immune response to viral antigens can lead to the formation of virus-antibody complexes capable of reacting with anti-immunoglobulins, rheumatoid factor, and the components of complement. (3) New antigens produced by viruses on infected cell surfaces can interact with specific antiviral antibody plus complement, thus causing cell destruction. (4) Recent findings suggest that sensitized lymphocytes can also react with virus-induced cell surface antigens and destroy the cell. Furthermore, cellmediated (or antibody-mediated) immune responses to viruses may result in the release or activation of biological mediators causing immunopathological
(5) An autoimmune response may be produced if the virus (a) releases host-cell antigens, (b) alters host-cell antigens and act as a " helper determinant ", or (c) depresses the host genome, thus increasing the production of embryonic or other antigens. In addition, the genetic makeup of the host, while not a mechanism of producing immunopathological damage, can influence the nature and severity of injury incurred during virus infection (Notkins et al., 1970). In some infections, such as lymphocytic choriomeningitis, the immune response of the host may be the principal cause of the pathological manifestations while in other infections it may be of less importance. In most if not all virus infections, the host's immune response probably contributes somewhat to the pathological picture. It should be emphasized, however, that in the majority of cases the overall effect of the immune response is more likely to be beneficial than harmful. Recent studies on virus-induced immunopathological reactions in domestic and experimental animals have led to the development of concepts and technical methods that may be useful in investigating certain viral diseases in man, including hepatitis. Progress in the field of viral immunopathology has been rapid, and it was felt that a summary and critical review of present knowledge would encourage its wider application to clinical problems. Only selected references have been included, since the breadth of the subject made a complete review of the literature impracticable. Suggestions for further lines of investigation in viral immunopathology in general and in viral hepatitis in particular will be offered in Part 2 of this Memorandum, to be published later.
EFFECTS OF VIRUSES ON THE IMMUNE SYSTEM
It has long been known that certain virus infections can alter the morphology of lymphoid organs. Electron microscopy studies have demonstrated the presence of virus particles in cells of the lymphoreticular system, such as macrophages, lymphocytes, neutrophils, thymocytes, Kupffer cells, and stem cells. More recent investigations have shown that certain viruses are able to replicate in macrophages (e.g., arboviruses, murine hepatitis virus, lactate dehydrogenase virus (LDV], and herpes simplex virus (HSV]) while others can replicate in lymphocytes (e.g., lymphocytic choriomeningitis virus [LCMV], leukaemia viruses, and Epstein-Barr virus [EBVD. Several viruses appear to replicate only in lympho-
cytes that have undergone blast transformation following exposure to specific antigen or phytohaemagglutinin. Not all infections of the immune system, however, result in cell destruction; some lead to a persistent infection. For example, infection with EBV can result in the establishment of a continuous lymphoid cell line in vitro, while infection with the leukaemia viruses may be followed by malignant transformation. Recent studies indicate that certain virus infections can affect the function of the immune system. These investigations have utilized the immune response to a variety of antigens unrelated to the infecting virus in order to evaluate immunologic function. Murine leukaemia viruses have received the most attention. These viruses usually depress the immune system, under certain circumstances to a significant extent (Dent, 1972). For example, the number of antibody-producing cells as determined by the haemolytic plaque test (Jeme) may reportedly be depressed by as much as 99 %. In general, infection prior to the injection of antigen was found to result in immunodepression, whereas infection after antigen administration had considerably less effect. The degree of immunodepression was dependent on the dose of virus and on the nature and concentration of the particular antigen. Moreover, some evidence has been adduced that the leukaemia viruses (particularly Friend virus) can exert " selectively " depressive effects, i.e. that they produce a greater depression of the 7S than of the 19S immune response. Selective effects also have been described in connexion with other viruses. Infection with Aleutian disease virus (ADV) can result in the appearance in the serum of an excess of monoclonal immunoglobulin. It also has been claimed that LDV and LCMV can produce an acute and " selective " depression of T cells, but these results need to be confirmed and extended. Several non-oncogenic viruses (e.g., ADV, LCMV, and Junin virus) are also able to depress the humoral immune response. In addition, certain viruses, such as LDV, LCMV, and Venezuelan equine encephalitis virus (VEEV), can prevent the development of experimentally-induced immunologic tolerance. Although most studies of viral effects have been concerned with the humoral immune response, recent investigations ofcell-mediated immunity and reticuloendothelial function demonstrate that these too can be depressed. For example, allograft rejection is profoundly depressed in animals infected with Gross leukaemia virus and mildly depressed in animals in-
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 1
fected with LDV. A number of viruses, including the Rauscher and Friend viruses and those causing measles and rubella, have been shown to inhibit blast transformation of lymphocytes (Dent, 1972). Not all viruses exert depressant effects on the immune system. Several, such as LDV and VEEV, can act as adjuvants and potentiate the immune response to certain antigens. A number of mechanisms have been postulated to explain the immunodepressive effect of certain virus infections (Allison, 1972; Notkins et al., 1970). These include (1) virus-induced changes in the uptake and processing of antigens, possibly by alteration of cell surfaces; (2) depression of nucleic acid and protein (antibody) synthesis; (3) destruction of antibody-producing cells or their precursors; (4) alteration of thymic function; (5) acceleration of immunoglobulin catabolism; (6) antigenic competition; and (7) lymphocytolysis as a result of increased adrenocortical secretion. Possible explanations of the immunologic enhancement associated with virus infections include (1) altered uptake and processing of antigens; (2) increase in the number of antibody-producing cells or their precursors; and (3) enhanced metabolism of antibody-producing cells. The effects of virus infections on immune function may have several important pathological repercussions. Virus-induced immunodepression might allow certain infections to persist, thereby adding to the antigenic load and increasing the likelihood of immunopathological consequences (e.g.. immunecomplex disease). Moreover, depression of the immune response might trigger or enhance the growth of certain tumours. Virus-induced potentiation of immune response might also have immunopathological consequences, such as the development of autoimmune disorders (WHO Scientific Group on Factors Regulating the Immune Response, 1970).
should be studied in greater detail, with morphologic changes perhaps serving as an indication of functional alterations. Since differences in terminology often make it difficult to assess reports of pathological changes in lymphoid tissue, all modifications of the lymphoid organs should be described according to standardized criteria. Efforts at standardization are currently being supported by the World Health Organization. (3) An attempt should be made to ascertain whether viruses can in fact exert selective effects on immune function, e.g., by depressing 7S versus 19S antibody, or by affecting T cell function as opposed to B cell function (Allison et al., 1971). The possibility should also be looked into that the immune response to the virus may itself be impaired if the infecting virus damages more or less selectively the cells responding to the viral antigens. If this proves to be the case, virus-induced immunodepression might conceivably be highly instrumental in prolonging certain virus infections, such as murine leukaemia, hepatitis, subacute sclerosing panencephalitis, or infections caused by LDV, LCMV, or ADV.
Recommendations (1) A systematic evaluation of the effects of viruses on immune function should be undertaken. A number of viruses should be studied and a standard set of immune function tests should be employed. Among the factors that deserve special investigation are antigen types (e.g., thymus-dependent versus non-thymus-dependent), antigen dose, and the time relationship between infection and antigen adminisstration. (2) The effects of virus infection on different cell types (e.g., macrophages, T and B lymphocytes)
It is well known that the persistence of antigenantibody complexes in the circulating blood can lead to serum sickness, as manifested by glomerulonephritis, polyarteritis, urticaria, arthralgia, and arthritis. Recently, it has been shown in animals that viruses can persist in the bloodstream in the form of virus-antibody complexes, and that the deposition of these complexes in the kidney can produce an immune-complex type of glomerulonephritis. Infectious virus-antibody complexes have been detected in the blood of animals with murine leukaemias and those infected with LDV, ADV, and LCMV (Mellors et al., 1969; Oldstone & Dixon, 1969; Notkins et al., 1966; Porter et al., 1969; Oldstone & Dixon, 1971b, respectively). Preliminary evidence suggests that infectious complexes also exist in the bloodstream of horses infected with equine infectious anaemia virus (EIAV) (McGuire et al., 1971). Immunopathological studies have revealed the presence of viral antigens, specific antiviral antibody, and complement in the kidneys of these animals (Oldstone & Dixon, 1971b). Severe glomerulonephritis has been found in LCMV carrier mice (Hotchin & Collins, 1964; Oldstone & Dixon, 1969, 1971b). The severity of the disease appears to be related to the strain of
the mouse, the amount of LCMV, and the amount of antiviral antibody (Oldstone & Dixon, 1969). Aleutian disease of mink also is characterized by severe glomerulonephritis (Porter et al., 1969). All mink appear to be susceptible to infection by ADV, but those homozygous for the Aleutian gene develop a more severe form of the disease, characterized by heavy deposition of virus, antibody, and complement in the glomeruli. However, relatively mild glomerular lesions are seen in mice infected with LDV. In humans, circulating Australia antigen can exist in the form of antigen-antibody complexes (Zuckerman, 1971). One case of immune-complex nephritis with deposition of Australia antigen, IgG, and complement in the glomeruli has been reported, and in 4 cases of hepatitis autopsy disclosed the presence of Australia antigen, IgG, IgM, and complement in glomerular capillaries. There is generally little evidence that vasculitis can be caused by virus-antibody complexes, but vascular lesions suggestive of polyarteritis nodosa and containing immunoglobulins have been reported late in the course of infection with ADV and EIAV. Recently, polyarteritis nodosa has been described in patients with circulating Australia antigen (Gocke et al., 1971); in one such case, Australia antigen, immunoglobulin, and complement were detected in the arterial wall (Gocke et al., op. cit.). In 5 cases of fatal hepatitis, Australia antigen, immunoglobulin, and complement were found in the intima of arterioles exhibiting changes typical of periarteritis. It has also been suggested that immune complexes may be causally involved in the urticaria and arthritis (Alpert et al., 1971) sometimes associated with hepatitis. Although deposition of circulating immune complexes appears to be the most likely explanation of these findings, the possibility has not been excluded that viral antibody might attach to viral antigens released locally from infected cells or to virus-induced antigens on the surface of infected cells (see section entitled " Antibody-mediated immunopathological injury "). In several autopsy studies of patients with various forms of hepatitis, intracellular and extracellular deposits of Australia antigen, immunoglobulins and complement were reportedly found in liver parenchymal and Kupffer cells (Nowoslawski et al., 1972). In these cases, immunoglobulins directed specifically against Australia antigen were eluted from the liver with 2.5 M thiocyanate. At present, however, there is very little information available to pinpoint the factors responsible for
producing virus-induced immune-complex disease. Whether the causal factor is the size of the complex, the nature of the viral antigen, the amount or type of antibody, the attachment of accessory factors such as complement (Winchester et al., 1971) or rheumatoid factor (Notkins, 1971; Winchester et al., 1971; Ziegenfuss et al., 1971), or the rate at which the antigen and antibody turn over (in the glomerular lesions) remains to be determined. To date, virus-induced immune-complex disease has been attributed to the deposition of virusantibody complexes during persistent virus infections (Oldstone & Dixon, 1971b). Conceivably, immunecomplex disease also could occur from the repeated deposition of such complexes during various acute and recurrent virus infections. It should be emphasized that, in addition to virion-antibody complexes, antibody bound to virus-induced membrane antigens, soluble viral antigens, and viral nucleoproteins might contribute to the pool of circulating immune complexes. The mechanism of tissue injury associated with deposition of virus-antibody complexes is presumably similar to that involved in the deposition of nonviral antigen-antibody complexes. It is known that activation of the complement sequence by immune complexes can effect the release of substances that have the capacity to increase vascular permeability, contract smooth muscle, and attract polymorphonuclear and mononuclear leucocytes. These factors would seem to play a role in the tissue injury associated with immune-complex disease. In addition, it has been postulated that immune complexes might activate components of the clotting system and thereby cause the deposition of fibrin.
Recommendations (1) The presence of immune complexes in the kidney, arterial walls, or other tissues should be confirmed by demonstrating viral antigens, specific antiviral antibody, and complement in the lesions by immunofluorescence. If, however, the antigen cannot be detected because antigenic sites have been saturated by antiviral antibody, the antibody should if possible be dissociated by standard techniques (e.g., acid buffer, pH 2.0-3.0). The eluted antigen or antibody may be characterized by immunodiffusion, complement fixation, virus neutralization, or other techniques. (2) Attempts should be made to recover and identify infectious virus from the kidney, extrarenal
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 1
tissue, and circulating blood by standard virus isolation techniques. To determine whether the isolated virus exists in the form of an infectious virusantibody complex, the anti-immunoglobulin neutralization technique should be used. (3) Efforts should also be made to detect noninfectious virus-antibody complexes in the circulation. Upon incubation with the Clq component of complement or rheumatoid factor (Winchester et al., 1971), these complexes may precipitate out demonstrably. Conversely, incubation in an acid buffer may dissociate the complexes and permit the viral antigens and specific antiviral antibody to be identified as described in (1) above. (4) If virus cannot be recovered by any of the above techniques, the animals should be immunized with isolated complexes and their sera tested for antibodies to a variety of viruses. (5) When DNA-anti-DNA complexes are present in the glomeruli, an endeavour should be made to distinguish between viral nucleic acids and nucleic acids of nonviral origin. (6) Since glomerulonephritis of differing degrees of severity can be produced in the same host by different viruses (e.g., LCMV versus LDV), attention should be focused on the factors involved in the initiation and production of immune-complex disease. It would be desirable to develop models to study the clearance of virus-antibody complexes from the bloodstream and the rate at which these complexes deposit and turn over in the kidney. (7) Animals with infections characterized by persistent or recurrent viraemia (e.g., feline leukaemia, African swine fever, hog cholera, and avian lymphomatosis) should be examined for antiviral antibody circulating virus-antibody complexes, and immunecomplex nephritis. (8) A major effort should be made to elucidate the role of immune complexes in the pathogenesis of viral hepatitis in man.
In the last decade it has been shown that the transformation of cells by oncogenic viruses results in the appearance of new antigens on the cell surface and that immune responses to these antigens may be involved in tumour rejection. Non-oncogenic viruses can also produce new antigens on the surfaces of infected cells, but the biological signi-
ficance of these antigens has received relatively little attention. Evidence is now beginning to emerge, however, suggesting that the interaction of specific antiviral antibody and complement with surface antigens induced by non-oncogenic viruses can lead to cell destruction and may contribute to the pathogenesis of the lesions associated with certain virus infections. In vivo, the best experimental evidence that antibody can play such a pathogenetic role comes from the demonstration that the passive administration of specific antiviral antibody to animals infected with LCMV (Oldstone & Dixon, 1970), ADV, or Japanese B encephalitis virus produces or intensifies the characteristic lesions associated with these infections. In addition, it has been speculated that the interaction of specific antiviral antibody and complement with antigens induced by the respiratory syncytial, measles, hepatitis, dengue, and equine infectious arteritis viruses may be partly responsible for the pathological picture seen in these infections. Another suggestion has been that the passive attachment of virus, antiviral antibody, and complement to the surface of platelets or erythrocytes may result in cell injury and might give rise to some of the haematologic abnormalities associated with virus infections, such as dengue shock syndrome (Russell, 1971)the most severe form of dengue haemorrhagic feverand equine infectious anaemia. The strongest evidence that antiviral antibody and complement can injure virus-infected cells has been produced by in vitro experiments. It has been shown that the infection of cells with viruses that do not produce cytologic injury (rabies (Wiktor et al., 1968), LCMV) (Oldstone & Dixon, 1971a), or with viruses that ultimately do cause cell damage (HSV, vacciniavirus, influenzavirus, Newcastle disease virus [NDV] (Brier et al., 1970) is followed by the appearance of new antigens on the surface of the infected cells and that the interaction of specific antiviral antibody and complement with these antigens can produce immunologic injury. In the absence of either specific antiviral antibody or complement, such injury does not occur. The degree of injury may depend on a number of factors (Brier et al., 1970), including (1) the density of viral antigens on the infected cell surfaces; (2) the inherent susceptibility of the cells to lysis by complement; (3) the nature and concentration of the antiviral antibody; (4) the ratio of complement-fixing to non-complement-fixing antibody in the particular serum; and (5) the presence of inhibitors, such as
anti-immunoglobulins or rheumatoid factor, that might block complement-fixing sites on the antiviral antibody. The appearance of viral antigens might in turn be related to other factors, such as the phase of the cell cycle or coinfection with a second virus. If a particular virus produces few or no new antigenic sites on the surface of cells or if these antigenic sites are far apart, complement-mediated cell destruction may not occur. If, however, the density of virus-specific antigens on the cell surface should rise during the course of an infection, this would increase the likelihood of complement-mediated cell destruction. Fluctuations in the density of viral antigens on the infected cell surface might contribute heavily to the pathogenesis of lesions associated with " slow virus " infections (Porter, 1971). Implications The attachment of antiviral antibody in sublytic concentrations to the surface of infected cells may conceivably be instrumental in deciding the fate of the cell. On the one hand, the attachment of antiviral antibody might accelerate phagocytosis of the infected cell by activated macrophages. On the other hand, antiviral antibody might prevent sensitized lymphocytes from recognizing or reacting with the viral antigens and thereby inhibit the cell-mediated immune response. In virus infections, this might prove to be the counterpart of "blocking" or " enhancing " antibody. Under certain circumstances the destruction of virus-infected cells by antiviral antibody and complement may be more beneficial than harmful to the host. Antibody-mediated cell destruction may be one of the mechanisms by which the host combats those viruses that tend to elude neutralization by spreading directly from cell to cell. Moreover, the destruction of cells that are actively producing virus would slow down viral replication and release or expose the infectious virus within the cell to neutralizing antibody. Thus, in virus infections, antibodymediated cell destruction may fulfil many of the functions that have been postulated for cell-mediated immunity and may serve as a complementary or supplementary defence mechanism. In addition, in vitro experiments suggest that the release of one or more chemotactic-generating factors (Brier et al., 1970) from infected cells and/or the interaction between antiviral antibody and viral antigens can activate the complement sequence and cause the release of mediators able to attract polymorphonuclear and mononuclear leucocytes.
Recommendations (1) Although many investigators have speculated that immunologic injury may contribute to the pathological picture in certain virus infections, it has been difficult to isolate and evaluate this phenomenon in vivo. The release of 51Cr from virusinfected cells by antiviral antibody and complement provides a simple, objective, and quantitative technique for studying immunologic injury in vitro. With this technique it should be possible to (a) investigate a variety of viruses; (b) evaluate virusinduced immunologic injury in different types of cell; (c) compare the roles of cytolytic and noncytolytic antibody in the serum of patients during the course of various virus infections; (d) determine whether biological mediators are released or activated as a result of the interaction of antiviral antibody and complement with viral antigens; and (e) investigate the relationship between antibodymediated and cell-mediated destruction of infected cells. (2) In vivo studies should be extended and experimental models developed. Additional studies should be conducted to evaluate the results of the passive administration of cytolytic antiviral antibody to infected animals with normal and depleted levels of complement. Efforts should be made to demonstrate the presence of antiviral antibody and complement on the surface of injured cells at the site of the lesion. Further thought should be given to the potential beneficial or harmful effects of passive protection with immunoglobulins containing cytolytic antiviral antibody or with vaccines (e.g., rabies vaccine) that might induce cytolytic antibody. (3) Attempts should be made to compare the in vitro and in vivo effects of antibody and complement on the lysis of virus-infected cells. Whether the attachment of nonlytic antibody to infected cells can inhibit the cell-mediated immune response should be investigated.
* * *
A. C. ALLISON, Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex, England W. I. B. BEVERIDGE, Veterinary Public Health Consultant, World Health Organization, Geneva, Switzerland W. C. COCKBURN, Chief Medical Officer, Virus Diseases, World Health Organization, Geneva, Switzerland JUNE EAST, Department of Environmental Carcinogenesis, Imperial Cancer Research Fund, Mill Hill,
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 1
H. C. GOODMAN, Chief Medical Officer, Immunology, World Health Organization, Geneva, Switzerland H. KOPROWSKI, The Wistar Institute of Anatomy and Biology, Philadelphia, Pa., USA P.-H. LAMBERT, World Health Organization Haematology Research Unit, Cantonal Hospital, Geneva, Switzerland J. J. vAN LOGHEM, Department of Immunopathology, University of Amsterdam, The Netherlands
P. A. MIESCHER, Haematology Division, Cantonal Hospital, Geneva, Switzerland C. A. MIMMs, Microbiology Department, The John Curtin School of Medical Research, Australian National University, Canberra City, Australia A. L. NOTKNS, Chief, Virology Section, National Institute of Dental Research, Bethesda, Md., USA G. TORRIGIANI, Medical Officer, Immunology, World Health Organization, Geneva, Switzerland
ETATS IMMUNOPATHOLOGIQUES INDUITS PAR LES VIRUS: MODLLES ANIMAUX ET RELATIONS AVEC LES MALADIES HUMAINES: 1. EFFETS DES VIRUS SUR LE SYSTEME IMMUNITAIRE, MALADIES DUES A DES IMMUNCO)APLEXES ET LtSIONS IMMUNOLCGIQUES PROVCQUVES PAR L'INTERMtDIA]RE DES ANTICORPS
On sait depuis longtemps que les virus produisent des lesions en endommageant et parfois en detruisant les cellules a l'int6rieur desquelles ils se multiplient. Plus recemment, on a decouvert que des alterations tissulaires peuvent aussi r6sulter d'interactions entre le virus et le systeme immunitaire de l'h6te. L'etude de diverses maladies des animaux a permis de deceler un certain nombre de mecanismes immunopathologiques responsables des lesions provoquees par des infections virales. Ces mecanismes - ainsi que les concepts et les techniques issus de ces recherches - scnt d6crits dans la lre et dans la 2e partie du present memorandum. Leurs consequences eventuelles au regard des maladies humaines sont examinees et plusieurs de leurs applications sont envisag6es. Un premier type de lesion immunologique est diu aux effets directs exerc6s par certains virus sur le systWme immunitaire. Des virus, notamment les virus des leuc& mies et des virus responsables d'i nfections de longue dur6e, diminuent la capacite de production des anticorps chez l'h6te; d'autres agissent en renforgant la reponse immunitaire a divers antigenes. Dans certains cas, il est manifeste que les virus inhibent 6galement l'immunite a support cellulaire. Cette action des virus sur la fonction immunitaire pourrait avoir de nombreuses et importantes cons& quences du point de vue clinique; c'est ainsi que l'affaiblissement de l'immunit6 serait susceptible de favoriser le d6veloppement de tumeurs. Les maladies dues a des immuncomplexes representent une autre forme de l6sion immun opathologique provoqu6e par des virus. Dans les infections virales persistantes, les anticorps specifiques se combinent parfois aux virus
ou aux antiggenes viraux pour former des immuncomplexes qui sont ensuite deposes dans divers endroits de l'organisme, et en particulier dans le rein. Dans cette dernie eventualite, on peut voir apparaitre ult6rieurement une glomerule-nephrite caus&e par la combinaison des immuncomplexes et du complement entrainant la liberation de substances qui lesent les tissus. Les depots d'immunocomplexes dans la paroi des petites arteres peuvent provoquer des lesions vasculaires rappelant celles de la periarterite noueuse. Enfin, un troisieme type de lesion immunologique est celui realise a l'intervention des anticorps. I1 s'agit de la reaction produite lors de la fixation des anticorps spcifiques sur les antigenes cellulaires de surface induits par les virus. Le complement, normalement present, peut alors leser les cellules et meme provoquer leur lyse. On connalt un certain nombre de virus, en dehors des virus oncogenes, qui produisent des antigenes a la surface des cellules dans lesquelles ils ont penere. L'exp6rimentation in vitro montre qu'en l'absence d'anticorps sp&cifiques ou de complement il ne se produit aucune l6sion immunologique. Dans certaines conditions, la destruction des cellules infectees par le virus se revele, en depit du dommage cause, favorable pour l'h6te en ralentissant ou en arretant la multiplication du virus. Dans d'autres cas, et notamment lorsqu'un grand nombre de cellules d'un organe vital sont atteintes, la lesion peut avoir des consequences graves et me me fatales. D'autres formes d'interaction entre les virus et le systeme immunitaire entrainant egalement des lesions sont decrites dans la 2e partie du mimorandum.
Allison, A. C. (1971) Immunity against viruses. In: The scientific basis of medicine, annual review for 1971, London, Athlone Press (in press) Allison, A. C. et al. (1971) Cooperating and controlling functions of thymus-derived lymphocytes in relation to autoimmunity, Lancet, 2, 135
Alpert, E. et al. (1971) The pathogenesis of arthritis associated with viral hepatitis, New Engl. J. Med.,
285, 185 Brier, A. M. (1972) Inhibition or enhancement of immunological injury of virus infected cells. Proc. nat. Acad. Sci. (Wash.) (in press) Brier, A. M. et al. (1970) Inflammation and herpes simplex virus: release of a chemotaxis-generating factor from infected cells, Science, 170, 1104 Dent, P. (1972) Immunodepression by oncogenic viruses, Progr. med. Virol., 14, 1 Gocke, D. J. et al. (1971) Vasculitis in association with Australia antigen. J. exp. Med., 134, 330S Hotchin, J. & Collins, D. N. (1964) Glomerulonephritis and late onset disease of mice following neonatal virus infection. Nature (Lond.), 203, 1357 McGuire, T. C. et al. (1971) Immunofluorescent localization of equine infectious anemia virus in tissue. Amer. J. Path., 62, 283 Mellors, R. C. et al. (1969) Further implication of murine leukaemia-like virus in the disorders of NZB mice, J. exp. Med., 129, 1045 Notkins, A. L. et al. (1966) Infectious virus-antibody complex in the blood of chronically infected mice, J. exp. Med., 124, 81 Notkins, A. L. et al. (1970) Effect of virus infections on the function of the immune system, Ann. Rev. Microbiol., 24, 525 Notkins, A. L. (1971) Infectious virus-antibody complexes: interaction with anti-immunoglobulins, complement, and rheumatoid factor, J. exp. Med., 134, 41S Nowoslawski, A. et al. (1972) Australia antigen and hepatitis: pathogenic considerations and practical implications, Recent Adv. clin. Path. (in press) Oldstone, M. B. A. & Dixon, F. J. (1969) Pathogenesis of chronic disease associated with persistent lymphocytic choriomeningitis viral infection. I. Relationship of antibody production to diease in neonatally infected mice. J. exp. Med., 129, 483
Oldstone, M. B. A. & Dixon, F. J. (1970) Pathogenesis of chronic disease associated with persistent lymphocytic choriomeningitis viral infection. II. Relationship of the anti-lymphocytic choriomeningitis immune response to tissue injury in chronic lymphocytic choriomeningitis disease, J. exp. Med., 131, 1 Oldstone, M. B. A. & Dixon, F. J. (1971a) The immune response in lymphocytic choriomeningitis viral infection. In: P. A. Miescher, ed., Immunophathology: VIth International Symposium, 1970, Basel, Schwabe, p. 391 Oldstone, M. B. A. & Dixon, F. J. (1971b) Immune complex disease in chronic viral infections. J. exp. Med., 134, 32S Porter, D. D. et al. (1969) The pathogenesis of Aleutian disease of mink. I. In vivo viral replication and the host antibody response to viral antigen. J. exp. Med., 130, 575 Porter, D. D. (1971) A quantitative view of the slow virus landscape, Progr. med. Virol., 13, 339 Russell, P. K. (1971) Immunopathologic mechanisms in dengue shock syndrome. In: B. Amos, ed., Proceedings of the First International Congress of Immunology, Washington, New York, Academic Press, pp. 831-838 WHO Scientific Group on Factors Regulating the Immune Response (1970) Wld Hlth Org. techn. Rep. Ser., No. 448 Wiktor, T. J. et al. (1968) Immune lysis of rabies virusinfected cells, J. Immunol., 101, 1271 Winchester, R. J. et al. (1971) Occurrence of y-globulin complexes in serum and joint fluid of rheumatoid arthritis patients: use of monoclonal rheumatoid factors as reagents for their demonstration, J. exp. Med., 134, 286S Ziegenfuss, J. F. Jr. et al. (1971) Rheumatoid factor and Australia antigen. New Eng. J. Med., 284, 1104 Zuckermann, A. J. (1971) The immunopathology of viral hepatitis associated with Australia antigen. In: P. A. Miescher, ed., Immunopathology: VIth International Symposium, 1970, Basel, Schwabe, p. 436
Virus-associated immunopathology: animal models and implications for human disease*
2. Cell-mediated immunity, autoimmune diseases, genetics, and implications for clinical research
Part 2 of this memorandum describes further mechanisms whereby the interaction of a virus with the host's immune system may lead to tissue damage. Cell-mediated immunity plays a vital role in promoting recovery from virus infections, but under some circumstances tissue damage may be caused by the reaction of immune cells with viral antigens. When mice are infected with lymphocytic choriomeningitis virus neonatally or as adults while receiving immunosuppressive drugs, widespread invasion of cells is seen but there is little overt disease. If, however, normal adults are infected or if immune cells are transfused into tolerant mice, cell injury and death follow. Viruses have long been suspected of contributing to the pathogenesis of autoimmune diseases. Antibodies directed against normal cell constituents have been reported in several virus infections. Viruses may conceivably unmask or release host antigens, alter host antigens and act as " helper determinants ", or perhaps in other ways provoke immune responses against normal body constituents. The immunopathological manifestations caused by viruses may also be influenced by the host's genetic makeup. Certain observations indicate that, in addition to controlling susceptibility to virus infection, genetic factors partly determine the effectiveness of the immune response. The memorandum calls attention to the possible implications of these concepts and findings for clinical research. Some of the diseases of animals and man that serve as models for studies of virus-associated immunopathology are briefly described. Part 1 of this memorandum on viral immunopathology (Bull. Wld Hlth Org., 1972) dealt with the direct effects of viruses on the immune system and their role in immune-complex disease and antibody-mediated immunological injury. In Part 2 the relationship of viruses to cell-mediated immunity and autoimmune diseases is examined, the genetic aspects of the host's response to virus infection are reviewed, and some implications of the new concepts and techniques in viral immunopathology for clinical research are explored. Annex 1 describes a number of animal and human disease models in use in this field.
tific Group on Cell-Mediated Immune Responses, 1969), promoting the sterilization of tissues and the resolution of lesions. Under some circumstances, however, the reaction of immune cells with viral antigens, either free or on the surface of infected cells, may cause tissue damage. The laboratory model of cell-mediated immunopathology that has received the most study is lymphocytic choriomeningitis virus (LCMV) infection of mice (Oldstone & Dixon, 1970a).
There is increasing evidence that in some virus infections the cell-mediated immune response plays a central role in recovery from infection (Allison, 1972; Blanden, 1971; Glasgow, 1970; WHO Scien* This memorandum was prepared by the signatories listed on page 270.
LCMV-induced immunopathological damage In adult mice infected with LCMV, viral replication occurs in many tissues, including the viscera and meninges. The infection is noncytopathic, and the animals remain free of clinical signs until the onset of an immune response 5-7 days after infection. Associated with this response are inflammatory cell infiltrations occurring at the visceral and meningeal sites of viral replication and leading to pathological changes, overt clinical signs, and death. Inhibition of the immune response by neonatal thymectomy,
irradiation, or the administration of antilymphocyte serum (ALS) (Mims & Tosolini, 1969; Wiktor & Koprowski, unpublished data) or cytotoxic drugs can prevent the acute disease. Mice infected with LCMV neonatally or congenitally also develop widespread infection but their immune response to the virus is poor. No clinical signs are seen in these animals during the early phase ofthe infection 1 and late in life chronic glomerulonephritis develops as a result of the deposition of virus-antibody complexes in the kidney (see Bull. Wld Hlth Org., 1972). Attempts have been made to determine whether it is the antibody-mediated or the cell-mediated component of the immune response that causes the pathological changes in mice infected as adults with LCMV. In vitro experiments have shown that when spleen cells from LCMV-immune mice are added to LCMV-infected cells, the latter will be destroyed (Lundstedt, 1969). Cell destruction is probably mediated by a cytotoxic factor that is released when spleen cells from LCMV-immune mice interact with LCMV or LCMV-infected mouse fibroblasts (Oldstone & Dixon, 1970b). The design of the corresponding in vivo experiments was as follows. Cyclophosphamide 2 was given to adult mice infected with LCMV (Cole et al., 1972), thus inhibiting the immune response, and a persistent clinically inapparent infection was established. When immune spleen cells were injected into these mice, the animals died and acute pathological changes comparable to these occurring in the natural infection were seen. Since the transfusion of immune serum produced less severe pathological changes, it was concluded that the injected cells had reacted with infected host cells to create a cell-mediated immunopathological condition. In congenitally infected carrier mice, in contrast, the transfer of immune spleen cells failed to
1 In addition to LCMV, some other microorganisms are known to produce less severe clinical signs in newborn than in older animals, e.g., the agents of many protozoal diseases and of heartwater. The latter is caused by a mycoplasma-like organism that grows in vascular endothelium and other sites. Infections of very young lambs or calves cause only mild clinical signs, even when numerous organisms are present for many months, whereas infections of older animals are severe and often fatal. The transfer of lymphocytes from old to young infected animals precipitates overt signs of disease in the latter. Although there may be other explanations-including maternal antibody-for the relative insusceptibility of young animals to some infectious diseases, the possible role of immunopathological reactions in adults with the disease should always be borne in mind. 2 2-bis(2 - chloroethyl) amino]tetrahydro - 2H- 1,3,2-oxazaphosphorine 2-oxide.
yield any clinical or histologic evidence of tissue damage. High titres of neutralizing and complementfixing antibody were produced in the recipient carrier mice so that, under these circumstances too, the animals remained free of clinical signs. Other experiments, however, have shown that LCMV antibody plus complement is cytotoxic for LCMV-infected cells in vitro (Oldstone & Dixon, 1971a), and tissue lesions have been detected after the injection of large amounts of antibody into carrier animals. The possible immunopathological action of antibody was also suggested by the finding that complement depletion by means of cobra venom made LCMV considerably less lethal for adult mice. However, mice deficient in the C5 component of complement showed normal susceptibility to LCMV. In summary, these results suggest that cellmediated immunity may be an important factor in LCMV-induced immunopathological injury, although they do not rule out the possibility that antibody may also play a role. Evidence of cell-mediated immunity in other virus infections Certain experiments have indicated that cells persistently infected with measles or mumps virus (Speel et al., 1968) may be destroyed in vitro by incubation with spleen cells obtained from specifically immunized animals, but these studies need to be confirmed and expanded. It has also been suggested that cell-mediated immune responses may be responsible for the rashes associated with certain infections (e.g., measles) and might account for the skin lesions seen in rabbits infected with Shope fibroma (Tompkins et al., 1970) and rabbit pox viruses. Indirect evidence from patients with immunologic deficiencies also points to the importance of cell-mediated immunity in certain virus infections, but in others (e.g., enterovirus infections) cellmediated immunity may be relatively insignificant. There is some evidence that sensitized lymphocytes after reacting specifically with viral antigen may release biological mediators, such as migration inhibition factor (MIF), lymphotoxin, interferon, and factors chemotactic for polymorphonuclear and mononuclear leucocytes. Some mediators also activate macrophages, which will then show an increased capacity to take up and kill bacteria, protozoa, and possibly viruses. In addition, activated macrophages may interact with viral antigens on the surface of infected cells and in this way, perhaps with the help
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
of cell-bound antibody, may play a role in the host's defence against virus infections (Tompkins et al., 1970) while at 'the same time contributing to the production of lesions (Allison, 1972). Recommendations
(1) In vitro experiments to test for cell-mediated cytotoxicity of the type described above should be carried out in more virus infections. Caution should be exercised in interpreting negative results, however, because much may depend on the type of infected target cell used. (2) The distribution and density of viral antigens on the surface of infected cells should be studied thoroughly, since these are the targets of-cell-mediated immunopathology. Immunofluorescence techniques are relatively simple to use; alternatively, the topographic localization of antigen can be elegantly studied (Aoki et al., 1970) at the ultrastructural level by the use of reconstituted antibody molecules reacting with both viral antigen and marker particles. (3) Since lysis of cells by sensitized lymphocytes might be prevented by a " blocking antibody " adsorbed on the surface of the target cells or by antigen-antibody complexes adsorbed on the sensitized lymphocytes or target cells (Hellstrom & Hellstr6m, 1970), it would be desirable to investigate this phenomenon in virus infections where cellmediated immunity may be involved. (4) Sensitized lymphocytes may exert much of their protective or other immunologic effects by recruiting activated macrophages into infected areas. Further studies are therefore needed of the role played by macrophages not only in the expression and pathological consequences of cell-mediated immunity but also in the induction of the immune response to virus infections. (5) Attempts should be made to assess the importance of cell-mediated immune responses in the pathogenesis of natural infections of animals. A useful approach would be to treat animals with cyclophosphamide, a drug that suppresses both antibody- and cell-mediated immunity (Cole et al., 1972), and then infect them with the virus to be studied. If cyclophosphamide treatment were found to decrease the pathogenicity of the infection without affecting viral growth in the tissues, then the respective parts played by immune cells and antibody could be investigated. Infected cyclophosphamide-treated animals could be given either cells or serum from
normal or immune donors and the ensuing pathological changes and disease could then be recorded. (6) Cells found to be immunocompetent, as shown by blast transformation after exposure to virus, should be transferred to cyclophosphamide-treated animals as in (5) with a view towards elucidating their role. (7) Since the mechanism of recovery from some infections, notably human hepatitis, is still obscure, an analysis of cell-mediated immunity against specific antigens should be made. One approach would be to obtain lymphocytes (Rosenberg et al., unpublished data) from patients recovering from hepatitis, expose the cells in vitro to Australia antigen, and determine whether they undergo blast transformation.
AUTOIMMUNE DISEASES 1
Autoimmunity is the general term used to describe an immune response, either antibody- or cellmediated, against normal body constituents. The presence of autoantibodies to certain host antigens, such as thyroglobulin, DNA, mitochondria, and microsomes, is relatively common, especially in older individuals and does not always lead to overt pathological changes. However, certain autoantibodies, particularly those directed against surface components of such cells as erythrocytes, frequently produce severe autoimmune disease. Other autoimmune diseases, such as Hashimoto's disease (a chronic and progressive thyroiditis), pernicious anaemia, and adrenalitis, are accompanied by infiltrations of mononuclear cells, and cell-mediated responses are believed to contribute to these pathological manifestations. The role of viruses in the etiology or pathogenesis of autoimmune disease has long been suspected (Lindenmann & Klein, 1967). When considering this problem, however, one must distinguish between immune reactions directed against virus-specified antigens and those directed against host antigens (true autoantigens). Such distinctions may be difficult to make in practice, especially where viruses are transmitted from mother to offspring and are present throughout life. Furthermore, while a virus can often be proved to be present, formal proof of its absence is far more difficult to obtain.
1 Further information on some diseases of animals in which autoimmune manifestations may be related directly or indirectly to an underlying virus infection (in some cases surmised but not proved) is given in Annex 1.
Theoretically, virus infections could precipitate autoimmune reactions in a variety of ways: (1) The virus itself might provide antigens (e.g., viral nucleoprotein) cross-reacting with host antigens; (2) viruses might unmask or release antigens from damaged cells-autoantibodies directed against soluble nuclear components have been found, for example, after infectious mononucleosis; (3) viruses might alter host-cell antigens and act as " helper determinants " (Allison et al., 1971); (4) viruses might derepress hostcell antigens (e.g., embryonic antigens); and (5) viruses might affect the proliferation or responses of immunocompetent cells or their precursors (see Bull. Wld Hlth Org., 1972). For many years it has been speculated that myxoviruses may cause autoimmune disease. These viruses contain neuraminidase, an enzyme known to produce antigenic modification in host cells. Incubation of erythrocytes with certain myxoviruses results in the unmasking of T-agglutinins, and immunization of animals with the altered red cells can raise serum titres of antibody to these antigens. In addition, elevated T-agglutinin levels have been observed following natural myxovirus infection, but no obvious autoimmune manifestations have been reported. Thus, the relationship between myxovirus infection and autoimmune disease remains unclear. It has been shown that animals recovered from tumours destroyed by lysis after infection with nononcogenic viruses (Koprowski et al., 1957), or immunized with homogenates (Lindenmann & Klein, 1967) are more resistant to subsequent challenge with viable uninfected tumour cells than are animals immunized with uninfected tumour homogenates. Several investigators have postulated that the virus modifies the surface antigens of the tumour by introducing' a " helper determinant " (Hirsch et al., 1968). These helper determinants would act in much the same way as a hapten to increase the host's ability to mount an effective immune response and reject the tumour. Similarly, it has been suggested that viruses acting as helper determinants might initiate an autoimmune response to antigens of the host's own cells. An alternative hypothesis is that host antigens released from virus-infected cells or host antigens incorporated into the envelope of the maturing virion might reach immunologically competent cells and stimulate the production of specific antibody. These hypotheses have been proposed to explain a number of diseases, including subacute thyroiditis and postinfection encephalitis, but firm experimental evidence is still lacking.
Antibodies to normal cell constituents have been reported in a variety of other diseases known or suspected to be caused by viruses. Antinuclear antibodies (ANA) have been observed in Aleutian disease of mink, equine infectious anaemia (Henson et al., 1970; Squire, 1968), systemic lupus erythematosus (SLE) in dogs and man (Lewis & Schwartz, 1971; Blomjous & Feltkamp-Vroom, 1971), hepatitis in man (Zuckerman, 1971), as well as in New Zealand Black (NZB) mice (Blomjous & Feltkamp-Vroom, 1971). Antibodies to erythrocytes have been reported in Aleutian disease, SLE, NZB mice and EIA (Squire, 1968). Antibodies to mitochondria and smooth muscle have been found in hepatitis, and antibody to thyroglobulin has been detected in dogs with SLE. In some of these conditions-e.g., SLE, a viral etiology has not been established, while in others it is still far from clear whether the autoimmune manifestations are a direct or an indirect consequence cf the virus infection. Even in the widely studied NZB syndrome, many questions still remain concerning the relationship between the Ctype particles observed in the tissue of these animals and the rise in antibody to erythrocytes and nucleic acids (East, 1970). Recently, several electron microscopy studies have detected tubular structures resembling the nucleocapsids of myxoviruses or paramyxoviruses in the glomeruli and synovia of patients with SLE (Melczer et al., 1962; Gyorky et al., 1969; Sinkovics, 1971), in the skin of patients with scleroderma and dermatomyositis (Norton et al., 1970). and in the thymus of patients with myasthenia gravis and Hashimoto's disease (Loghem, 1965). Although these diseases are believed to have an autoimmune component, it is not known how their pathogenesis may be related to the virus-like particles.
Recommendations (1) A clear distinction should be made between immune responses to viral antigens (which should not be considered under the topic of autoimmunity) and immune responses to host or modified host antigens. (2) Assay methods should be developed and standardized for the detection and quantification of both the humoral and the cellular immune response to host antigens. (3) The ability of a virus to cause autoimmune disease should be determined by inoculating animals with a cell-free preparation of purified virions.
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
(4) In diseases of unknown etiology where autoimmune manifestations have been demonstrated or are strongly suspected, a search for a viral etiology should be initiated. In humans, SLE, rheumatoid arthritis and subacute thyroiditis appear to be good ecandidates for study (see below). In animals, canine (Lewis & Schwartz, 1971) and feline SLE (Slauson et al., 1971) should receive further attention.
Genetic differences in the susceptibility of animals to virus infections have been well established. Genetic factors linked to the major histocompatibility loci of the mouse may determine the susceptibility of the animals to infection with, e.g., LCMV and some of the oncogenic viruses (WHO Scientific Group on the Genetics of the Immune Response, 1968). When mice of the H-2k haplotype are exposed to oncogenic viruses such as the Gross (WHO Scientific Group on the Genetics of the Immune Response, 1968), Friend, Rauscher, or Moloney virus, they develop leukaemia, whereas mice of the H-2b haplotype show much less sensitivity to these viruses. Susceptibility to various types of RNA avian leucosis virus is also genetically determined (WHO Scientific Group on the Genetics of the Immune Response, 1968), and a single dominant factor seems to account for the resistance of one strain of mice to influenza virus (WHO Scientific Group on the Genetics of the Immune Response, 1968). A single gene also is responsible for the resistance of mice to group B arbovirus infections (Hanson & Koprowski, 1969). That there are genetic differences in the immune response of the host to a variety of nonviral antigens has also been well documented. In some cases, the magnitude of the immune responses to defined antigens is under the control of allelic genes that are closely linked to major histocompatibility genes. It is possible that the nature and magnitude of the immune response to viral antigens may likewise be under genetic control and may affect immunopathological manifestations. Although genetic control has not been documented with viral antigens, certain animal experiments are consistent with this possibility. The association of the IR-1 gene with the H-2k haplotype may indicate that " genetic resistance " to oncogenic viruses is expressed by the magnitude of the immune response of the animal host (Fed. Proc., 1972). Similarly, the genetic resistance of C57BL mice to ectromelia virus is thought to be reflected by their greater immunologic reactivity to
the virus (Schell, 1960). Conversely, the weaker immune response of C57BL mice to LCMV is believed to be responsible for the less severe LCMVinduced immunopathological lesions in this strain (Tosolini & Mims, 1971; Mims & Tosolini, 1969). In man, there is some evidence that differences in the immune response may be a factor in susceptibility to hepatitis (Blumberg et al., 1969; Ceppellini et al., 1970), Indian childhood cirrhosis (Nyak et al., 1972), and subacute sclerosing panencephalitis (SSPE) (Lischner et al., 1972), but it is not certain whether the depressed immune response is genetically determined and precedes the infection or whether it is a result of the infection.
Recommendations (1) In view of the correlation in mice between H-2 type and susceptibility to RNA virus and the analogy between H-2 and human histocompatibility antigens (HL-A), studies are now in progress to search for an association between HL-A phenotype and susceptibility to diseases with a suspected viral etiology, such as Hodgkin's disease and acute leukaemia. The preliminary studies on SLE should be continued and extended to include rheumatoid arthritis, myasthenia gravis, and eventually other autoimmune diseases of possible viral etiology. (2) Tests for cell-mediated immunity are not well standardized, as shown by the conflicting reports not only on SSPE (Lischner et al., 1972), but also Hodgkin's disease and other conditions. It is recommended that tests be carried out under uniform conditions, using internationally standardized reagents (Bull. Wld Hlth Org., 1971). The World Health Organization may be able to facilitate the establishment and dissemination of standard tests for cell-mediated immunity. Such tests should be undertaken, for instance, in regions where the incidence of Indian childhood cirrhosis is high (see above). (3) Studies to investigate possible linkage relationships between hereditary diseases and genetic markers present in blood cells and serum proteins should be encouraged.
IMPLICATIONS FOR CLINICAL RESEARCH
Examples of each of the principal virus-associated immunopathological mechanisms discussed in Parts 1 and 2 of this memorandum are known in experimental animals and provide an opportunity for detailed
analysis of the factors involved. While it would not be possible to conduct similar research on human subjects, the knowledge acquired in animal studies may be applicable to human disease. In certain virus infections of man, immunopathological complications are suspected of contributing to the disease syndrome. These infections include hepatitis, virus-related haemorrhagic fevers (dengue almost certainly, and perhaps Argentinian haemorrhagic fever), respiratory syncytial virus infection, infectious mononucleosis, and possibly SSPE. Moreover, immunopathological mechanisms may be involved in causing the skin lesions-e.g., rashesassociated with some of the common virus infections of man, such as measles and vaccinia. Conversely, there are some recognized immunopathological diseases in which viruses are suspected of playing a role. For instance, in immune-complex glomerulonephritis of man the antigen may ultimately prove to be viral in origin. Virus-like particles have been found in patients with SLE (as well as in a dog with SLE); these may be involved in the pathogenesis of the disease syndrome. More extensive investigations are needed to clarify the role of viruses in the etiology of these diseases. Viruses have also been suspected in the etiology of rheumatoid arthritis, but thus far no proof of this has been obtained. The chronic arthritis associated with chlamydial disease in animals and with Reiter's disease in man may have immunopathological components but it is unlikely that these diseases are useful models for rheumatoid arthritis. A possible viral etiology involving immunopathological mechanisms has also been suggested for endocrine diseases, e.g., subacute thyroiditis, and haemolytic anaemia. In conclusion, the theoretical concepts and technical methods summarized in this memorandum may
be usefully applied to the study of suspected immunopathological manifestations in human disease, including autoimmune reactions. The experimental finding that a disease can be prevented or attenuated by the use of immunosuppressive drugs strongly implies thatit has an immunopathological component. However, caution should be exercised in attempting to use immunosuppression in human patients, because of the risk of increasing the ability of the virus to replicate and thereby disseminate the infection.
A. C. ALLISON, Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex, England W. I. B. BEVERIDGE, Veterinary Public Health Consultant, World Health Organization, Geneva, Switzerland W. C. COCKBURN, Chief Medical Officer, Virus Diseases, World Health Organization, Geneva, Switzerland JUNE EAST, Department of Environmental Carcinogenesis, Imperial Cancer Research Fund, Mill Hill, London NW7 18D, England H. C. GOODMAN, Chief Medical Officer, Immunology, World Health Organization, Geneva, Switzerland H. KOPROWSKI, The Wistar Institute of Anatomy and Biology, Philadelphia, Pa., USA P.-H. LAMBERT, World Health Organization Haematology Research Unit, Cantonal Hospital, Geneva, Switzerland J. J. VAN LOGHEM, Department of Immunopathology, University of Amsterdam, Netherlands P. A. MLESCHER, Haematology Division, Cantonal Hospital, Geneva, Switzerland C. A. MIMMs, Microbiology Department, The John Curtin School of Medical Research, Australian National University, Canberra City, Australia A. L. NOTKINS, Chief, Virology Section, National Institute of Dental Research, Bethesda, Md., USA G. TORRIGIANI, Medical Officer, Immunology, World Health Organization, Geneva, Switzerland
RtSUMPtTATS IMMUNOPATHOLOGIQUES INDUITS PAR LES VIRUS: MODALES' ANIMAUX ET RELATIONS
AVEC LES MALADIES.HUMAINES: 2. IMMUNITE A SUPPORT CELLULAIRE, MALADIES AUTO-IMMUNES, GENETIQUE ET CONStQUENCES POUR LA RECHERCHE CLINIQUE
Dans cette deuxieme partie du memorandum, on decrit d'autres mecanismes par lesquels l'interaction d'un virus et du systeme immunitaire de 1'hote est susceptible de provoquer des lesions tissulaires. L'immunite a support cellulaire joue un role vital dans la r6sistance aux infections virales et dans le processus de guerison mais, dans certains cas, la reaction entre les cellules immunes et les antigenes viraux, libres
ou fixes a la surface des cellules infectees, peut leser gravement les tissus et meme entrainer la mort. L'infection de souris (pendant la p6riode neonatale ou, a 1'etat adulte, apr6s administration de medicaments immunosuppresseurs) par le virus de la choriomeningite lymphocytaire determine une invasion massive des cellules par le virus, mais les animaux ne pr6sentent que tres peu de sympt6mes cliniques de maladie pendant de longues
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
p6riodes. Si on leur administre ensuite par transfusion des cellules immunes, les l6sions tissulaires apparaissent et la mort survient, la r6action etant similaire a celle qui succede a l'infection de la souris adulte normale par le meme virus. On pressent depuis longtemps que les virus jouent un r6le dans l'etiologie ou la pathog6nie de certaines maladies auto-immunes, et les r6sultats de recherches recentes plaident en faveur de cette hypothese. On a signale l'apparition d'anticorps dirig6s contre les constituants normaux de la cellule au cours de plusieurs infections virales. On admet, comme explications les plus plausibles, que a) certains antigenes viraux seraient semblables aux antigenes de l'h6te; b) les virus pourraient r6veler la pr6sence d'antigenes cellulaires ou les liberer; c) les virus pourraient modifier les antigenes cellulaires de l'h6te et/ou se comporter comme des * determinants accessoires *. La microscopie electronique a recemment detecte des structures de type viral dans les tissus d'ani-
maux et de malades atteints de certaines affections a composante immunologique, comme le lupus erythemateux diss6mine et la maladie de Hashimoto. Les differences g6netiques entre individus, au sein d'une espece, peuvent influencer les manifestations immunopathologiques dues aux virus. D'apres certaines observations, il apparait que les facteurs g6netiques controlent non seulement la receptivite a l'infection virale - la receptivite a l'infection par les virus de la leuc6mie, par exemple, est liee it certains antiganes h6reditaires d'histocompatibilite - mais encore qu'ils conditionnent en partie l'aptitude de l'hote a edifier une reponse immumtaire a l'egard des virus et d'autres antigenes. Le memorandum formule des recommandations relatives aux investigations futures dans le domaine de l'immunopathologie chez l'homme et chez des modeles animaux et suggere differentes orientations pour la recherche. Certaines affections humaines et animales A composante immunologique sont brievement evoquees.
Allison, A. C. (1972) Immunity against viruses. In: The scientific basis of medicine, annual review for 1972, London, Athlone Press, p. 49 Allison, A. C. et al. (1971) Cooperating and controlling functions of thymus-derived lymphocytes in relation to autoimmunity, Lancet, 2, 135 Aoki, T. et al. (1970) G (Gross) and H-2 cell surface antigens: location on gross leukaemia cells by electron microscopy with visually labelled antibody. Proc. Nat. Acad. Sci. (Wash.), 65, 569 Blanden, R. V. (1971 Mechanisms of recovery from a generalized viral infection: mousepox. II. Passive transfer of recovery mechanisms with immune lymphoid cells, J. exp. Med., 133, 1074 Blomjous, F. J. E. M. & Feltkamp-Vroom, T. M. (1971) Hidden antinuclear antibodies in seronegative SLE patients and in NZB and NZB x NZW F1 mice, Europ. J. Immunol., 1, 396 Blumberg, B. S. et al. (1969) Hepatitis and Australia antigen: autosomal recessive inheritance of susceptibility to infection in humans, Proc. Nat. Acad. Sci. (Wash.), 62, 1108 Bull. Wld Hlth Org., 1971, Primary immunodeficiencies, 45, 125 Ceppellini, R. et al. (1970) High frequency and family clustering of an antigen in some Italian populations. In: Australia ed epatite antigene virale, Turin, Minerva Medica Cole, G. A. et al. (1972) Lymphocytic choriomeningitis virus: pathogenesis of acute central nervous system disease (in press) East, J. (1970) Immunopathology and neoplasms in New Zealand Black (NZB) and SJL/J mice, Progr. exp. Tumor Res. (Basel), 13, 84
Fed. Proc., 1972, Biological significance of histocompatibility antigens: report of an NIH-WHO Conference, 31, 1087 Glasgow, L. A. (1970) Cellular immunity in host resistance to viral infections, Arch. int. Med., 126, 125 Gyorkey, F. et al. (1969) Systemic lupus erythematosus and myxovirus, New Engl. J. Med., 280, 333 Hanson, B. & Koprowski, H. (1969) Interferon mediated natural resistance of mice to arbo B virus infection Microbios, 1B, 51 Hellstrom, K. E. & Hellstrom, I. (1970) Immunological enhancement as studied by cell culture techniques, Ann. Rev. Microbiol., 24, 343 Henson, J. B. et al. (1970) Recent research on the virology, serology and pathology of equine infectious anaemia. In: Proceedings of the 2nd International Congress on Equine Infectious Diseases, Paris, Basle, Karger, p. 178 Koprowski, H. et al. (1957) Enhancement of susceptibility to viruses in neoplastic tissues, Texas Rep. Biol. Med., 15, 559 Lewis, R. M. & Schwartz, R. S. (1971) Canine systemic lupus erythematosus: genetic analysis of an established breeding colony, J. exp. Med., 134, 417 Lindenmann, J. & Klein, P. A. (1967) Viral oncolysis: increased immunogenicity of host cell antigen associated with influenza virus, J. exp. Med., 126, 93 Lischner, H. W. et al. (1972) New Engl. J. Med., 286,786 Loghem, J. J. van (1965) Viral infection and idiopathic autoimmune diseases, a hypothesis, Vox. Sang. (Basel), 10, 1 Lundstedt, C. (1969) Interaction between antigenically different cells: virus-induced cytotoxicity by immune lymphoid cells in vitro, Acta. path. microbiol. scand., 75, 139
Melczer, M. et al. (1962) Data on the etiology of lupus erythematosus, Orv. Hetil, 103, 581-584 Mims, C. A. & Tosolini, F. A. (1969) Pathogenesis of lesions in lymphoid tissues of mice infected with lymphocytic choriomeningitis (LCM) virus, Brit. J. exp. Path., 50, 584 Norton, W. L. et al. (1970) Endothelial inclusions in dermatomyositis, Ann. rheum. Dis., 29, 67 Nyak, N. C. et al. (1972) a-Fetoprotein in Indian childhood cirrhosis, Lancet, 1, 68-69 Oldstone, M. B. A. & Dixon, F. J. (1970a) Pathogenesis of chronic disease associated with persistent lymphocytic choriomeningitis viral infection. HI. Relationship of the anti-lymphocytic choriomeningitis immune response to tissue injury in chronic lymphocytic choriomeningitis disease, J. exp. Med., 131, 1 Oldstone, M. B. A. & Dixon, F. J. (1970b) Tissue injury in lymphocytic choriomeningitis viral infection: virusinduced immunologically specific release of a cytotoxic factor from immune lymphoid cells, Virology, 42, 805 Oldstone, M. B. A. & Dixon, F. J. (1971) The immune response in lymphocytic choriomeningitis viral infection. In: P. A. Miescher, ed., Immunopathology: VIth International Symposium, 1970, Basel, Schwabe, p. 391 Schell, K. (1960) Studies on the innateresistance of mice to infection with mousepox. I. Resistance and antibody production, Aust. J. exp. Biol. med. Sci., 38, 271
Sinkovics, J. G. (1971) Virus-like particles in systemic lupus erythematosus, New Engl. J. Med., 284, 107 Slauson, D. 0. et al. (1971) Naturally occurring immune complex glomerulonephritis in the cat, J. Path. Bact., 103, 131 Speel, L. F. et al. (1968) An immuno-cytopathogenic interaction between sensitized leukocytes and epithelial cells carrying a persistent noncytocidal myxovirus infection, J. Immunol., 101, 409 Squire, R. A. (1968) Equine infectious anaemia: a model of immunoproliferative disease, Blood, 32, 157 Tompkins, W. A. F. et al. (1970) An in vitro measure of cellular immunity to fibroma virus, J. Immunol., 104, 502 Tosolini, F. A. & Mims, C. A. (1971) Effect of murine strain and viral strain on the pathogenesis of lymphocytic choriomeningitis infection and a study of footpadi responses, J. infect. Dis., 123, 134 WHO Scientific Group on Cell-Mediated Immune Responses (1969) Wld Hlth Org. techn. Rep. Ser., No. 423 WHO Scientific Group on the Genetics of the Immune Response (1968) Wld Hith Org. techn. Rep. Ser., No. 402 Zuckerman, A. J. (1971) The immunopathology of viral hepatitis associated with Australia antigen. In: P. A. Miescher, ed., Immunopathology: VIth International Symposium, Basel, Schwabe, p. 436
Murine autoimmune haemolytic anaemia
Haemolytic disease begins to develop in some mice of the NZB strain at the age of 4-5 months; direct Coombs reactions increase in incidence and intensity until the majority of animals are positive at age one year. These serologic reactions are accompanied by anaemia of insidious onset and variable degree and by progressive reticulocytosis and splenomegaly. The circulating autoantibody reacts with the erythrocytes of normal strains of mice in the indirect Coombs test. Impairing the cell-mediated immune responses by neonatal thymectomy neither delays nor prevents Coombs conversion, which shows that the reaction is antibody-mediated. Persistent high levels of IgM are found. C-type viruses, very similar to known viruses of the murine leukaemia group, are regularly observed in conventional and germ-free NZB embryos and in animals of the NZB strain throughout life. Presumably, the virus is transmitted via the germ-cells or placenta, or both, in the same way as the indi-
genous Gross virus of other spontaneously "highly leukaemic strains ". The NZB virus possesses antigens of the Gross type. The fact that germ-free NZB mice also develop autoimmune haemolytic anaemia suggests that the stimulus precipitating autoimmunity is intrinsic and might be related to the animals' own endogenous murine leukaemia virus. There is still no convincing experimental evidence that the virus in the form of cell-free filtrates can reproduce the autoimmune disease in either syngeneic or allogeneic recipients. Male or female NZBs can, nevertheless, transmit the disease to their hybrid offspring when mated with normal partners, and since the transmission patterns cannot be easily interpreted in simple genetic terms a viral etiology must still be suspected. Coombs reactions do not develop in other highly leukaemic strains of mice. This implies that the NZBs react differently to their Gross leukaemia virus, as indicated by the observation that they produce cytotoxic and possibly virus-neutralizing antibody. If their thymocytes are sensitized to viral antigens, a helper
VIRUS-ASSOCIATED IMMUNOPATHOLOGY. 2
effect may contribute to autoimmunity. However, on the basis of present evidence, it is still a matter for conjecture whether the virus contributes to the development of autoimmunity in NZB mice.
Murine SLE Moderate to severe membranous glomerulo- Human SLE nephritis with proteinuria develops in both germThe distinctive features of human SLE are the free and conventional NZB mice as they age, but anti-DNA antibodies and the immune complexes only a few experience severe histologic damage and containing DNA present in the kidneys. Tubular renal failure. However the lesions of F1 (NZBx structures resembling the nucleocapsids of myxo- or New Zealand White [NZWD hybrids are more severe, paramyxoviruses have frequently been observed by the majority of F1 females dying within a year. electron microscopy in the endothelial cells of renal The hybrids spontaneously develop high titres of glomeruli and, less often, in the lymphocytes and circulating anti-DNA, which together with DNA synovia of patients with SLE. Similar particles have and complement accumulates in the mesangium and been found in skin biopsies of patients with scleroglomerular capillaries. Antibodies to RNA also derma and dermatomyositis, in the thymus of patients circulate in young animals. Immunization of very with myasthenia gravis or Hashimoto's disease, and young hybrids with single stranded DNA can hasten in the stomach of one person with pernicious anaethe appearance of ANA and cause a fatal acceleration mia. The particles are situated in the cytoplasm of of the glomerulonephritis. Synthetic double-stran- epithelial and endothelial cells and fibroblasts; they ded RNA without adjuvant provokes the formation may be localized around the nucleus or lie free in of anti-RNA and/or anti-DNA complexes and, with the dilated cisternae of the endoplasmic reticulum. adjuvant, exacerbates the kidney disease and forms There is evidence that they contain RNA. complexes in the kidneys. Other strains spontaneously develop ANA, some in very high incidence, and are able to produce speci- Canine SLE fic ANA when suitably immunized. The titres of A canine counterpart of human SLE has been ANA obtained vary with the strain, but high titres described, characterized by LE cells, circulating are sometimes seen. The severe kidney disease of ANA, and fatal glomerulonephritis, associated with the NBZ x NZW F1 hybrids is only one aspect of a severe Coombs-positive haemolytic anaemia often their intrinsic hyperreactivity to some specific anti- accompanied by thrombocytopenic purpura. Polygenic stimuli, including DNA. A contribution arthritis, antibody to thyroglobulin, rheumatoid by the endogenous Gross virus is suggested by the factor, and hyperglobulinaemia are also found. A fact that glomerulonephritis still develops in germ- breeding colony of affected dogs has now been free NZB mice, in which type-specific viral antigen established, and the offspring of affected parents, or and anti-viral antibody are also present in the glo- an affected mother, exhibit multiple serologic abnormerular deposits. The most likely explanation is malities and thymic lesions but no clinical signs as that the Gross virus triggers or reinforces the auto- yet of SLE. The data obtained to date do not point immune reaction, resulting in glomerulonephritis. to a simple pattern of genetic inheritance, and the However, the problem has yet to be solved, and the possibility of a vertically transmitted infectious agent source of the DNA is still uncertain. In view of the has to be considered. One dog examined has had presence of an RNA-dependent DNA polymerase virus-like particles in renal glomerular endothelium in C-type particles, it is conceivable that the RNA similar to those reported in human SLE. An SLEin the virions may be transcribed into DNA pro- like condition has also been observed in cats. viruses, thus providing material which either induces or reacts with ANA. Equine infectious anaemia There is increasing evidence that two factors are involved in the pathogenesis of the lupus type of This is a disease produced by a virus transmitted glomerulonephritis in mice. Mice carrying a large mechanically by insects and characterized by vascular amount of Gross virus develop anti-DNA antibody lesions, anaemia, and glomerulonephritis. Splenoand a lupus-type glomerulonephritis, provided they megaly and lymphadenopathy are accompanied by
have a good immune reactivity to DNA-like antigens. In contrast, in NZB x NZW F1 hybrids infected with lactic dehydrogenase virus, depression of natural Gross virus infection leads to an inhibition of the renal disease.
proliferation of atypical plasma cells; serum IgM increases concomitantly with clinical episodes but high titres may persist in chronic cases. The virus,
growing in cultured lymphocytes, is very similar although not identical in ultrastructure to C-type particles of leukaemia viruses. The infected horses are C3-deficient, and complement coats the erythrocytes of some animals at various stages of the disease. It is possible that virus antibody-complexes on the surface of the erythrocytes account for the presence of complement, in which case there is no need to postulate the presence of true autoimmunity. This condition would then be considered among the immune-complex diseases.
Mycoplasma pneumoniae infection in man is regularly followed by the transient appearance of cold autoantibody with specificity against the I or i blood group antigen. It has also been reported that antibodies to some cell components of lung tissue are present, and this finding would be worth confirming. Antibodies with anti-I specificity have been reproduced in one experiment by inoculating M. pneumoniae into rabbits. Claims that rheumatoid arthritis is related to infection by M. fermentans have not been substantiated, and there is as yet no strong evidence for a viral etiology of this disease.
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