Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.

Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection.

The course of mouse cytomegalovirus (MCMV) infection was compared between wild-type and mutant C57BL / 6 (B6) mice deficient in either RAG-2, perforin, granzyme A, granzyme B or combinations thereof at two time points post infection (p. i.). At day 15 p. i., virus titers were similarly elevated in salivary glands of all mutant, but not wild-type B6 mice and undetectable in lung and spleen tissues of any of the mouse strains. Significant pathological alterations were only seen in salivary glands and spleen from RAG2(- / -), but not in those from other mice whereas few inflammatory foci were observed in lung tissues of all mice except B6. At day 30 p. i., elevated virus titers were observed only in salivary glands, lung and spleen from RAG2(- / -), but in none of the other mice, and were accompanied by extended pathological alterations in all three organs. The data extend previous reports on the critical role of NK / CD8(+) T cells in the early control of MCMV infection by showing that both perforin and granzymes A / B contribute to viral elimination in salivary glands; however, neither of the three molecules alone seem to be indispensable for the final control of infection.

Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes.

Analysis of perforin-deficient mice has identified the cytolytic pathway and perforin as the preeminent effector molecule in T cell-mediated control of virus infections. In this paper, we show that mice lacking both granzyme A (gzmA) and granzyme B (gzmB), which are, beside perforin, key constituents of cytolytic vesicles, are as incapable as are perforin-deficient mice of controlling primary infections by the natural mouse pathogen ectromelia, a poxvirus. Death of gzmAxgzmB double knockout mice occurred in a dose-dependent manner, despite the expression of functionally active perforin and the absence of an intrinsic defect to generate splenic cytolytic T cells. These results establish that both gzmA and gzmB are indispensable effector molecules acting in concert with perforin in granule exocytosis-mediated host defense against natural viral pathogens.

Resolution of experimental and tick-borne Borrelia burgdorferi infection in mice by passive, but not active immunization using recombinant OspC.

Vaccination with outer surface protein A (OspA) of Borrelia burgdorferi prevents subsequent infection and disease in both laboratory animals and humans with high efficacy. OspA-based immunity, however, does not affect established infection due to the loss of OspA expression in the vertebrate host. We show here that repeated passive transfer of mouse and/or rabbit immune sera to recombinant GST-OspC fusion protein resulted in a dose-dependent resolution (1) of fully established arthritis and carditis as well as infection in needle-challenged C.B-17 SCID and (2) of infection in both experimentally and tick-infected BALB/c mice. Unexpectedly, active immunization of disease-susceptible AKR/N mice with GST-OspC only led to prevention but not resolution of disease and infection, in spite of high serum titers of OspC-specific Ab and the expression of ospC in tissue-derived spirochetes. The data suggest that the efficacy of OspC antibody-mediated immunity depends on the immunological history of the recipient and/or environment-dependent regulation of OspC surface expression by spirochetes in vivo. The results encourage further attempts to develop therapeutic vaccination protocols against Lyme disease.

Perforin is essential for control of ectromelia virus but not related poxviruses in mice.

Lack of perforin renders the relatively resistant mouse strain C57BL/6 highly susceptible to the natural mouse pathogen ectromelia virus, a cytopathic orthopoxvirus. This is indicated by increased mortality, elevated virus titers and pathology in liver and spleen, and increased levels of liver enzymes in blood. Cowpox virus on the other hand is more virulent in the presence of perforin than in its absence. An additional lack of granzyme A which together with perforin is a constituent of cytoplasmic granules from cytotoxic T cells increases the virulence of cowpox virus.

Therapeutic passive vaccination against chronic Lyme disease in mice.

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with Borrelia burgdorferi. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 microg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of B. burgdorferi resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 microg OspC-specific Abs) were repeatedly given (4x in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.

Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

Prednisolone reduces experimental arthritis, and inflammatory tissue destruction in SCID mice infected with Borrelia burgdorferi.

Glucocorticosteroids (GC) are widely used as anti-inflammatory agents. The effects of Prednisolone on the development of Borrelia (B.) burgdorferi-induced clinical arthritis and organ inflammation was studied in severe combined immunodeficiency (SCID) mice. The drug was administered orally at a dose of 3, 10 and 30 mg/kg, starting shortly before experimental infection of the mice. A dose dependent inhibition of arthritic joint swelling was observed. Full protection was obtained with 30 mg/kg until 21 days after infection, subsequently, mild joint swelling developed but progression and severity of the disease was considerably less than in the other treated as well as in the untreated mice. Inhibition of clinical arthritis coincided with reduction of inflammatory cell infiltration in the joints, liver and muscle. Prednisolone was ineffective when application was initiated after arthritis was fully developed, i.e., 22 days after infection. Since the activated endothelium plays a critical role in development of inflammatory lesions, the expression of the cellular adhesion molecules (CAMs) E-selectin, P-selectin, ICAM-1 and VCAM-1 was determined in vitro using the bEnd3 endothelial cell line. Stimulation with a sonicated B. burgdorferi preparation in the presence of the water-soluble compound Prednisolone-21-hemisuccinate considerably reduced expression of ICAM-1, and marginally also of E-selectin, whereas the level of P-selectin and VCAM-1 remained unaltered. Thus, downregulation of ICAM-1 might be a critical factor in Prednisolone-mediated inhibition of B. burgdorferi-induced inflammation; the flare up of the disease after the initial protection indicates that additional therapy, e.g. with antibiotics, is necessary.

Studies on early events of Borrelia burgdorferi-induced cytokine production in immunodeficient SCID mice by using a tissue chamber model for acute inflammation.

Lyme arthritis, one of the common features of Borrelia burgdorferi infection in the human, is associated with the production of various monocyte derived cytokines. To investigate the expression and regulation of cytokines during the acute phase of spirochete induced inflammation, a perforated Teflon chamber was implanted under the dorsal skin of severe combined immunodeficiency (SCID) and immunocompetent co-isogenic C.B-17 mice. The histology of the surrounding chamber tissue exhibited sterile inflammation with several features reminiscent of an inflamed synovium, i.e. infiltration of polymorphonuclear and mononuclear cells, fibroblast-like cells and neovascularization. The experimental inoculation of Borrelia burgdorferi into the chamber resulted in the production of TNF-alpha, IL-1 and IL-6 into the chamber exudate, in both the immunodeficient, disease susceptible SCID and the immunocompetent, disease resistant C.B-17 mice. Peak levels of TNF-alpha were reached at 2 hours and of IL-1 and IL-6 at 6 hours after infection; by 24 hours, cytokine levels were only marginal (IL-1, IL-6) or non-detectable (TNF-alpha). Experimental infection by s.c. injection distant from the tissue chamber led to colonization of the spirochetes into the chamber, suggesting a tropism of the bacteria for this tissue. Thus, this model provides a system for studying acute events of Borellia burgdorferi induced cytokine regulation in a complex cellular, synovium-like environment that the bacterium encounters in vivo.

Protection against Borrelia burgdorferi infection in SCID mice is conferred by presensitized spleen cells and partially by B but not T cells alone.

Borrelia burgdorferi induces spirochetemia, arthritis, carditis and myositis in SCID mice but not in immunocompetent co-isogenic C.B-17 mice. The contribution of naive or presensitized B and T cells in the control of spirochetal infection has now been analysed in SCID mice reconstituted with unselected spleen cells or enriched B or T cell populations thereof and subsequently challenged with B. burgdorferi. It is shown that SCID mice were protected (i) completely against disease (arthritis, carditis, myositis) by unselected spleen cells previously sensitized either to intact spirochetes or to recombinant outer surface protein A (OspA), (ii) to a large extent by mixtures of enriched spirochete-specific B and T cells, (iii) partially by equivalent preparations of presensitized B cells or by naive spleen or B cells, and (iv) not at all by presensitized or naive T cells alone. The degree of protection transferred was similar for the corresponding lymphocyte populations presensitized either to viable spirochetes or to recombinant OspA and correlated mainly with serum levels of B. burgdorferi-specific antibodies, in particular those to OspA/OspB. The capacity of enriched presensitized or naive B cells alone to generate specific antibodies of the isotypes IgM, IgG2b and IgG3, and to confer partial protection to SCID mice upon challenge with B. burgdorferi is most probably due to a B cell mitogen(s) associated with the spirochetes. These data further emphasize the important role of B cells and antibodies in the control of B. burgdorferi infection in mice, and suggest that T cells are critically involved in the optimal generation of protective antibody responses but not in the direct elimination of spirochetes from the host.

Immune sera to individual Borrelia burgdorferi isolates or recombinant OspA thereof protect SCID mice against infection with homologous strains but only partially or not at all against those of different OspA/OspB genotype.

The outer surface proteins OspA and OspB of Borrelia burgdorferi have recently been demonstrated to be major target proteins for protective antibodies in mice against infection with the homologous spirochaetal strain. However, it has become clear from a variety of studies that B. burgdorferi isolates of different geographical origin and/or sources are heterogeneous and that they can be divided into at least six subgroups according to their distinct OspA/OspB genotypes. In order to analyse cross-protection between these subgroups we have now generated immune sera to various isolates of B. burgdorferi with different OspA/OspB genotypes. We show that passive immunization with antisera specific for whole spirochaetes or recombinant OspA of one spirochaetal isolate protects severe combined immunodeficiency mice against infection with strains of the corresponding OspA/OspB genotype but only partially or not at all against infection with isolates expressing distinct OspA/OspB genotypes. The incomplete protection mediated by individual antisera against independent isolates of B. burgdorferi suggests that an effective subunit vaccine against Lyme disease should consist of a mixture of OspA structures covering the heterogeneity of this protein within the species B. burgdorferi.

Myositis in mice inoculated with Borrelia burgdorferi.

The authors describe the appearance of myositis in immunocompetent and immunodeficient mice after subcutaneous inoculation with Borrelia burgdorferi by histology and immunohistology. Experimental infection of mice 1) causes inflammation of striated but not smooth muscles, 2) affects the entire musculoskeletal system, and 3) is characterized by perivascular and interfacicular infiltration of mononuclear leukocytes in the striated muscle leading to necrosis as well as disruption of muscle fibers. The lesions found in striated muscle specimens were most pronounced in immunodeficient (SCID), less severe in T-cell-deficient nu/nu (BALB/c, C57BL/6) and marginal to moderate or almost not present in immunocompetent AKR/N and C.B-17 mice, respectively.

Recombinant outer surface protein a from Borrelia burgdorferi induces antibodies protective against spirochetal infection in mice.

The outer surface protein A (OspA) of Borrelia burgdorferi was isolated in its native form from strains ZS7 and B31 and as a recombinant protein from strain ZS7. Amino acid sequence analysis of internal peptides of native OspA (strain ZS7) revealed identity with the sequence deduced from the OspA gene. Repeated immunization of C57BL/6 and C.B-17 mice with any of the three OspA structures resulted in the generation of monospecific hyperimmune sera reactive with both native and recombinant OspA. Upon transfer of immune sera specific for either native OspA (strain B31) or recombinant OspA (strain ZS7) but not of those reactive with the recombinant 41-kDa flagellin-associated antigen, severe combined immunodeficient (SCID) mice were completely protected against infection with strain ZS7. The finding that monoclonal antibodies to OspA and to OspB but not to non-outer surface spirochetal structures such as flagellin, p20, p65, and p70 conferred protection in SCID mice makes OspA (and possibly OspB) a promising candidate vaccine against Lyme disease.

A mouse model for Borrelia burgdorferi infection: pathogenesis, immune response and protection.

Viable Borrelia burgdorferi (B. burgdorferi) organisms induce a chronic infection associated with arthritis, carditis and hepatitis in severe combined immunodeficiency (scid) mice but not in most of the adult mice from the various immunocompetent inbred strains tested. Furthermore, we have found that experimental inoculation of normal mice with B. burgdorferi organisms leads to the generation of antibodies and T cells specific for various spirochetal antigens including the outer surface proteins A and B (OspA, OspB) as well as flagellin. The assumption of a protective role of the immune response during B. burgdorferi infection in mice is supported by our recent findings that passively transferred B. burgdorferi-specific immune mouse sera as well as monoclonal antibodies to OspA are able to prevent the development of the disease in scid mice. We show now that purified OspA protein both in its native and recombinant form is immunogenic and that the antibodies generated are able to confer protection to scid mice against B. burgdorferi infection.

Lyme borreliosis in the severe combined immunodeficiency (scid) mouse manifests predominantly in the joints, heart, and liver.

The authors describe the histopathologic evolution of Lyme disease in severe combined immunodeficiency (scid) and normal C.B-17 and C57BL/6 mice inoculated with Borrelia burgdorferi. Starting on day 7 after inoculation, all scid mice infected subcutaneously in the tail with a low-passage European tick isolate of B. burgdorferi had clinical evidence of arthritis characterized by reddening and swelling of tibiotarsal joints. Later on, other joints, ie, metatarsal and ulnacarpal joints were also affected. The infection of scid mice resulted in a persistent spirochetemia and the development of a multisystem disease with chronic progressive inflammation of joints, heart, and liver. Major histopathologic alterations included 1) severe joint lesions, characterized by the presence of hyperplastic inflamed synovial lining cells associated with the erosion and destruction of cartilage and/or bone; 2) pancarditis with infiltrations of mononuclear cells in the endocardium, myocardium, and pericardium; and 3) hepatitis with mononuclear cell infiltrations confined to the portal field and central vein, granulomatous reactions, and eventually the development of liver fibrosis. In addition, smaller more confined lesions were found in kidneys, lung, brain, and striated muscle. The inflammatory infiltrates in the various organs were associated mostly with Mac-1+ cells, largely monocytes and macrophages, as well as some polymorphonuclear leukocytes, but not B and T lymphocytes. Infective spirochetes could be readily isolated from blood and joints and were found at the site of inoculum and the myocardium. In contrast, subcutaneous inoculation of normal C.B-17 or C57BL/6 mice with spirochetes in general did not result in clinical signs of arthritis. Only 10% to 20% of the C57BL/6 mice, but none of the C.B-17 mice, showed clinical evidence of oligoarthritis, which appeared not before day 36 after inoculation. In general, the infection of normal mice resulted in minimal lesions in various organs, and no spirochetes could be visualized or reisolated from their tissues. The data demonstrate that Lyme borreliosis may develop in mice in the absence of detectable specific B and T cells and thus suggest an immunologic control of the disease in this species. The scid mouse model therefore can be used to define the components of the immune system responsible for the suppression and/or the progression of the disease.

Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice.

We have recently shown that viable Borrelia burgdorferi organisms induce a chronic infection associated with arthritis and carditis in severe combined immunodeficiency (scid) mice but not in immunocompetent mice. The disease is similar to that found in patients suffering from Lyme disease. We now show that B. burgdorferi-specific immune mouse sera as well as a monoclonal antibody to the spirochetal outer surface antigen A (31 kDa) but not monoclonal antibodies specific for the 41-kDa antigenic component of the periplasmic flagella are able to prevent (or mitigate) the development of the disease in scid mice when passively transferred at the time of the bacterial inoculation. The identification of a B. burgdorferi-associated protective antigen suggests that the corresponding spirochetal protein should be tested as a vaccine against Lyme disease.

Lyme carditis in immunodeficient mice during experimental infection of Borrelia burgdorferi.

Recently, we described the severe combined immunodeficiency (scid) mouse as a laboratory model for B. burgdorferi infection. Scid mice inoculated with the virulent low-passage tick isolate Borrelia burgdorferi ZS7 developed a severe pancarditis involving endocardium, myocardium and epicardium in the absence of functional B- or T-cells. Soon after inoculation perivascular infiltration was observed, later diffuse infiltration of the interstitium of the subendocardial and subepicardial areas was seen. The infiltrate was mainly mononuclear and predominantly composed of Mac-1+ cells. Concomitantly, fibroblast proliferation and augmented collagen deposition occurred in the interstitium. This was associated with the presence of B. burgdorferi organisms. The histopathological and ultrastructural findings observed in scid mice resemble those observed in human Lyme carditis. The data emphasize the suitability of the scid mouse as a model in which to study the role of the immune system in the pathogenesis of Lyme carditis.

The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis.

We report that the spirochete B. burgdorferi induces progressive polyarthritis and carditis in mice with severe combined immunodeficiency syndrome (scid) but not in normal C.B-17 mice. The onset and severity of the disease were dependent on (a) the viability; (b) the infectivity; and (c) the dose of inoculated B. burgdorferi organisms. Infective spirochetes were isolated from both blood and joints of inoculated scid mice. These findings suggest that B. burgdorferi-induced chronic arthritis and carditis in mice develops independently of lymphocyte function and makes the scid mouse an attractive laboratory model to study the role of the immune system in experimental Lyme Borreliosis.

Demonstration of antigen-specific T cells and histopathological alterations in mice experimentally inoculated with Borrelia burgdorferi.

Antigen-specific T-cell responses and histopathological changes were studied in mice experimentally inoculated with Borrelia burgdorferi B31. Inbred mice with different H-2 haplotypes and/or different genetic backgrounds were inoculated with B. burgdorferi organisms and tested for antigen-specific T-cell responses in vivo (delayed-type hypersensitivity [DTH]) and in vitro (T-cell proliferation). Comparable DTH responses were found after inoculation with either inactivated (in the presence of adjuvants) or viable microorganisms in all mouse strains, except BALB/c, irrespective of the H-2 haplotype (b, d, k, or s) tested and the sex of the animals. Moreover, in mice presensitized to B. burgdorferi, DTH responses could be induced only with antigen preparations derived from the corresponding strain but not with those obtained from either related spirochetes such as Treponema phagedenis and Leptospira interrogans or unrelated bacteria such as Mycobacterium tuberculosis. T cells isolated from lymph nodes or spleens of mice previously sensitized to B. burgdorferi but not those from naïve mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation. Histopathological examination of mice inoculated with viable B. burgdorferi organisms revealed significant perivascular infiltrates consisting mainly of mononuclear and a few polymorphonuclear leukocytes in different organs (brain, heart, lungs, liver, and kidneys) and the appearance of giant multinucleated cells within the spleen similar to those found in human skin specimens of patients suffering from cutaneous manifestations of Lyme disease. Our findings suggest that mice are a suitable animal model with which to study the immune response to B. burgdorferi and the pathogenesis of Lyme disease.