REVIEWS OF INFECTIOUS DISEASES • VOL. 12, SUPPLEMENT 7 • SEPTEMBER-OCTOBER 1990 © 1990 by The University of Chicago. All rights reserved. 0162-0886/90/1205-0043$02.00
Virologic and Pathogenetic Aspects of Cytomegalovirus Infection Jane E. Grundy
From the Department of Virology, Royal Free Hospital School of Medicine. Hampstead, London. England
Cytomegalovirus (CMV), a member of the herpesvirus group, must be considered a successful infectious agent in that it causes widespread infection but under normal conditions rarely kills its host. Furthermore, the virus is not eliminated from the body after primary infection but persists in the form of a low-grade chronic infection or remains in a latent state, such that reactivation or increased excretion of virus at a later stage allows further transmission of the virus to new hosts. The relatively low pathogenicity of the virus in normal individuals is a testimony to the successful outcome of the interaction between the host's immune system and the virus. However, a number of host and viral factors can affect this interaction. The most important is a shift in favor of the virus when the host's immune system is compromised or immature, such that infection can be associated with disease. The purpose of this review is to consider the viral and host factors that are associated with pathology so that appropriate preventative or therapeutic measures can be designed.
CMV as a Pathogen CMV excretion is not synonymous with CMV disease. The virus can be found at many sites in the body Please address requests for reprints to Dr. Jane E. Grundy, Department of Virology, Royal Free Hospital School of Medicine, Hampstead, London NW3 2QG, England.
but seems to cause disease only at some of these, and then only in certain patient groups. The role of CMV as a human pathogen at various sites has recently been reviewed [1], and the conclusions from that review for the various patient groups are outlined in table 1. The criteria used for assigning a pathologic role for CMV in each instance [1] were the following: (1) that CMV be found at the site of disease in the absence of other pathogens; (2) that disease be seen in those individuals with the highest virus titers; (3) that effective antiviral therapy suppresses the disease; (4) that animal models reproduce the disease; and (5) that immunity to the virus prevents or modifies the disease. Pathogenicity was accepted if some of these criteria were met [1]. It was noted that although CMV frequently infects the salivary gland and the kidney, disease at those sites is not usually observed [1]. The relationship of CMV infection in renal transplant recipients to acute transplant glomerulopathy is a subject of controversy [2]. CMV infection of the liver, retina, or gut was deemed to be associated with pathology in all patient groups in which it was observed [1]. Infection of the brain with CMV was considered to be associated with pathology in congenitally infected infants [1], while in patients with AIDS the contribution of CMV to pathology in dual infections with the human immunodeficiency virus (HIV) remains to be resolved. The mechanism by which CMV causes disease at the above sites was considered to be direct viral repliS711
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Cytomegalovirus (CMV) is a ubiquitous agent that rarely causes disease in immunocompetent humans but is an important cause of morbidity and mortality in the immunocompromised. A number of host and viral factors are associated with pathology following CMV infection. CMV can be found at many sites in the body but only causes disease at some of these and then only in certain patient populations. In some situations the mechanism underlying disease is direct viral replication, but in others, particularly CMV pneumonitis in allogeneic transplant recipients, an immunopathologic basis is strongly implicated. An important factor in the pathogenesis of infection and the expression of symptomatic disease is the source of CMV infection-whether it arises from an exogenous source or is due to reactivation of latent endogenous virus. Exogenous infection can occur in previously seronegative individuals or in those with prior exposure to the virus. In renal transplant recipients both types of exogenous infection have been associated with disease. Another factor that affects the interaction betweenCMV and its host is the modulating effect of the virus on the host immune response, the mechanism of which remains unknown.
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Table 1. Pathogenicity of CMV at various sites in different patient groups.
Site of infection
TX,A,C TX,A,C TX,A,C TX,A,C TX,A
C A
Lung
TX A
No No Yes Yes Yes Yes Yes? Yes No?
Mechanism
Viral Viral Viral Viral
replication replication replication? replication
? Immunopathology
NOTE. TX = transplant recipients; A = patients with AIDS; C = congenitally infected infants; ? = information presently available is inconclusive.
cation [1], since antiviral chemotherapy reduced disease at those sites in transplant recipients and patients with AIDS (criterion 3) and since disease was associated with high virus titers in congenitally infected infants (criterion 2). The contribution ofimmunopathology to CMV lesions in the gut is unknown at the present time. The mechanism of any CMV-associated pathology in the brain of patients with AIDS might be related to a synergistic interaction with HIV [I, 3].
CMV as a Pathogen in the Lung The ability of CMV to act as a pathogen in the lung is well established for recipients of allogeneic renal [4], liver [5], heart [6], or bone marrow transplants [7], where an interstitial pneumonitis with a high fatality rate is seen. However, this is not the case for other patient groups. In one study, no cases of CMV pneumonitis were observed in 100recipients of syngeneic bone marrow from identical twin donors [8] but 67 (190/0) were seen in 351 recipients of allogeneic bone marrow, despite a similar incidence of CMV infection in the two groups [8]. The incidence of CMV pneumonitis was also reported to be low (2 0J0) in 143 recipients of autologous bone marrow [9, 10], as compared with an incidence of 170/0 in 386 recipients of allogeneic marrow in the same study [7, 9, 10]. Again, the incidence of CMV infection was similar in the two groups. Another study reported a similarly low incidence of CMV pneumonitis in autologous bone marrow recipients [11]. The total incidence
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Salivary gland Kidney Liver Retina Gut Brain
Presence Patient of group(s) with infection disease at site at site
of pneumonitis and the incidence of idiopathic pneumonitis are also lower in recipients of syngeneic or autologous bone marrow than in recipients of allogeneic bone marrow [7-11]. One explanation for the lower incidence of CMV pneumonitis in recipients of autologous or syngeneic marrow is that the extent of immunosuppression produced by the pretransplantation conditioning is less than that in recipients of allogeneictransplants. However, in the study of autologous vs, allogeneic transplantation referred to above [7, 9], patients in both groups weretreated with busulfan plus cyclophosphamide or with total body irradiation plus cyclophosphamide, and no association was found between the pretransplantation conditioning and the development of CMV pneumonitis [7]. The incidence of CMV pneumonitis in allogeneic transplant recipients receiving busulfan plus cyclophosphamide was 130J0 and that of patients receiving total body irradiation plus cyclophosphamide was 180/0, while for autologous recipients the figures were 3 % and 60/0, respectively [7]. Thus, the lower incidence of CMV pneumonitis in recipients of autologous vs. allogeneic marrow cannot be ascribed to differences in pretransplantation conditioning. The situation in patients with AIDS is quite different; in these patients the virus often is found in the lungs during an episode of pneumonitis but almost always in the presence of another pathogen [lla]. A collaborative study involving the Royal Free Hospital [l1a] has recently shown that although patients were treated only for the other pathogen, usually Pneumocystiscarin ii, the mortality of the patients with CMV and a second pathogen was no different from that in patients in whom only the second pathogen was detected. Thus, no additional mortality was attributable to CMV in the lungs of patients with AIDS, a finding in sharp contrast to that in allogeneic transplant recipients, in whom mortality due to CMV pneumonitis is high. Thus, since none of the criteria for pathogenicity outlined above has been satisfied, the role of CMV as a pathogen in the lungs of patients with AIDS cannot be substantiated at present. It is therefore apparent that the pathogenesis of CMV infection in the lungs of recipients of allogeneic transplants, who develop interstitial pneumonitis, differs markedly from that seen in recipients of syngeneic or autologous bone marrow transplants or in patients with AIDS, who experience little or no pathology. Since it has been shown that the differ-
CMV Pathogenesis
ence found in allogeneic and autologous transplant recipients is not due to differences in immunosuppression [7], some effect induced by transplanted allogeneic cellsmust be responsible. Data from the murine model shed some light on this issue and will be discussed here.
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Murine models for interstitial pneumonitis induced by MCMV GvH PLUS MCMV MODEL:
)kz~MCMV i.p. ~...----parental
....fatal interstitial pneumonitis spleen
cells Lv.
adult unirradiated F1 hybrid mouse
A fatal interstitial pneumonitis has been induced in mice infected with murine CMV (MCMV) in two different models (figure 1). In the graft-vs.-host (GvH) plus MCMV model [12], adult unirradiated F 1 hybrid mice simultaneously challenged with parental spleen cells and MCMV died of interstitial pneumonitis, whereas mice given either challenge alone survived. The virus titers in the lungs of mice given MCMV alone did not differ from those in mice given MCMV with a GvH challenge [12]. Thus, the development of interstitial pneumonitis in this model was not related to viral replication but to some component of the immune response involved in the GvH reaction. Indeed, if ganciclovir was given 1 day after the challenge with parental spleen cells and MCMV, viral replication in the lung was undetectable but pneumonitis occurred with the same frequency [15]. MCMV was found to increase the severity of the GvH reaction [12] and to decrease the number of cells necessary to induce GvH reaction [16]. Analysis of the cells in the lungs obtained by bronchoalveolar lavage from mice with interstitial pneumonitis showed an influx of Thy 1.2-positive cells of donor origin [17]. Depletion of Thy 1.2positive cells from the donor inoculum prevented the development of interstitial pneumonitis (J. D. Shanley,personal communication). Thus, it was proposed that some immune response, triggered by MCMV and mediated by donor lymphocytes, that was directed against recipient cells in the lung was involved in the pathogenesis of the pneumonitis [12, 15-17]. The second model, the cyclophosphamide model [13], showed that interstitial pneumonitis could be induced by intranasal infection with MCMV at the same time as the ip administration of one dose of cyclophosphamide. With continued administration of cyclophosphamide, virus titers in the lung increased but no pneumonitis was seen [13]. The development of pneumonitis was influenced by host genetic factors; a susceptible H-2 genotype was a prerequisite for viral replication in the lung but was not
CYCLOPHOSPHAMIDE MODEL:
.... interstitial pneumonitis
adult mouse of particular genotype TOTAL BODY IRRADIATION MODEL: y irradiation .... fatal infection with lung pathology Az4'''-=_MCMVLPI. adult BALB/c mouse
Figure 1. Two models of induced interstitial pneumonitis are illustrated: the GvH plus MCMV model and the cyclophosphamide model (seetext and [12] and [13], respectively. for details). The third model illustrated (see text and [14] for details) induces a fatal disseminated infection with some lung pathology (i.pl. = interplantar).
sufficient for the induction of pneumonitis, for which additional host genetic factors were necessary [18]. The lungs of mice with pneumonitis showed an increase in Thy 1.2-positive lymphocytes [19]. Thus, in this model, as in the GvH-plus-MCMV model, interstitial pneumonitis was not related to viral replication in the lung per se but required some component of the host immune response. Indeed, a reduction in viral replication in the lung to undetectable levelsby treatment with ganciclovir had no effect on the lung pathology [20].Furthermore, the central role of T cells was demonstrated by studies with T celldeficient athymic (nude) mice [21]. MCMV infection of nude mice resulted in extensive viral replication in the lungs but not in diffuse pneumonia until the terminal stages of infection, even if cyclophosphamide was given [21]. However, when T cell function was reconstituted with syngeneic cells before the mice were challenged with MCMV and cyclophosphamide, pneumonitis developed [21]. Another model that results in some lung pathology has recently commanded much attention. Mice that had received 6 Gy total body irradiation were given MCMV in the footpad and subsequently de-
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Mechanism of CMV Pneumonitis: Clues from Murine Models
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CMV Pneumonitis as an Immunopathologic Condition
Twoimportant features emerge from the two models of interstitial pneumonitis: first, that the development of pneumonitis is unrelated to viral replication and, second, that some component of the host T cell response, in one case a component involved with the GvH reaction, is required. These features are also found in human CMV pneumonitis; pneumonitis in allogeneic bone marrow transplant recipients is associated with GvH disease [24],and ganciclovir therapy for patients with established CMV pneumonitis successfully reduced the virus titers in the lung
to undetectable levelsbut failed to prevent death due to pneumonitis [25].Such an outcome suggests a dissociation of viral replication from pathology. On the basis of these and other observations from clinical and experimental studies, a hypothesis was constructed for the pathogenesis of CMV pneumonitis [26]. In brief, my colleagues and I proposed that in allogeneic transplant recipients, CMV pneumonitis is an immunopathologic condition associated with a T cell response to a virally induced antigen in the lung. Candidate antigens were the CMV immediate early or early proteins, since these can be expressed in the absence of complete viral replication, and HLA antigens, since CMV has been shown to enhance expression of class I HLA molecules in vitro [27]and the cytotoxic T cell response to alloantigens has been shown to be enhanced during MCMV infection in vivo [28, 29]. The above hypothesis was considered to be consistent with the failure of effective antiviral agents such as ganciclovir in the treatment of CMV pneumonitis (a finding paralleled in the mouse model) since ganciclovir, which acts on viral DNA replication, would not prevent the expression of CMV immediate early or early proteins [26]. However, a recent report suggested that ganciclovir inhibited CMV replication by a virostatic mechanism [30] and resistance to inhibition by ganciclovir is greater in viral protein synthesis than in viral infectivity or viral DNA synthesis [31]. Thus CMV late viral proteins may still be expressed in the tissues of patients (or mice) treated with ganciclovir.The hypothesis should therefore be extended to include CMV late proteins as candidate antigens for stimulation of the pathologic host cell response in the lung. The precise nature of the pathologic T cell response involved is not yet clear; however, preliminary data from the mouse model suggest that a delayed-type hypersensitivity T cell might be responsible [26] and thus, perhaps, that cytokines playa role. Studies of interferon in the bronchoalveolar lavage fluid of patients with CMV pneumonitis failed to detect interferon-y but did demonstrate the presence of acid-labile interferon-a in patients with a poor prognosis [32]. A pathologic role for acid-labile interferon-a has been suggested in autoimmune disease [32]. Further studies of the role of other cytokines in the development of CMV pneumonitis are required. It is of interest that the pneumonitis episode occurs after the antigen-presenting cells of the
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veloped a fatal disseminated infection that included viral replication in the lung [14]. The authors described a moderate focal interstitial pneumonia, with widened alveolar septae containing intranuclear inclusions. They concluded that the virus was replicating in interstitial cells, pneumocytes, and endothelial cells but noted that cellular infiltration was virtually absent. The transfer of sensitized T lymphocytes reduced virus titers in liver, spleen, and lung, and the animals survived. Further studies demonstrated that T cells with specificity for immediate early MCMV proteins, but not structural proteins, wereable to transfer such protection [22]. Although some lung pathology was seen in this model, it cannot be considered a model of interstitial pneumonitis since the mice also had an overwhelming disseminated infection, and indeed the cause of death in this model was recently shown to be a failure to generate hematopoietic stem cells [23]. However, salient features of the model merit discussion here. First, it is clear that any lung pathology seen was due to viral replication and not to any cellular infiltrate. Second, reconstitution with syngeneic sensitized T cells protected the mice from lethal infection, coincident with a reduction in virus titers in the lung, and - although the authors did not discuss the effect of such reconstitution on lung pathology the fact that the mice survived indicates that any effect on the lungs was obviously not associated with death. This is reminiscent of the clinical findings in humans that only allogeneic, and not syngeneic or autologous, bone marrow transplantation was associated with the development of interstitial pneumonitis.
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CMV Pathogenesis
Role of Antibody in the Lungs of Patients with CMV Pneumonitis CMV hyperimmune globulin has been used in the treatment of established CMV pneumonitis, apparently with beneficial effects. In an open study [26], mortality, which historically is 1'\J85070, was reduced to 50070. Patients who did not respond to this therapy were already being ventilated before treatment was initiated, suggesting that prompt treatment was necessary. These results ,,;ere not confirmed by another group using a different hyperimmune globulin preparation [35], but an average of 8.2 days (range, 2-30 days) had elapsed between the onset of pneumonitis and the initiation of treatment, a delay that may explain the poor results obtained. The mechanism by which antibody might reduce mortality from CMV pneumonitis has been discussed in detail elsewhere [26]. It was considered unlikely that it was due to neutralization of virus by antibody since the hyperimmune globulin used could not neutralize virus obtained directly from patients' body fluids [36] - although it had good neutralizing titers against virus harvested from cell cultures. The inability to neutralize virus from body fluids was thought to be due to the protective effect of host I3rmicroglobulin bound to the viral envelope of CMV in body fluids [36]. It is surprising that exogenous CMV antibodies from pooled donors could reduce mortality since it has recently been shown that patients with CMV pneumonitis had high levels of locally generated antibody in their lungs at the time of their episode of pneumonitis [37, 38]. This suggests either that factors other than CMV antibodies contained in the hyperimmune globulin mediate the protective effect or that the class, subclass, or specificity of CMV antibodies generated locally in the lungs of patients with CMV pneumonitis is different in some way from that present in the serum of seropositive blood donors.
CMV and Graft Rejection Graft rejection might be considered another manifestation of CMV-associated disease. There are clinical data both for [39] and against [40] a role for CMV in provoking graft rejection. In vitro CMV can enhance the expression of class I HLA antigens, partly through the release of interferon [27]. Enhancement of allogeneic antigens, mismatched between donor and recipient, could feasibly contribute to graft rejection. In mice, MCMV infection in vivo has been shown to enhance the host cytotoxic T cell response to alloantigens [28,29]. However, the clinical data linking episodes of CMV infection and rejection are largely circumstantial. In addition, one assay historically used for monitoring rejection, the measurement of levels of 132-microglobulinin urine, has recently been shown to give artifactually high results, mimicking rejection, when CMV is present in the urine specimen assayed [41]. The issue is further complicated by the fact that treatment for rejection relies on increased immunosuppression, while the dose of immunosuppressive agent is usually lowered during episodes of CMV infection. Thus, treatment for either condition can lead to exacerbation of the other, making interpretation of the links between the two on a mechanistic level virtually impossible.
Effect of the Type of CMV Infection on Pathogenicity An important factor in the pathogenesis of infection and the expression of symptomatic disease is whether CMV infection arises from an exogenous source of virus or from reactivation of latent endogenous virus. Table 2 summarizes the types of CMV infection associated with disease in different patient groups. Infection has been categorized as primary, denoting infection of a seronegative individual with exogenous virus; reactivation, denoting reactivation of latent endogenous virus in seropositive individuals; or reinfection, denoting infection with a new strain of CMV from an exogenous source. Damage to the fetus following either primary infection in the mother or presumed reactivation in mothers seropositive before conception has been described [42] (although it must be noted in the latter case that the distinction between reinfection and reactivation was not made). CMV disease in apparently healthy individuals - manifest as CMV mono-
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marrow transplant recipient have been replaced by those of donor origin [33]. This finding suggests that histocompatibility (presumably for class II antigens) between T cells, which will be of donor origin, and antigen-presenting cells may be necessary for the induction of pathology. This is remarkably similar to the situation recently described in liver transplant recipients, in whom CMV infection was associated with the ''vanishing bile duct syndrome" if donor and recipient were matched at the HLA-DR locus [34].
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Table 2. Relationship between type of CMV infection and expression of CMV disease in various groups. Disease association*
Group
+ +
Reactivation
Reinfection
NA
NA ?
+
+ + + + + ?
+ + + + ?
? ? ?
?
* + and - = association and absence of association, respectively. NA = not applicable to this patient group; ? = information presently available is inconclusive. t This refers to the type of infection in the mother that results in disease in the fetus.
nucleosis [43]- is a feature of primary infection only, thus demonstrating that in immunocompetent individuals, primary infection induces protective immunity. In all groups of seronegative transplant recipients, primary infection, due to transmission of CMV by the donor organ or blood products, has been associated with disease [4-7]. CMV infection in seropositivetransplant recipients has been associated with disease following all types of transplantation, but the question of whether this is due to reactivation, reinfection, or both has been pursued only in the case of renal transplantation [44-46]. Restriction enzyme analysis of viral DNA was used to type the strain of CMV excreted after renal transplantation and hence to distinguish between reactivation and reinfection [44-46]. The results showed that disseminated infection and symptomatic illness (including CMV pneumonitis) were associated with reinfection [46] and that reactivation of latent recipient virus was accompanied by asymptomatic shedding of virus at restricted sites [46]. This finding suggests that prior immunity is not protective against new strains of virus but can protect against the homologous reactivated strain. This difference in protection may be due to important antigenic differencesamong strains, although it was noted that differences in the sites of viral replication following reactivation of endogenous infection, vs. reinfection from donor organ or blood products, may influence the outcome [46]. In seropositive recipients of liver [5] and heart [6]
Mechanism of the Effect of CMV on Host Immune Responses CMV is reputed to be immunosuppressive in both humans and mice [48], although enhanced immune responses have been described and the virus induces a variety of autoantibodies [40,48]. Although much attention has focused on the mechanism of such immunosuppression, it remains an enigma. The low frequency of infection of lymphocytes and monocytes during acute CMV infection argues for an indirect mechanism of immunosuppression [45, 49]. The induction by CMV of a soluble inhibitor of interleukin-l was recently described [50];however, this find-
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Fetus Mother to fetus t Healthy adults Transplant recipient Kidney Liver Heart Bone marrow Patients with AIDS
Primary infection
transplants and, as discussed earlier, of allogeneic bone marrow [7], serious CMV disease is seen even when donors are seronegative. Although it is possible that reinfection with exogenous virus from blood products is partly responsible, it is likely that many instances of this disease are associated with reactivation of endogenous recipient virus. Thus, in these patients prior immunity does not prevent dissemination of reactivated virus or the expression of disease. Since the majority of homosexual patients with HIVinfection are seropositive for CMV [47], in this group CMV disease is associated with either reactivation or reinfection. Prospective studies will be needed to differentiate between these two types of infection. Although CMV disease can be associated with primary infection, reactivation, or reinfection in particular patient groups, the incidence of disease associated with the different types of infection differs in some circumstances. For example, there is evidence that congenitally infected babies born to previously seropositive mothers are less severely damaged than are those born to mothers who contracted a primary infection during pregnancy [39]. Similarly, in renal transplant recipients the incidence of disease associated with reinfection is lower than that accompanying primary infection [46]. It is thus apparent that in particular patient groups prior infection does not protect against disease due to infection with exogenous or endogenous virus. It is of some importance that this lack of protection extends not only to diseaseassociated with viral replication (e.g., brain damage in congenital infection) but also to disease associated with immunopathology (e.g., CMV pneumonitis in transplant recipients).
CMV Pathogenesis
References 1. Griffiths PO, Grundy JE. The status of CMV as a human pathogen. Epidemiol Infect 1988;100:1-15 2. BoyceNW, Hayes K, Gee 0, Holdsworth SR, Thomson NM, Scott 0, Atkins RC. Cytomegalovirus infection complicating renal transplantation and its relationship to acute transplant glomerulopathy. Transplantation 1988;45:706-9 3. Skolnik PR, Kosloff BR, Hirsch MS. Bidirectional interactions between human immunodeficiency virus type 1 and cytomegalovirus. J Infect Dis 1988;157:508-14 4. Rubin RH, Tolkoff-Rubin NE, Oliver 0, Rota TR, Hamilton J, Betts RF, Pass RF, Hillis W, Szmuness W, Farrell ML, Hirsch MS. Multicenter seroepidemiologic study of
the impact of cytomegalovirus infection on renal transplantation. Transplantation 1985;40:243-9 :5. Singh N, Dummer JS, Kusne S, Breinig MK, Armstrong JA, Makowka L, Starzl TE, Ho M. Infections with cytomegalovirus and other herpes viruses in 121 liver transplant recipients: transmission by donated organ and the effect of OKT3 antibodies. J Infect Dis 1988;158:124-31 6. Gorensek MJ, Stewart RW, Keys TF, McHenry MC, Goormastic M. A multivariate analysis of the risk of cytomegalovirus infection in heart transplant recipients. J Infect Dis 1988;157:515-22 7. Wingard JR, Mellits ED, Sostrin MD,Chen DY-H,Burns WH, Santos GW, Vriesendorp HM, Beschorner WE, Saral R. Interstitial pneumonitis after allogeneicbone marrow transplantation. Nine-year experience at a single institution. Medicine 1988;67:175-86 8. Appelbaum FR, Meyers JD, Fefer A, Flournoy N, Cheever MA, Greenberg PO, Hackman R, Thomas ED. Nonbacterial nonfungal pneumonia following marrow transplantation in 100identical twins. Thmsplantation 1982;33:265-8 9. Wingard JR, Sostrin MB, Vriesendorp HM, Mellits ED, Santos GW, Fuller DJ, Braine HG, YeagerAM, Burns WH, Sara! R. Interstitial pneumonitis followingautologous bone marrow transplantation. Transplantation 1988;46:61-5 10. Wingard JR, Chen DY-H,Burns WH, Fuller OJ, Braine HG, YeagerAM, Kaiser H, Burke PJ, Graham ML, Santos GW, Saral R. Cytomegalovirus infection after autologous bone marrow transplantation with comparison to infection after allogeneic bone marrow transplantation. Blood 1988; 71:1432-7 u. Valteau 0, Hartmann 0, Benhamou E, Caillaud JM, Brugieres L, Beaujean F, Patte C, Flamant F, Lemerle J. Nonbacterial nonfungal interstitial pneumonitis following autologous bone marrow transplantation in children treated with high-dose chemotherapy without total-body irradiation. Transplantation 1988;45:737-40 l1a. Millar AD, Patou G, Miller RF, Grundy JE, Katz DR, Weller IV, Semple SJ. Cytomegalovirus in the lungs of patients with AIDS: respiratory pathogen or passenger? Am Rev Respir Dis 1990;141:1474-7 12. Grundy JE, Shanley JD, Shearer GM. Augmentation of graft versus host reaction by cytomegalovirus infection resulting in interstitial pneumonitis. Transplantation 1985;39: 548-53 13. Shanley JD, Pesanti EL, Nugent KM. The pathogenesis of pneumonitis due to murine cytomegalovirus. J Infect Dis 1982;146:388-96 14. Reddehase MJ, Weiand F, Munch K, Jonjic S, Luske A, KoszinowskiVH. Interstitial murine cytomegalovirus pneumonia after irradiation: characterization of cells that limit viral replication during established infection of the lungs. J Virol 1985;55:264-73 15. Shanley JD, Pomeroy C, Via CS, Shearer GM. Interstitial pneumonitis during murine cytomegalovirus infection and graft-versus-host reaction: effect of ganciclovir therapy. J Infect Dis 1988;158:1391-4 16. Via CS, Shanley JD, Weatherly BR, Lang P, Shearer GM. Altered threshold for the induction of graft-versus-host immunodeficiency following murine cytomegalovirus infection. Transplantation 1988;46:298-302 17. Shanley JD, Via CS, Sharrow SO, Shearer GM. Interstitial
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ing could not be reproduced when mycoplasma-free strains of CMV were used, and the authors concluded that the phenomenon was due to mycoplasma contamination [51]. Another possibility is that the virus itself exerts a direct immunosuppressive action, triggering alterations in immune reactivity through a direct interaction with lymphoid cells, without even limited viral replication being required. This proposal is consistent with studies in the mouse [52, 53] that indicate MCMV can be directly immunosuppressive in vitro and that the peak of immunosuppression [54] correlates with peak virus titers. A direct effect of human CMV on the induction of immune responses has also been suggested in several studies [55, 56]. A possible, although highly speculative mechanism by which CMV could alter immune reactivity is by directly binding to lymphoid cells, leading to some triggering of the cell. The basis for this proposal is the widely reported ability of monoclonal antibodies to ~z-microglobulin (the light chain of the class I HLA dimer) to induce lymphocyte activation, with both enhancement and suppression of immune responses having been described [57-60]. Since CMV has been reported to bind Ih-microglobulin [33, 58], the cross-linking of class I HLA molecules by multipoint binding of the virus to more than one light chain might be expected to trigger the cell in the same way as does cross-linking by antibodies to ~z-micro globulin, resulting in abnormal immune reactivity. Such a situation is analogous to the well-known triggering of B cells by antibodies to immunoglobulin, a phenomenon in vitro that mimics the cross-linking of cell-surface immunoglobulin by antigen. Although this model for a direct action of CMV is highly speculative, it is testable. The solution to the intriguing question of how CMV alters host immune responses thus awaits further investigation.
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32.
33. Reittie JE, Poulter LW, Prentice HG, Burns J, Drexler HG, Balfour B, Clarke J, Hoffbrand AV, McGee JO, Brenner MK. Differential recovery of phenotypically and functionally distinct circulating antigen-presenting cells after allogeneic marrow transplantation. Transplantation 1988; 45:1084-91 34. O'Grady JG, Alexander GJM, Sutherland S, Donaldson PT, Harvey F, Portmann B, Calne RY, Williams R. Cytomegalovirus infection and donor/recipient HLA antigens: interdependent co-factors in pathogenesis of vanishing bileduct syndrome after liver transplantation. Lancet 1988;2: 302-5 35. Reed EC, Bowden RA, Dandliker PS, Gleaves CA, Meyers JD. Effect of cytomegalovirus immunoglobulin in marrow transplant recipients with viral pneumonia. J Infect Dis 1987;156:641-5 36. McKeating JA, Griffiths PO, Grundy JE. Cytomegalovirus in urine specimens has host 131 microglobulin bound to the viral envelope: a mechanism of evading the host immune response? J Gen Virol 1987;68:785-92 37. Milburn HJ, Grundy JE, du Bois RM, Prentice HG, Griffiths PD. Humoral immune responses within the lung of bone marrow transplant recipients studied by bronchoalveolar lavage. Clin Exp ImmunoI1988;72:309-14 38. Milburn HJ, Grundy JE, du Bois RM, Prentice HG, Griffiths PD. Is the measurement of virus-specific antibody in the lungs of transplant recipients with cytomegalovirus pneumonitis of diagnostic or prognostic value? J Med Virol 1988;26:197-206 39. May AG, Betts RF, Freeman RB, Andrus CH. An analysis of cytomegalovirus infection and HLA antigen matching on the outcome of renal transplantation. Ann Surg 1978; 187:110-7 40. Armstrong JA, Evans AS, Rao N, Ho M. Viral infections in renal transplant recipients. Infect Immun 1976;14:970-5 41. Grundy JE, McKeating JA, Sanderson AR, Griffiths PD. Cytomegalovirus and 131 microglobulin in urine specimens: reciprocal interference in their detection is responsible for artifactually high levels of urinary 131 microglobulin in infected transplant recipients. Transplantation 1988;45:1075-9 42. Stagno S, Pass RF, Dworsky ME, Henderson RE, Moore EG, Walton PO, Alford CA. Congenital cytomegalovirus infection: the relative importance of primary and recurrent maternal infection. N Engl J Med 1982;306:945-9 43. Kantor GL, Goldberg LS, Johnson BL Jr, Derechin MM, Barnett EV. Immunologic abnormalities induced by postperfusion cytomegalovirus infection. Ann Intern Med 1970;73:553-8 44. Grundy JE, Super M, Griffiths PD. Reinfection of a seropositive allograft recipient by cytomegalovirus from donor kidney. Lancet 1986;1:159-60 45. Grundy JE, Super M, Lui S,SwenyP, Griffiths PD. The source of cytomegalovirus infection in seropositive renal allograft recipients is frequently the donor kidney. Transplant Proc 1987;19:2126-8 46. Grundy JE, Lui SF, Super M, Berry NJ, Sweny P, Fernando ON, Moorhead J, Griffiths PD. Symptomatic cytomegalovirus infection in seropositive kidney recipients: reinfection with donor virus rather than reactivation of recipient virus. Lancet 1988;2:132-5 47. Berry NJ, Burns OM, Wannamethee G, Grundy JE, Lui SF,
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pneumonitis during murine cytomegalovirus infection and graft-versus-host reaction. Characterization of bronchoalveolar lavage cells. Transplantation 1987;44:658-62 Shanley JD. Host genetic factors influence murine cytomegalovirus lung infection and interstitial pneumonitis. J Gen Virol 1984;65:2121-8 Shanley JD, Ballas ZK. Alteration of bronchoalveolar cells during murine cytomegalovirus interstitial pneumonitis. Am Rev Respir Dis 1985;132:77-81 Shanley JD, Pesanti EL. The relation of viral replication to interstitial pneumonitis in murine cytomegalovirus lung infection. J Infect Dis 1985;151:454-8 Shanley JD. Murine cytomegalovirus pneumonitis in T cell deficient nude mice. In: Abstracts of the 12th Herpesvirus Workshop, Philadelphia, 1987 Reddehase MJ, Mutter W, Miinch K, Buhring H-J, Koszinowski UH. CD8-positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity. J Virol 1987;61:3102-8 Mutter W, Reddehase MJ, Busch FW, Biihring H-J, Koszinowski UH. Failure in generating hemopoietic stem cells is the primary cause of death from cytomegalovirus disease in the immunocompromised host. J Exp Med 1988;167:1645-58 Meyers JD, Flournoy N, Thomas ED. Risk factors for cytomegalovirus infection after human marrow transplantation. J Infect Dis 1986;153:478-88 Shepp DH, Dandliker Ps, de Miranda P, Burnette TC, Cederberg OM, Kirk LE, Meyers JD. Activity of 9-[-2-hydroxy1-(hydroxymethyl)ethoxymethyl] guanine in the treatment of cytomegalovirus pneumonia. Ann Intern Med 1985; 103:368-73 Grundy JE, Shanley JD, Griffiths PD. Is cytomegalovirus interstitial pneumonitis in transplant recipients an immunopathological condition? Lancet 1987;1:996-9 Grundy JE, Ayles HM, McKeating JA, Butcher RG, Griffiths PO, Poulter LW. Enhancement of class 1 HLA antigen expression by cytomegalovirus: role in amplification of virus infection. J Med Virol 1988;25:483-95 Grundy JE, Shearer GM. The effect of cytomegalovirus infection on the host response to foreign and hapten-modified self histocompatibility antigens. Transplantation 1984; 37:484-90 Grundy JE, Reid MF. The effect of primary and secondary infection with cytomegalovirus on the host response to alloantigens. Transplant Proc 1985;17:592-4 Teich SA, Castle J, Friedman AH, Siroty W, Orellana J, Schmitterer M. Active cytomegalovirus particles in the eyes of an AIDS patient being treated with 9-[2-hydroxy-l-(hydroxymethyl)ethoxymethyl] guanine (Ganciclovir), Br J Ophthalmol 1988;72:293-8 Rasmussen L, Chen PT, Mullenax JG, Merigan TC. Inhibition of human cytomegalovirus replication by 9-(1,3dihydroxy-2-propoxymethyl)guanine alone and in combination with human interferons. Antimicrob Agents Chemother 1984;26:441-5 Lever AML, Milburn HJ, du Bois RM, Griffiths PD, Grundy JE. Presence of a partially acid-labile alpha-interferon in bronchoalveolar lavage fluid of patients with cytomegalovirus pneumonitis is associated with a poor prognosis. J Med Virol 1988;24:67-73
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CMV Pathogenesis
48. 49.
51.
52.
53.
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58.
59.
60.
61.
netically resistant and susceptible mice. Infect Immun 1982;36:235-42 Schrier RD, Rice GPA, Oldstone MBA. Suppression of natural killer cell activity and T cell proliferation by fresh isolates of human cytomegalovirus. J Infect Dis 1986;153: 1084-91 Wahren B, Ljungman P, Paulin T, Ringden O. Enhancive and suppressive effects of cytomegalovirus on human lymphocyte responses in vitro. J Virol 1986;58:909-13 Ringden 0, Moller E. B cell mitogenic effects on human lymphocytes of rabbit anti-human Bl-microglobulin. Scand J Immunol 1975;4:171-9 Ringden O. Activation of human lymphocyte subpopulations by rabbit anti-human Bl-microglobulin and by lipopolysaccharide. Scand J Immunol 1976;5:891-900 Ostberg L, Lundblom JB, Peterson PA. Bl-microglobulin on the cell surface: specificity of inhibition of the mixed leukocyte reaction and mitogenic properties of antibodies against the two HLA antigen polypeptide chains. Eur J Immunol 1976;6:108-13 Chouaib S, Welte K, Dupont B. Differential effect of antiBrmicroglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction. J Immunol 1985;134:940-8 Grundy JE, McKeating JA, Griffiths PD. Cytomegalovirus strain AD169 binds Bl microglobulin in vitro after release from cells. J Gen Virol 1987;68:777-84
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Prentice HG, Griffiths PD. Seroepidemiologic studies on the acquisition of antibodies to cytomegalovirus, herpes simplex virus, and human immunodeficiency virus among general hospital patients and those attending a clinic for sexually transmitted diseases. J Med ViroI1988;24:385-93 Griffiths PO, Grundy JE. Molecular biology and immunology of cytomegalovirus. Biochem J 1987;241:313-24 Thrtinen LW, Saltzman R, Jordan MC, Haase AT. Interactions of human cytomegalovirus with leukocytes in vivo: analysis by in situ hybridisation. Microbial Pathogen 1989;7:135-45 Rodgers BC, Scott OM, Mundin J, Sissons JGP. Monocytederived inhibitor of interleukin 1 induced by human cytomegalovirus. J Virol 1985;55:527-32 Scott OM, Rodgers BC, Freeke C, Bruiter J, Sissons JGP. Human cytomegalovirus and monoeytes: limited infection and negligibleimmunosuppression in normal mononuclear cells infected in vitro with mycoplasma free strains. J Gen Virol 1989;70:685-94 Ho M. Role of specific cytotoxic lymphocytes in cellular immunity against murine cytomegalovirus. Infect Immun 1980;27:767-76 SinickasVG,Ashman RB, Blanden RV. The cytotoxicresponse to murine cytomegalovirus. I. Parameters in vivo. J Gen Virol 1985;66:747-55 Allan JE, Shellam GR, Grundy (Chalmer) JE. Effect of murine cytomegalovirus infection on mitogen responses in ge-
Virologic and Pathogenetic Aspects of Cytomegalovirus Infection Jane E. Grundy
From the Department of Virology, Royal Free Hospital School of Medicine. Hampstead, London. England
Cytomegalovirus (CMV), a member of the herpesvirus group, must be considered a successful infectious agent in that it causes widespread infection but under normal conditions rarely kills its host. Furthermore, the virus is not eliminated from the body after primary infection but persists in the form of a low-grade chronic infection or remains in a latent state, such that reactivation or increased excretion of virus at a later stage allows further transmission of the virus to new hosts. The relatively low pathogenicity of the virus in normal individuals is a testimony to the successful outcome of the interaction between the host's immune system and the virus. However, a number of host and viral factors can affect this interaction. The most important is a shift in favor of the virus when the host's immune system is compromised or immature, such that infection can be associated with disease. The purpose of this review is to consider the viral and host factors that are associated with pathology so that appropriate preventative or therapeutic measures can be designed.
CMV as a Pathogen CMV excretion is not synonymous with CMV disease. The virus can be found at many sites in the body Please address requests for reprints to Dr. Jane E. Grundy, Department of Virology, Royal Free Hospital School of Medicine, Hampstead, London NW3 2QG, England.
but seems to cause disease only at some of these, and then only in certain patient groups. The role of CMV as a human pathogen at various sites has recently been reviewed [1], and the conclusions from that review for the various patient groups are outlined in table 1. The criteria used for assigning a pathologic role for CMV in each instance [1] were the following: (1) that CMV be found at the site of disease in the absence of other pathogens; (2) that disease be seen in those individuals with the highest virus titers; (3) that effective antiviral therapy suppresses the disease; (4) that animal models reproduce the disease; and (5) that immunity to the virus prevents or modifies the disease. Pathogenicity was accepted if some of these criteria were met [1]. It was noted that although CMV frequently infects the salivary gland and the kidney, disease at those sites is not usually observed [1]. The relationship of CMV infection in renal transplant recipients to acute transplant glomerulopathy is a subject of controversy [2]. CMV infection of the liver, retina, or gut was deemed to be associated with pathology in all patient groups in which it was observed [1]. Infection of the brain with CMV was considered to be associated with pathology in congenitally infected infants [1], while in patients with AIDS the contribution of CMV to pathology in dual infections with the human immunodeficiency virus (HIV) remains to be resolved. The mechanism by which CMV causes disease at the above sites was considered to be direct viral repliS711
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Cytomegalovirus (CMV) is a ubiquitous agent that rarely causes disease in immunocompetent humans but is an important cause of morbidity and mortality in the immunocompromised. A number of host and viral factors are associated with pathology following CMV infection. CMV can be found at many sites in the body but only causes disease at some of these and then only in certain patient populations. In some situations the mechanism underlying disease is direct viral replication, but in others, particularly CMV pneumonitis in allogeneic transplant recipients, an immunopathologic basis is strongly implicated. An important factor in the pathogenesis of infection and the expression of symptomatic disease is the source of CMV infection-whether it arises from an exogenous source or is due to reactivation of latent endogenous virus. Exogenous infection can occur in previously seronegative individuals or in those with prior exposure to the virus. In renal transplant recipients both types of exogenous infection have been associated with disease. Another factor that affects the interaction betweenCMV and its host is the modulating effect of the virus on the host immune response, the mechanism of which remains unknown.
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Table 1. Pathogenicity of CMV at various sites in different patient groups.
Site of infection
TX,A,C TX,A,C TX,A,C TX,A,C TX,A
C A
Lung
TX A
No No Yes Yes Yes Yes Yes? Yes No?
Mechanism
Viral Viral Viral Viral
replication replication replication? replication
? Immunopathology
NOTE. TX = transplant recipients; A = patients with AIDS; C = congenitally infected infants; ? = information presently available is inconclusive.
cation [1], since antiviral chemotherapy reduced disease at those sites in transplant recipients and patients with AIDS (criterion 3) and since disease was associated with high virus titers in congenitally infected infants (criterion 2). The contribution ofimmunopathology to CMV lesions in the gut is unknown at the present time. The mechanism of any CMV-associated pathology in the brain of patients with AIDS might be related to a synergistic interaction with HIV [I, 3].
CMV as a Pathogen in the Lung The ability of CMV to act as a pathogen in the lung is well established for recipients of allogeneic renal [4], liver [5], heart [6], or bone marrow transplants [7], where an interstitial pneumonitis with a high fatality rate is seen. However, this is not the case for other patient groups. In one study, no cases of CMV pneumonitis were observed in 100recipients of syngeneic bone marrow from identical twin donors [8] but 67 (190/0) were seen in 351 recipients of allogeneic bone marrow, despite a similar incidence of CMV infection in the two groups [8]. The incidence of CMV pneumonitis was also reported to be low (2 0J0) in 143 recipients of autologous bone marrow [9, 10], as compared with an incidence of 170/0 in 386 recipients of allogeneic marrow in the same study [7, 9, 10]. Again, the incidence of CMV infection was similar in the two groups. Another study reported a similarly low incidence of CMV pneumonitis in autologous bone marrow recipients [11]. The total incidence
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Salivary gland Kidney Liver Retina Gut Brain
Presence Patient of group(s) with infection disease at site at site
of pneumonitis and the incidence of idiopathic pneumonitis are also lower in recipients of syngeneic or autologous bone marrow than in recipients of allogeneic bone marrow [7-11]. One explanation for the lower incidence of CMV pneumonitis in recipients of autologous or syngeneic marrow is that the extent of immunosuppression produced by the pretransplantation conditioning is less than that in recipients of allogeneictransplants. However, in the study of autologous vs, allogeneic transplantation referred to above [7, 9], patients in both groups weretreated with busulfan plus cyclophosphamide or with total body irradiation plus cyclophosphamide, and no association was found between the pretransplantation conditioning and the development of CMV pneumonitis [7]. The incidence of CMV pneumonitis in allogeneic transplant recipients receiving busulfan plus cyclophosphamide was 130J0 and that of patients receiving total body irradiation plus cyclophosphamide was 180/0, while for autologous recipients the figures were 3 % and 60/0, respectively [7]. Thus, the lower incidence of CMV pneumonitis in recipients of autologous vs. allogeneic marrow cannot be ascribed to differences in pretransplantation conditioning. The situation in patients with AIDS is quite different; in these patients the virus often is found in the lungs during an episode of pneumonitis but almost always in the presence of another pathogen [lla]. A collaborative study involving the Royal Free Hospital [l1a] has recently shown that although patients were treated only for the other pathogen, usually Pneumocystiscarin ii, the mortality of the patients with CMV and a second pathogen was no different from that in patients in whom only the second pathogen was detected. Thus, no additional mortality was attributable to CMV in the lungs of patients with AIDS, a finding in sharp contrast to that in allogeneic transplant recipients, in whom mortality due to CMV pneumonitis is high. Thus, since none of the criteria for pathogenicity outlined above has been satisfied, the role of CMV as a pathogen in the lungs of patients with AIDS cannot be substantiated at present. It is therefore apparent that the pathogenesis of CMV infection in the lungs of recipients of allogeneic transplants, who develop interstitial pneumonitis, differs markedly from that seen in recipients of syngeneic or autologous bone marrow transplants or in patients with AIDS, who experience little or no pathology. Since it has been shown that the differ-
CMV Pathogenesis
ence found in allogeneic and autologous transplant recipients is not due to differences in immunosuppression [7], some effect induced by transplanted allogeneic cellsmust be responsible. Data from the murine model shed some light on this issue and will be discussed here.
S713
Murine models for interstitial pneumonitis induced by MCMV GvH PLUS MCMV MODEL:
)kz~MCMV i.p. ~...----parental
....fatal interstitial pneumonitis spleen
cells Lv.
adult unirradiated F1 hybrid mouse
A fatal interstitial pneumonitis has been induced in mice infected with murine CMV (MCMV) in two different models (figure 1). In the graft-vs.-host (GvH) plus MCMV model [12], adult unirradiated F 1 hybrid mice simultaneously challenged with parental spleen cells and MCMV died of interstitial pneumonitis, whereas mice given either challenge alone survived. The virus titers in the lungs of mice given MCMV alone did not differ from those in mice given MCMV with a GvH challenge [12]. Thus, the development of interstitial pneumonitis in this model was not related to viral replication but to some component of the immune response involved in the GvH reaction. Indeed, if ganciclovir was given 1 day after the challenge with parental spleen cells and MCMV, viral replication in the lung was undetectable but pneumonitis occurred with the same frequency [15]. MCMV was found to increase the severity of the GvH reaction [12] and to decrease the number of cells necessary to induce GvH reaction [16]. Analysis of the cells in the lungs obtained by bronchoalveolar lavage from mice with interstitial pneumonitis showed an influx of Thy 1.2-positive cells of donor origin [17]. Depletion of Thy 1.2positive cells from the donor inoculum prevented the development of interstitial pneumonitis (J. D. Shanley,personal communication). Thus, it was proposed that some immune response, triggered by MCMV and mediated by donor lymphocytes, that was directed against recipient cells in the lung was involved in the pathogenesis of the pneumonitis [12, 15-17]. The second model, the cyclophosphamide model [13], showed that interstitial pneumonitis could be induced by intranasal infection with MCMV at the same time as the ip administration of one dose of cyclophosphamide. With continued administration of cyclophosphamide, virus titers in the lung increased but no pneumonitis was seen [13]. The development of pneumonitis was influenced by host genetic factors; a susceptible H-2 genotype was a prerequisite for viral replication in the lung but was not
CYCLOPHOSPHAMIDE MODEL:
.... interstitial pneumonitis
adult mouse of particular genotype TOTAL BODY IRRADIATION MODEL: y irradiation .... fatal infection with lung pathology Az4'''-=_MCMVLPI. adult BALB/c mouse
Figure 1. Two models of induced interstitial pneumonitis are illustrated: the GvH plus MCMV model and the cyclophosphamide model (seetext and [12] and [13], respectively. for details). The third model illustrated (see text and [14] for details) induces a fatal disseminated infection with some lung pathology (i.pl. = interplantar).
sufficient for the induction of pneumonitis, for which additional host genetic factors were necessary [18]. The lungs of mice with pneumonitis showed an increase in Thy 1.2-positive lymphocytes [19]. Thus, in this model, as in the GvH-plus-MCMV model, interstitial pneumonitis was not related to viral replication in the lung per se but required some component of the host immune response. Indeed, a reduction in viral replication in the lung to undetectable levelsby treatment with ganciclovir had no effect on the lung pathology [20].Furthermore, the central role of T cells was demonstrated by studies with T celldeficient athymic (nude) mice [21]. MCMV infection of nude mice resulted in extensive viral replication in the lungs but not in diffuse pneumonia until the terminal stages of infection, even if cyclophosphamide was given [21]. However, when T cell function was reconstituted with syngeneic cells before the mice were challenged with MCMV and cyclophosphamide, pneumonitis developed [21]. Another model that results in some lung pathology has recently commanded much attention. Mice that had received 6 Gy total body irradiation were given MCMV in the footpad and subsequently de-
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Mechanism of CMV Pneumonitis: Clues from Murine Models
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CMV Pneumonitis as an Immunopathologic Condition
Twoimportant features emerge from the two models of interstitial pneumonitis: first, that the development of pneumonitis is unrelated to viral replication and, second, that some component of the host T cell response, in one case a component involved with the GvH reaction, is required. These features are also found in human CMV pneumonitis; pneumonitis in allogeneic bone marrow transplant recipients is associated with GvH disease [24],and ganciclovir therapy for patients with established CMV pneumonitis successfully reduced the virus titers in the lung
to undetectable levelsbut failed to prevent death due to pneumonitis [25].Such an outcome suggests a dissociation of viral replication from pathology. On the basis of these and other observations from clinical and experimental studies, a hypothesis was constructed for the pathogenesis of CMV pneumonitis [26]. In brief, my colleagues and I proposed that in allogeneic transplant recipients, CMV pneumonitis is an immunopathologic condition associated with a T cell response to a virally induced antigen in the lung. Candidate antigens were the CMV immediate early or early proteins, since these can be expressed in the absence of complete viral replication, and HLA antigens, since CMV has been shown to enhance expression of class I HLA molecules in vitro [27]and the cytotoxic T cell response to alloantigens has been shown to be enhanced during MCMV infection in vivo [28, 29]. The above hypothesis was considered to be consistent with the failure of effective antiviral agents such as ganciclovir in the treatment of CMV pneumonitis (a finding paralleled in the mouse model) since ganciclovir, which acts on viral DNA replication, would not prevent the expression of CMV immediate early or early proteins [26]. However, a recent report suggested that ganciclovir inhibited CMV replication by a virostatic mechanism [30] and resistance to inhibition by ganciclovir is greater in viral protein synthesis than in viral infectivity or viral DNA synthesis [31]. Thus CMV late viral proteins may still be expressed in the tissues of patients (or mice) treated with ganciclovir.The hypothesis should therefore be extended to include CMV late proteins as candidate antigens for stimulation of the pathologic host cell response in the lung. The precise nature of the pathologic T cell response involved is not yet clear; however, preliminary data from the mouse model suggest that a delayed-type hypersensitivity T cell might be responsible [26] and thus, perhaps, that cytokines playa role. Studies of interferon in the bronchoalveolar lavage fluid of patients with CMV pneumonitis failed to detect interferon-y but did demonstrate the presence of acid-labile interferon-a in patients with a poor prognosis [32]. A pathologic role for acid-labile interferon-a has been suggested in autoimmune disease [32]. Further studies of the role of other cytokines in the development of CMV pneumonitis are required. It is of interest that the pneumonitis episode occurs after the antigen-presenting cells of the
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veloped a fatal disseminated infection that included viral replication in the lung [14]. The authors described a moderate focal interstitial pneumonia, with widened alveolar septae containing intranuclear inclusions. They concluded that the virus was replicating in interstitial cells, pneumocytes, and endothelial cells but noted that cellular infiltration was virtually absent. The transfer of sensitized T lymphocytes reduced virus titers in liver, spleen, and lung, and the animals survived. Further studies demonstrated that T cells with specificity for immediate early MCMV proteins, but not structural proteins, wereable to transfer such protection [22]. Although some lung pathology was seen in this model, it cannot be considered a model of interstitial pneumonitis since the mice also had an overwhelming disseminated infection, and indeed the cause of death in this model was recently shown to be a failure to generate hematopoietic stem cells [23]. However, salient features of the model merit discussion here. First, it is clear that any lung pathology seen was due to viral replication and not to any cellular infiltrate. Second, reconstitution with syngeneic sensitized T cells protected the mice from lethal infection, coincident with a reduction in virus titers in the lung, and - although the authors did not discuss the effect of such reconstitution on lung pathology the fact that the mice survived indicates that any effect on the lungs was obviously not associated with death. This is reminiscent of the clinical findings in humans that only allogeneic, and not syngeneic or autologous, bone marrow transplantation was associated with the development of interstitial pneumonitis.
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CMV Pathogenesis
Role of Antibody in the Lungs of Patients with CMV Pneumonitis CMV hyperimmune globulin has been used in the treatment of established CMV pneumonitis, apparently with beneficial effects. In an open study [26], mortality, which historically is 1'\J85070, was reduced to 50070. Patients who did not respond to this therapy were already being ventilated before treatment was initiated, suggesting that prompt treatment was necessary. These results ,,;ere not confirmed by another group using a different hyperimmune globulin preparation [35], but an average of 8.2 days (range, 2-30 days) had elapsed between the onset of pneumonitis and the initiation of treatment, a delay that may explain the poor results obtained. The mechanism by which antibody might reduce mortality from CMV pneumonitis has been discussed in detail elsewhere [26]. It was considered unlikely that it was due to neutralization of virus by antibody since the hyperimmune globulin used could not neutralize virus obtained directly from patients' body fluids [36] - although it had good neutralizing titers against virus harvested from cell cultures. The inability to neutralize virus from body fluids was thought to be due to the protective effect of host I3rmicroglobulin bound to the viral envelope of CMV in body fluids [36]. It is surprising that exogenous CMV antibodies from pooled donors could reduce mortality since it has recently been shown that patients with CMV pneumonitis had high levels of locally generated antibody in their lungs at the time of their episode of pneumonitis [37, 38]. This suggests either that factors other than CMV antibodies contained in the hyperimmune globulin mediate the protective effect or that the class, subclass, or specificity of CMV antibodies generated locally in the lungs of patients with CMV pneumonitis is different in some way from that present in the serum of seropositive blood donors.
CMV and Graft Rejection Graft rejection might be considered another manifestation of CMV-associated disease. There are clinical data both for [39] and against [40] a role for CMV in provoking graft rejection. In vitro CMV can enhance the expression of class I HLA antigens, partly through the release of interferon [27]. Enhancement of allogeneic antigens, mismatched between donor and recipient, could feasibly contribute to graft rejection. In mice, MCMV infection in vivo has been shown to enhance the host cytotoxic T cell response to alloantigens [28,29]. However, the clinical data linking episodes of CMV infection and rejection are largely circumstantial. In addition, one assay historically used for monitoring rejection, the measurement of levels of 132-microglobulinin urine, has recently been shown to give artifactually high results, mimicking rejection, when CMV is present in the urine specimen assayed [41]. The issue is further complicated by the fact that treatment for rejection relies on increased immunosuppression, while the dose of immunosuppressive agent is usually lowered during episodes of CMV infection. Thus, treatment for either condition can lead to exacerbation of the other, making interpretation of the links between the two on a mechanistic level virtually impossible.
Effect of the Type of CMV Infection on Pathogenicity An important factor in the pathogenesis of infection and the expression of symptomatic disease is whether CMV infection arises from an exogenous source of virus or from reactivation of latent endogenous virus. Table 2 summarizes the types of CMV infection associated with disease in different patient groups. Infection has been categorized as primary, denoting infection of a seronegative individual with exogenous virus; reactivation, denoting reactivation of latent endogenous virus in seropositive individuals; or reinfection, denoting infection with a new strain of CMV from an exogenous source. Damage to the fetus following either primary infection in the mother or presumed reactivation in mothers seropositive before conception has been described [42] (although it must be noted in the latter case that the distinction between reinfection and reactivation was not made). CMV disease in apparently healthy individuals - manifest as CMV mono-
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marrow transplant recipient have been replaced by those of donor origin [33]. This finding suggests that histocompatibility (presumably for class II antigens) between T cells, which will be of donor origin, and antigen-presenting cells may be necessary for the induction of pathology. This is remarkably similar to the situation recently described in liver transplant recipients, in whom CMV infection was associated with the ''vanishing bile duct syndrome" if donor and recipient were matched at the HLA-DR locus [34].
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Table 2. Relationship between type of CMV infection and expression of CMV disease in various groups. Disease association*
Group
+ +
Reactivation
Reinfection
NA
NA ?
+
+ + + + + ?
+ + + + ?
? ? ?
?
* + and - = association and absence of association, respectively. NA = not applicable to this patient group; ? = information presently available is inconclusive. t This refers to the type of infection in the mother that results in disease in the fetus.
nucleosis [43]- is a feature of primary infection only, thus demonstrating that in immunocompetent individuals, primary infection induces protective immunity. In all groups of seronegative transplant recipients, primary infection, due to transmission of CMV by the donor organ or blood products, has been associated with disease [4-7]. CMV infection in seropositivetransplant recipients has been associated with disease following all types of transplantation, but the question of whether this is due to reactivation, reinfection, or both has been pursued only in the case of renal transplantation [44-46]. Restriction enzyme analysis of viral DNA was used to type the strain of CMV excreted after renal transplantation and hence to distinguish between reactivation and reinfection [44-46]. The results showed that disseminated infection and symptomatic illness (including CMV pneumonitis) were associated with reinfection [46] and that reactivation of latent recipient virus was accompanied by asymptomatic shedding of virus at restricted sites [46]. This finding suggests that prior immunity is not protective against new strains of virus but can protect against the homologous reactivated strain. This difference in protection may be due to important antigenic differencesamong strains, although it was noted that differences in the sites of viral replication following reactivation of endogenous infection, vs. reinfection from donor organ or blood products, may influence the outcome [46]. In seropositive recipients of liver [5] and heart [6]
Mechanism of the Effect of CMV on Host Immune Responses CMV is reputed to be immunosuppressive in both humans and mice [48], although enhanced immune responses have been described and the virus induces a variety of autoantibodies [40,48]. Although much attention has focused on the mechanism of such immunosuppression, it remains an enigma. The low frequency of infection of lymphocytes and monocytes during acute CMV infection argues for an indirect mechanism of immunosuppression [45, 49]. The induction by CMV of a soluble inhibitor of interleukin-l was recently described [50];however, this find-
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Fetus Mother to fetus t Healthy adults Transplant recipient Kidney Liver Heart Bone marrow Patients with AIDS
Primary infection
transplants and, as discussed earlier, of allogeneic bone marrow [7], serious CMV disease is seen even when donors are seronegative. Although it is possible that reinfection with exogenous virus from blood products is partly responsible, it is likely that many instances of this disease are associated with reactivation of endogenous recipient virus. Thus, in these patients prior immunity does not prevent dissemination of reactivated virus or the expression of disease. Since the majority of homosexual patients with HIVinfection are seropositive for CMV [47], in this group CMV disease is associated with either reactivation or reinfection. Prospective studies will be needed to differentiate between these two types of infection. Although CMV disease can be associated with primary infection, reactivation, or reinfection in particular patient groups, the incidence of disease associated with the different types of infection differs in some circumstances. For example, there is evidence that congenitally infected babies born to previously seropositive mothers are less severely damaged than are those born to mothers who contracted a primary infection during pregnancy [39]. Similarly, in renal transplant recipients the incidence of disease associated with reinfection is lower than that accompanying primary infection [46]. It is thus apparent that in particular patient groups prior infection does not protect against disease due to infection with exogenous or endogenous virus. It is of some importance that this lack of protection extends not only to diseaseassociated with viral replication (e.g., brain damage in congenital infection) but also to disease associated with immunopathology (e.g., CMV pneumonitis in transplant recipients).
CMV Pathogenesis
References 1. Griffiths PO, Grundy JE. The status of CMV as a human pathogen. Epidemiol Infect 1988;100:1-15 2. BoyceNW, Hayes K, Gee 0, Holdsworth SR, Thomson NM, Scott 0, Atkins RC. Cytomegalovirus infection complicating renal transplantation and its relationship to acute transplant glomerulopathy. Transplantation 1988;45:706-9 3. Skolnik PR, Kosloff BR, Hirsch MS. Bidirectional interactions between human immunodeficiency virus type 1 and cytomegalovirus. J Infect Dis 1988;157:508-14 4. Rubin RH, Tolkoff-Rubin NE, Oliver 0, Rota TR, Hamilton J, Betts RF, Pass RF, Hillis W, Szmuness W, Farrell ML, Hirsch MS. Multicenter seroepidemiologic study of
the impact of cytomegalovirus infection on renal transplantation. Transplantation 1985;40:243-9 :5. Singh N, Dummer JS, Kusne S, Breinig MK, Armstrong JA, Makowka L, Starzl TE, Ho M. Infections with cytomegalovirus and other herpes viruses in 121 liver transplant recipients: transmission by donated organ and the effect of OKT3 antibodies. J Infect Dis 1988;158:124-31 6. Gorensek MJ, Stewart RW, Keys TF, McHenry MC, Goormastic M. A multivariate analysis of the risk of cytomegalovirus infection in heart transplant recipients. J Infect Dis 1988;157:515-22 7. Wingard JR, Mellits ED, Sostrin MD,Chen DY-H,Burns WH, Santos GW, Vriesendorp HM, Beschorner WE, Saral R. Interstitial pneumonitis after allogeneicbone marrow transplantation. Nine-year experience at a single institution. Medicine 1988;67:175-86 8. Appelbaum FR, Meyers JD, Fefer A, Flournoy N, Cheever MA, Greenberg PO, Hackman R, Thomas ED. Nonbacterial nonfungal pneumonia following marrow transplantation in 100identical twins. Thmsplantation 1982;33:265-8 9. Wingard JR, Sostrin MB, Vriesendorp HM, Mellits ED, Santos GW, Fuller DJ, Braine HG, YeagerAM, Burns WH, Sara! R. Interstitial pneumonitis followingautologous bone marrow transplantation. Transplantation 1988;46:61-5 10. Wingard JR, Chen DY-H,Burns WH, Fuller OJ, Braine HG, YeagerAM, Kaiser H, Burke PJ, Graham ML, Santos GW, Saral R. Cytomegalovirus infection after autologous bone marrow transplantation with comparison to infection after allogeneic bone marrow transplantation. Blood 1988; 71:1432-7 u. Valteau 0, Hartmann 0, Benhamou E, Caillaud JM, Brugieres L, Beaujean F, Patte C, Flamant F, Lemerle J. Nonbacterial nonfungal interstitial pneumonitis following autologous bone marrow transplantation in children treated with high-dose chemotherapy without total-body irradiation. Transplantation 1988;45:737-40 l1a. Millar AD, Patou G, Miller RF, Grundy JE, Katz DR, Weller IV, Semple SJ. Cytomegalovirus in the lungs of patients with AIDS: respiratory pathogen or passenger? Am Rev Respir Dis 1990;141:1474-7 12. Grundy JE, Shanley JD, Shearer GM. Augmentation of graft versus host reaction by cytomegalovirus infection resulting in interstitial pneumonitis. Transplantation 1985;39: 548-53 13. Shanley JD, Pesanti EL, Nugent KM. The pathogenesis of pneumonitis due to murine cytomegalovirus. J Infect Dis 1982;146:388-96 14. Reddehase MJ, Weiand F, Munch K, Jonjic S, Luske A, KoszinowskiVH. Interstitial murine cytomegalovirus pneumonia after irradiation: characterization of cells that limit viral replication during established infection of the lungs. J Virol 1985;55:264-73 15. Shanley JD, Pomeroy C, Via CS, Shearer GM. Interstitial pneumonitis during murine cytomegalovirus infection and graft-versus-host reaction: effect of ganciclovir therapy. J Infect Dis 1988;158:1391-4 16. Via CS, Shanley JD, Weatherly BR, Lang P, Shearer GM. Altered threshold for the induction of graft-versus-host immunodeficiency following murine cytomegalovirus infection. Transplantation 1988;46:298-302 17. Shanley JD, Via CS, Sharrow SO, Shearer GM. Interstitial
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ing could not be reproduced when mycoplasma-free strains of CMV were used, and the authors concluded that the phenomenon was due to mycoplasma contamination [51]. Another possibility is that the virus itself exerts a direct immunosuppressive action, triggering alterations in immune reactivity through a direct interaction with lymphoid cells, without even limited viral replication being required. This proposal is consistent with studies in the mouse [52, 53] that indicate MCMV can be directly immunosuppressive in vitro and that the peak of immunosuppression [54] correlates with peak virus titers. A direct effect of human CMV on the induction of immune responses has also been suggested in several studies [55, 56]. A possible, although highly speculative mechanism by which CMV could alter immune reactivity is by directly binding to lymphoid cells, leading to some triggering of the cell. The basis for this proposal is the widely reported ability of monoclonal antibodies to ~z-microglobulin (the light chain of the class I HLA dimer) to induce lymphocyte activation, with both enhancement and suppression of immune responses having been described [57-60]. Since CMV has been reported to bind Ih-microglobulin [33, 58], the cross-linking of class I HLA molecules by multipoint binding of the virus to more than one light chain might be expected to trigger the cell in the same way as does cross-linking by antibodies to ~z-micro globulin, resulting in abnormal immune reactivity. Such a situation is analogous to the well-known triggering of B cells by antibodies to immunoglobulin, a phenomenon in vitro that mimics the cross-linking of cell-surface immunoglobulin by antigen. Although this model for a direct action of CMV is highly speculative, it is testable. The solution to the intriguing question of how CMV alters host immune responses thus awaits further investigation.
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