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* Institute for Experimental Immunology, University Hospital, Zurich, Switzerland; and
Unidad de Investigación Médica en Inmunoquímica, Hospital de Especialidades, México City, México
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been observed that macrophage depletion reduces the host capacity to control viral infections (13, 14). Depletion of MZM can be experimentally induced by the administration of clodronate encapsulated in liposomes (15, 16, 17). Using this experimental approach, it has been shown that such mice fail to control a systemic lymphocytic choriomeningitis virus (LCMV) infection (18). However, long-term studies are difficult to carry out in MZM-depleted mice because the MZM are replenished and because repeated treatment with clodronate liposomes results in a high mortality rate with severe side effects resulting in noninterpretable results. Nevertheless, studies in MZM-depleted mice and in op-/- mice revealed that MZM play a role in controlling Listeria monocytogenes infections (19, 20). Additionally, earlier studies had shown that LCMV-infected MZM were destroyed by virus-specific CTLs and caused systemic immunosuppression (21). Non- or low-cytopathic viruses such as LCMV differ from cytopathic viruses such as vesicular stomatitis virus (VSV) in that the former are usually eliminated by CTLs, whereas neutralizing Abs induced during the course of the primary immune response usually control the latter. Common to both viruses is that they are initially recruited to lymphoid organs by natural Abs, where they are thought to be removed by MZM to reduce dissemination of virus to vital organs and at the same time enhance induction of an immune response (22, 23, 24). The present study used op-/- mice to evaluate the contribution of MZM to the elimination of a non- or low-cytopathic virus vs a cytopathic virus and to investigate the effect of impaired removal of infectious virus on antiviral immunity and disease manifestation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from the breeding colony of the Institut für Labortierkunde (Faculty of Veterinary Medicine, University Zurich-Irchel, Zurich, Switzerland). Experiments were conducted with age- and sex-matched animals kept under specific pathogen-free conditions. Heterozygous op/+ mice were obtained from The Jackson Laboratory (Bar Harbor, ME), embryo-derived, back-crossed onto C57BL/6 for two generations, and kept under specific pathogen-free conditions according to institutional guidelines. Heterozygous breeding pairs were identified by PCR as previously described (5). Inspecting them for the lack of incisors identified homozygous op-/- offspring. They were fed powdered mouse food and water supplemented with 5% glucose. Heterozygous and wild-type littermates were separated and sacrificed as early as possible from op-/- littermates to increase the survival rate of op-/- mice and to obtain a sufficient number of homozygotes. For this reason, i.e., lack of littermates, all experiments were conducted with age-matched (date of birth within the range of 2 wk) C57BL/6 control mice. Blood samples from experimental op-/- mice were checked for the presence of H-2b by FACS analysis. Despite the possibility that inbred mouse strains may show altered kinetics of virus clearance, our longstanding experience with transgenic and knockout mouse lines generated on mixed genetic backgrounds showed that the immune responses against LCMV and VSV were identical to C57BL/6 after the first, and even more so the second, backcross to C57BL/6; thus, C57BL/6 were considered to serve as appropriate controls in our experiments.
VSV-IND (Mudd-Summers isolate) was originally obtained from D. Kolakofsky (University of Geneva, Geneva, Switzerland) and was grown on BHK cells in MEM with 5% FCS to virus stocks containing 109 PFU/ml. LCMV-WE was originally provided by F. Lehmann-Grube (University Hamburg, Hamburg, Germany) and was grown on L-929 cells for 48 h in MEM with 5% FCS after infection with an initial multiplicity of infection of 0.01.
VSV neutralization assay
Mice were bled from the retroorbital cavity at the indicated time points. Serial 2-fold dilutions of serum samples (previously diluted 1/40) were mixed with equal volumes of VSV containing 500 PFU/ml and the mixtures were incubated for 90 min at 37°C in an atmosphere containing 5% CO2. A total of 100 µl of the mixture was transferred onto Vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. Monolayers were overlaid with 100 µl of DMEM containing 1% methylcellulose and incubated for 24 h at 37°C, after which the overlay was removed and the monolayer was fixed and stained with 0.5% crystal violet dissolved in 5% formaldehyde, 50% ethanol, and 4.25% NaCl. The dilution reducing the number of plaques by 50% was taken as titer (25). To determine IgG titers, undiluted serum was treated with an equal volume of 0.1 M 2-ME in MEM tissue culture medium for 1 h at room temperature.
Determination of VSV and LCMV titers in tissues
Mice were sacrificed by cervical dislocation. Brain tissue was homogenized in balanced salt solution, and serial 3-fold dilutions were plated on Vero cell monolayers in 96-well plates. Viral titers were calculated by multiplying counted plaques by the dilution factor. Mice without detectable virus in the brain (<103 PFU per brain) were scored negative (26, 27). LCMV titers of organs of infected mice were determined in an infectious focus formation assay as described previously (28). Briefly, spleen homogenates were serially diluted 10-fold and grown on an MC57G cell monolayer for 48 h under an overlay of 1% methylcellulose in DMEM. Cells were fixed with 4% formalin in PBS, and infectious foci were detected by intracellular LCMV staining of infected cells with the rat anti-LCMV mAb VL-4.
Cytotoxicity assay
The cytolytic activity of spleen cells was tested in a 51Cr release assay as described previously (29). Briefly, EL-4 target cells were coated with LCMV-derived peptide gp33–41 (30) at concentrations of 10-6 M. A total of 104 target cells were incubated in 96-well round-bottom plates with serial 3-fold dilutions of spleen effector cells starting at an E:T ratio of 70:1 in a final volume of 200 µl. After 5 h of incubation at 37°C, 70 µl of supernatant was harvested and radioactivity determined in a gamma counter, and the percentage of specific release was calculated.
Secondary in vitro restimulation
A total of 5 x 106 spleen cells and 2 x 105 LCMV-infected peritoneal exudate macrophages were cocultured in 24-well tissue culture plates in a total volume of 2 ml IMDM supplemented with 10% FCS, penicillin/streptomycin, glutamine, and 5 x 10-5 M 2-ME. Macrophages were obtained by injecting mice i.p. with 1 ml thioglycolate on day -6 and with 200 PFU LCMV on day -4. After 5 days, 1 ml medium was removed and 100 µl of these standard cultures were diluted in duplicate (four steps, 3-fold dilutions) in MEM supplemented with 2% FCS in 96-well round-bottom plates and specific cytotoxicity was determined as described above.
Immunohistochemistry
Freshly removed organs were immersed in HBSS and snap-frozen in liquid nitrogen. Tissue sections of 5-µm thickness were cut in a cryostat, placed on siliconized glass slides, air dried, fixed with acetone for 10 min, and stored at -70°C. Second-stage affinity-purified polyclonal anti-Ig antisera were diluted in Tween-buffered saline (TwBS; pH 7.4) containing 5% normal mouse serum. All other dilutions were made in TwBS alone. Incubations were performed at room temperature for 30 min; TwBS was used for all washing steps. Alkaline phosphatase was visualized using naphthol AS-BI phosphate and New Fuchsin as substrate, which yields a red precipitate. Endogenous alkaline phosphatase was blocked by levamisole. All color reactions were performed at room temperature for 15 min with reagents from Sigma-Aldrich (St. Louis, MO). Sections were counterstained with hemalum. Coverslips were mounted with glycerol and gelatin. Stainings for the cell differentiation markers MOMA-1 (mouse metallophilic macrophages) and 4C11 (follicular dendritic cells (FDC)) were performed exactly as previously described (31). The detection of VSV-Ag has been described previously (32).
Footpad swelling reaction
Mice were injected into the hind footpad with 100 PFU LCMV WE in 30 µl of MEM supplemented with 2% FCS. The footpad thickness was measured with a spring-loaded caliper (Kroeplein; Schluchtern, Hessen, Germany). Values were taken as the mean of both hind footpads (29, 33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the capacity of op-/-
mice to mount an immune response against non- or low-cytopathic LCMV,
mice were infected via the footpad (i.f.) with LCMV. After footpad
infection, immunocompetent mice develop a footpad swelling reaction due
to a biphasic CD8+ and CD4+
T cell response (29, 33). The strength and the kinetics of
the swelling reaction correlate with the induction and recruitment of T
cells to the site of infection. Naive mice show a strong swelling
reaction on day 8 after infection, whereas LCMV-immune mice immediately
clear the virus and therefore fail to generate a strong swelling
reaction. To assess priming and homing of T cells in
op-/- mice, 100 PFU LCMV WE were
injected into the footpad of op-/-
and C57BL/6 control mice (Fig. 1
). While
C57BL/6 mice generated a strong swelling reaction on day 8,
op-/- mice failed to produce a
swelling reaction, suggesting that in
op-/- mice LCMV either did not
induce a T cell response or induced T cells did not migrate to the
footpad. To evaluate whether op-/-
mice generated LCMV-specific CTL responses measurable in vitro,
cytotoxicity assays were performed with spleen cells from
op-/- and C57BL/6 mice infected i.f.
with LCMV (Fig. 2A). The data
show that op-/- mice generated
slightly reduced but still strong CTL responses. Thus, the lack of a
footpad swelling reaction in op-/-
mice cannot be explained by the lack of CTL induction but may rather
reflect the failure of CTL homing to the footpad.
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A possible explanation for the homing deficiency of CTL into the
footpad is that virus spreads throughout all organs and recruits CTL
away from the site of infection. To assess this possibility, virus
titers in op-/- mice were determined
in different organs of mice infected i.f. with 100 PFU LCMV (Fig. 2
B). LCMV titers were 10- to 50-fold increased in thymus,
spleen, and liver of op-/- mice
compared with C57BL/6 controls, suggesting that
op-/- mice could not efficiently
control virus spread during the acute phase. This observation was even
more pronounced when 100 PFU LCMV were injected i.v., in which case
virus titers in op-/- mice were
100–10,000 times higher than in C57BL/6 controls: thymus
op, 2.68 x 106 ± 9.1 x
105 PFU; thymus B6, 1.1 x
104 ± 8.9 x 103 PFU;
liver op, 4.9 x 107 ± 2.6
x 106 PFU; liver B6, 3.1 x
103 ± 2.9 x 103 PFU;
spleen op, 2.5 x 107 ± 2.2
x 107 PFU; spleen B6, 1.2 x
104 ± 1 x 104 PFU.
Taken together, uncontrolled virus dissemination may lead to increased
Ag loads in peripheral organs and deviation of CTL into these
organs.
After LCMV infection op-/- mice developed a generalized immunopathology
Mice that cannot efficiently control virus spread show signs of generalized immunopathological disease. To evaluate whether op-/- mice eventually controlled LCMV infection, they were infected i.f. with LCMV and clinical symptoms of generalized immunopathology were monitored. In contrast to C57BL/6 mice, all LCMV-infected op-/- mice developed generalized immunopathology characterized by a hunched back, ruffled fur, and lethargy: 9 of 25 op-/- mice succumbed to the infection between days 12 and 18, and the remaining mice recovered completely from clinical symptoms by days 21–28. Thus, op-/- mice are immunocompromised and have difficulties controlling virus spread despite being able to generate significant primary CTL responses.
Impaired virus clearance in op-/- mice correlates with the lack of LCMV-specific CTL
To determine whether op-/-
mice, which recover from transient immunopathology, eliminated the
virus or became virus carriers due to CTL exhaustion
(34, 35, 36), a total of 11
op-/- mice were injected with LCMV
into the footpad, virus titers were determined in the organs between
days 21 and 28, and the presence of CTL was assessed in secondary in
vitro assays. Three of the op-/-
mice died with severe clinical symptoms of generalized immunopathology.
Of the remaining eight op-/- mice,
six mice failed to eliminate LCMV from all organs (Fig. 3A) and two mice completely
cleared the virus below detectable levels (data not shown). After in
vitro restimulation, LCMV-specific CTL could be recovered from the mice
that had eliminated the viruses (Fig. 3B), whereas no
LCMV-specific CTL could be recovered from the carrier mice (Fig. 3C). Thus, elimination of virus correlated with CTL
activity, whereas impaired virus clearance correlated with the absence
of LCMV-specific CTL.
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To evaluate the potential of
op-/- mice to control infection by a
cytopathic virus, 2 x 106 PFU VSV IND were
injected i.v. and neutralizing Ab titers were monitored. Fig. 4A shows that
op-/- mice mount normal neutralizing
IgM and IgG Ab responses. However, surprisingly, mice succumb to lethal
VSV infection between days 8 and 11 and VSV can readily be isolated
from the brain of op-/- mice but not
from control C57BL/6 mice (Fig. 4B, expt. 1).
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B, expt. 2). The data show that
op-/- mice infected with a low dose
of VSV controlled the infection and did not succumb to VSV-mediated
encephalitis. VSV Ag is efficiently recruited to the spleen
Recruitment of VSV by natural Abs to secondary lymphoid organs
plays a key role in controlling disease by reducing virus spread and
enhancing induction of an immune response (22, 38).
op-/- mice lack metallophilic MZM in
the spleen and subcapsular macrophages in the lymph nodes, and have
normal FDC populations (Fig. 5, upper panels). To investigate whether VSV was efficiently
recruited to the spleen, op-/- mice
were infected i.v. with 5 x 108 PFU VSV. No
difference in VSV titers was found in the spleens after 6 h of
infection. Titers were >2.1 x 108 PFU per
gram of organ in both op-/- and
control mice. Consistent with this, cryosections revealed that VSV Ag
was recruited to the spleen. However, while VSV Ag was predominantly
found in the marginal zone of C57BL/6 mice, VSV Ag could be found in
the red pulp and the white pulp of
op-/- mice. Moreover, the Ag largely
colocalized with FDC in op-/- mice
(Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early CTL and B cell responses against LCMV and VSV were largely unimpaired in op mice. The small reduction in the early day 8 cytotoxic activity of spleen cells against LCMV in op-/- mice is probably a consequence of virus dissemination and CTL extravasation into peripheral infected tissues rather than inefficient CTL priming. This view is consistent with the observation that op-/- mice failed to generate a footpad swelling reaction, probably due to CTL recruitment to many sites in addition to the infected footpad (40).
All op-/- mice went through a critical phase of generalized immunopathology, which one-third of the mice did not survive. In a small proportion of the op-/- mice an efficient CTL response dominated over virus spread, virus was cleared, and LCMV-specific CTL could be recovered. However, most of the mice failed to completely clear LCMV from all organs (virus persisted particularly in the kidney) and no LCMV-specific CTL could be recovered, indicating that CTL had been exhausted.
It has previously been observed that T cell-dependent LCMV clearance involves cooperation between T cells and probably macrophages (14). In addition, an earlier study by Seiler et al. (18) investigated the role of MZM in immunity against LCMV. The authors depleted MZM by administration of liposome-encapsulated clodronate (15, 16, 17) and reported that CTL responses were exhausted in all mice. There was no mention of generalized immunopathology, most likely because the treatment of mice with clodronate liposomes is stressful to the animal and clinical symptoms may have been masked due to this harsh treatment. In this work we show that three clinical outcomes are possible: 1) virus elimination accompanied by intact LCMV-specific CTL responses, 2) virus persistence and CTL exhaustion, or 3) death as a consequence of virus spread and immunopathological CTL response.
After infection with VSV, op-/- mice generated normal neutralizing (IgM plus IgG) and IgG responses; nevertheless, mice eventually succumbed to disease. Analysis of VSV titers in the brain revealed that virus spread could not be controlled in these mice and that VSV had disseminated to the brain. This was apparent with an infectious dose of 2 x 106 PFU of VSV; however, a 100-fold lower dose was controlled. Natural Abs and complement have been implicated in directing Ag to lymphoid organs to accelerate and enhance immune responses (22, 38, 41); however, the precise mechanism by which virus spread is inhibited and influenced by MZM is unknown. VSV is sensitive to type I IFNs and thus early IFN responses generated by MZM may account for this inhibition. In op-/- mice, VSV Ag was recruited into the white pulp of the spleen and colocalized with FDC, while in C57BL/6 mice Ag localized to the marginal zone. Thus, Ag recruitment was still possible in op-/- mice, but to a different site. Additional experiments will be required to identify by which receptors VSV Ag is captured on FDC. Nevertheless, it seems clear that the Ag absorbance and removal capacity of the FDC are not sufficient to prevent viral dissemination.
op-/- mice have been shown to generate normal MLR T cell responses and normal B cell responses in vivo against OVA (12). The present study is consistent with these findings. However, despite generating normal T and B cell responses, op-/- mice were susceptible to viral infection. This points to the importance of the architecture of lymphoid organs for controlling viral infections in vivo (42, 43, 44, 45). Crucial functions of structural components of lymphoid organs may not be uncovered if nonreplicating Ags are used. Although such experimental systems provide us with important information, they usually disclose rather a narrow window of the complex processes that are triggered by infections.
Taken together this study shows that the early innate immune system provides an important preparatory phase for the adaptive immune system: cooperation between the innate and the adaptive immune system shifts the balance between induction of an immune response and virus spread in favor of the immune response. This is particularly important for cytopathic viruses where virus spread usually leads to death, while non- or low-cytopathic viruses, depending on the relative balance between immune response and virus spread, can establish a virus carrier state without harming the host if extensive immunopathology is avoided.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephan Oehen at the current address: Cytos Biotechnology, Wagistrasse 25, 8952 Zurich, Switzerland. E-mail address: oehen{at}cytos.com
3 Current address: Infektiologie, University Hospital, Zürich, Switzerland.
4 Abbreviations used in this paper: op, osteopetrotic; VSV, vesicular stomatitis virus; LCMV, lymphocytic choriomeningitis virus; MZM, marginal zone macrophage; i.f., via the footpad; FDC, follicular dendritic cell.
Received for publication January 14, 2002. Accepted for publication May 30, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice. Nature 382:462.[Medline]
-deficient mice: a critical requirement for TNF in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184:1397.This article has been cited by other articles:
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  J. Louten, N. van Rooijen, and C. A. Biron Type 1 IFN Deficiency in the Absence of Normal Splenic Architecture during Lymphocytic Choriomeningitis Virus Infection. J. Immunol., September 1, 2006; 177(5): 3266 - 3272. [Abstract] [Full Text] [PDF]
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H. C. Probst and M. van den Broek Priming of CTLs by Lymphocytic Choriomeningitis Virus Depends on Dendritic Cells J. Immunol., April 1, 2005; 174(7): 3920 - 3924. [Abstract] [Full Text] [PDF]
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S. Camelo, A. Shanley, A. S. P. Voon, and P. G. McMenamin The Distribution of Antigen in Lymphoid Tissues following Its Injection into the Anterior Chamber of the Rat Eye J. Immunol., May 1, 2004; 172(9): 5388 - 5395. [Abstract] [Full Text] [PDF]
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P. Bjorck Dendritic Cells Exposed to Herpes Simplex Virus In Vivo Do Not Produce IFN-{alpha} after Rechallenge with Virus In Vitro and Exhibit Decreased T Cell Alloreactivity J. Immunol., May 1, 2004; 172(9): 5396 - 5404. [Abstract] [Full Text] [PDF]
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M. A. Nolte, R. Arens, M. Kraus, M. H. J. van Oers, G. Kraal, R. A. W. van Lier, and R. E. Mebius B Cells Are Crucial for Both Development and Maintenance of the Splenic Marginal Zone J. Immunol., March 15, 2004; 172(6): 3620 - 3627. [Abstract] [Full Text] [PDF]
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P. Aichele, J. Zinke, L. Grode, R. A. Schwendener, S. H. E. Kaufmann, and P. Seiler Macrophages of the Splenic Marginal Zone Are Essential for Trapping of Blood-Borne Particulate Antigen but Dispensable for Induction of Specific T Cell Responses J. Immunol., August 1, 2003; 171(3): 1148 - 1155. [Abstract] [Full Text] [PDF]
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