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Journal of Virology, November 2001, p. 10467-10471, Vol. 75, No. 21
Laboratory for Clinical and Molecular
Virology, The University of Edinburgh, Edinburgh EH9 1QH, United
Kingdom
Received 29 May 2001/Accepted 26 July 2001
Tachykinins function not only as neurotransmitters but also as
immunological mediators. We used infection of tachykinin-deficient (PPT-A The tachykinin family of
neurotransmitters not only is involved in both central and peripheral
nervous system function but also has a role in inflammation (termed
neurogenic inflammation) and adaptive immunity (9, 16).
The most characterized member of the family is substance P (SP). This
is encoded by the preprotachykinin A (PPT-A) locus.
Alternative splicing of PPT-A RNA and processing of the
propeptide yields, in addition to SP, neurokinin A and neuropeptides K
and The demonstration that most immunocytes producing SP also express its
receptor led to the hypothesis that SP not only acts as a mediator of
the cross talk between the nervous and immune systems but also is
biologically involved in the direct interaction between immune cells in
a paracrine and/or autocrine fashion, independently of sensory nerves
or neurogenic inflammation (10, 11).
A number of studies have shown that viruses (e.g., respiratory
syncytial virus) can induce SP and neurogenic inflammation, particularly in the lungs, in the context of a respiratory challenge (19, 26). However, there are no published data on the
influence of SP in combating virus infection. The aim of this study was to assess the role of tachykinins in the host response to virus infection using murine gammaherpesvirus infection of mice as a model system.
Infection of laboratory mice by murine gammaherpesvirus 68 (MHV-68;
International Committee on Taxonomy of Viruses name, murid herpesvirus
4) (30) is a well-characterized system for the study of
gammaherpesvirus pathogenesis and, via the use of transgenic mice, for
the study of host components important in combating infection (for
reviews, see references 17 and 20).
Intranasal inoculation of mice with MHV-68 results in a productive
infection in the lungs (23) that is resolved at about day
10 postinfection (p.i.) by the action of CD8+ T
cells (7). The virus then persists in a latent form in
epithelial cells at this site (21). MHV-68 spreads to the
spleen during the subsequent viremia, where it becomes latent in B
lymphocytes, macrophages, and dendritic cells (8, 24, 29).
Establishment of latency in the spleen is associated with marked
splenomegaly and mononucleosis that resembles that caused by primary
infection of humans by Epstein-Barr virus (25).
Splenomegaly is driven by CD4+ T cells (7,
27) and is dependent on the presence of MHV-68-infected B cells
in the spleen (29, 31). The resolution of splenomegaly is
achieved by CD8+ T cells (7), which
are also important in the long-term control of persistent infection
(4, 21).
To elucidate the part played by tachykinins after MHV-68 infection, we
used transgenic mice with lesions in the PPT-A locus (PPT-A Infectious virus is recoverable from
PPT-A
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10467-10471.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Role of Tachykinins in the Host Response to Murine
Gammaherpesvirus Infection
ABSTRACT
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Abstract
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References
/) mice and wild-type controls
with murine gammaherpesvirus to assess the role of tachykinins in the
host response to a virus infection. Although infection was ultimately
controlled in PPT-A/ mice, there were
higher titers of infectious virus in the lungs, accompanied by a more
rapid influx of inflammatory cells. Clearance of latently infected
cells from the spleen was also delayed. This is the first report of the
direct influence of tachykinins in the host response to a virus infection.
TEXT
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Abstract
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References
. A major physiological source of SP is primary sensory neurons,
whose cell bodies in the dorsal root ganglia produce SP and transport
it to peripheral and central sites, where it is stored and later
released from nerve endings. However, cells of the immune system such
as T cells, monocytes-macrophages, and dendritic cells can also produce
SP (12). The effects of SP are mediated by signaling
through the G protein-coupled receptor NK-1 or NK-2 (13).
A number of cell types involved in inflammation and immunity express
the NK-1 receptor, including T lymphocytes, B lymphocytes, monocytes,
macrophages, neutrophils, and mast cells (10).
Consequently, SP can enhance immunoglobulin production by B cells,
stimulate mitogen-induced proliferation of lymphocytes, and induce the
production of several important inflammatory cytokines, including
interleukin 1 (IL-1), IL-2, IL-6, gamma interferon (IFN-), and tumor
necrosis factor alpha (for a review, see reference 18).
/) such that coding sequences for
SP and neurokinin A were both deleted (3). It has been
shown that these mice express none of the tachykinins encoded by
PPT-A. Aside from modulation of several nociceptive
parameters, these mice have no obvious deleterious phenotypic
abnormalities and have been used to address the role of tachykinins in
other neuronal challenge models (1, 14). The genetic
background for these mice is CD1. There are no data on the course of
MHV-68 infection in this strain. However, with minor variations, the
typical course of infection described above has been observed in all
strains tested to date (17). Wild-type (wt) CD1 mice were
therefore used as controls and were purchased from Harlan United
Kingdom (Bicester, England). MHV-68 (strain g2.4) was grown and titers
were determined on BHK-21 cells, and the virus was used to infect mice
exactly as described previously (23). Groups of
PPT-A/ and wt mice were infected
intranasally with 4 × 105 PFU of MHV-68 at
4 to 5 weeks of age. The mice were then monitored over a period of 79 days p.i. for clinical signs, histopathological changes, and the levels
of infectious and latent viruses in the lungs and spleens. The whole
experiment was performed twice, with comparable results.
/ but not wt mouse lungs.
To
assess the extent of virus replication in lungs, groups of four mice
were euthanized by CO2 asphyxiation at days 3, 5, 7, 10, and 14 p.i. The amount of infectious virus was then
measured by plaque titration (23). No infectious
virus was detected in the lungs of wt mice at any time p.i. In two
separate experiments, means of 2.3 and 2.5 log10
PFU per mouse were observed in the lungs of
PPT-A/ mice, but only at day 7 p.i.; actual values were 1.6, 3, 2.7, and 2 log10
PFU in experiment 1 and 2.4, 2, 2.9, and 2.6 log10 PFU in experiment 2.
/ mice at
day 7 p.i. indicates that tachykinins play a role in the host
control of virus infection in the lungs.
Splenomegaly is unaffected, but latently infected cells are cleared
less efficiently in PPT-A/ mice.
To
investigate changes in the spleen following infection, samples were
obtained from groups of four mice at 10, 14, 21, 28, 38, and 79 days
p.i. The number of cells present in the spleen was ascertained, and the
amount of latent virus present in the spleen was measured by using an
infective-center (i.c.) assay as described previously
(15). As shown in Fig 1A,
there was a characteristic transient rise in the numbers of splenocytes (splenomegaly) in both wt and PPT-A/
mice that peaked at day 14 p.i. However, there was no significant difference between the spleen cell numbers in the two groups of mice,
as determined by two-way analysis of variance (ANOVA)
(P = 0.18). Figure 1B shows that concomitant with
splenomegaly, there was a rise in the i.c. numbers in the spleens of
both wt and PPT-A/ animals. In the wt
mice, the increase peaked at day 14 p.i. and rapidly resolved to a
near-baseline level by day 21 p.i. In the PPT-A/ mice, the i.c. numbers increased
to a level similar to that in wt mice up to day 14 p.i. However,
after this time, the i.c. numbers in
PPT-A/ mice remained at the same level
through 21 days p.i. and then decreased less rapidly to a level that
was still higher than that in wt mice at day 79 p.i. The higher
i.c. level in the PPT-A/ mice than in
the wt mice was statistically significant at days 21 and 28 p.i.
(P = <0.001 and P = <0.01,
respectively), as determined by two-way ANOVA with Bonferroni
posttests. Similar results were obtained in a repeat of this
experiment, with the higher i.c. levels in
PPT-A/ mice being statistically significant
at days 21 and 28 p.i. (data not shown).
|
) or
PPT-A
) mice were infected
intranasally with 4 × 105 PFU of MHV-68 and
euthanatized at the indicated days p.i. (A). Mean total numbers of
splenocytes and standard error for four mice per group are shown for
each time point. (B) Latent virus in the spleens, as determined by an
i.c. assay. Infectious virus was not seen in the spleens in this
experiment. Mean i.c. numbers per spleen and standard error for four
mice per group are shown for each time point. Statistically significant
differences between points on the curves were determined by two-way
ANOVA with Bonferroni posttests and are indicated by asterisks above
the relevant points.
/ mice,
MHV-68 trafficked to the spleen and was associated with a rise in
latently infected splenocytes that was entirely typical of that seen in
other strains of mice. However, the observed delay in the i.c. decline
in PPT-A/ mice indicates that
tachykinins play a role in the host response to control cells that are
latently infected with MHV-68 in the spleen.
Pathological changes in lungs following MHV-68 infection.
Although small, there were consistently repeatable differences between
the levels of virus in the lungs of wt and
PPT-A/ mice. Also, the lung is an area
where SP is known to influence host responses to virus infection
(19, 26). As a preliminary step in assessing the influence
of tachykinins in the response to MHV-68 in the lungs, we performed a
sequential histopathological study. Groups of four mice were
euthanatized at 7 and 14 days p.i. and perfused through the left
ventricle with phosphate-buffered saline followed by Formol saline.
Sections cut from paraffin-embedded blocks were then stained with
hematoxylin and eosin and examined by microscopy. The results are shown
in Fig. 2. In wt mice at day 7 p.i.
there were mild lymphoid cell infiltrates around blood vessels, in
alveolar spaces, and in alveolar septa. These findings are
typical of the response seen at this time after MHV-68 infection (15, 22). In PPT-A/ mice,
however, there was unusually severe inflammation of lung parenchyma
consisting of more florid alveolar exudate and greater thickening of
alveolar septa by lymphocytes than were seen in wt mice. At day 14 p.i., this parenchymal inflammation had largely resolved in
PPT-A/ mice, with the persistence of
perivascular lymphoid infiltrates and small numbers of lymphocytes in
thickened alveolar septa. In contrast, in the wt mice at the same time
p.i., there were extensive intra-alveolar exudate of both macrophages
and lymphocytes and extensive interstitial fibrosis. In addition, there
was also evidence of emphysema, indicating some breakdown of
architecture. Fibrosis was confirmed by the presence of collagen
fibrils when serial sections were stained with Masson trichrome.
No such staining was observed in sections derived from
PPT-A/ mice.
|
receptor-deficient mice
(6). Taken together with the unusually low level of
infectious virus recovered from the lungs, the pathological findings
are suggestive of a vigorous host response that is reactive to MHV-68
infection in this strain, resulting in highly effective control of
virus replication but also in tissue damage and inappropriate repair
(fibrosis). This first report of infection of CD1 mice with MHV-68
indicates that it may be a good mouse model for future studies of the
treatment of virus-associated pulmonary fibrosis.
The kinetics of the appearance of reactive inflammatory cells in
PPT-A/ mice were far more rapid than
those in wt mice, but the control of MHV-68 productive replication was
less effective. There could be a number of possible reasons for these
findings. First, tachykinins could be involved in a host response that
does not involve inflammatory cells (e.g., interferon or apoptosis). In
this context, it is interesting to note the role of SP as a regulatory
molecule upstream of the well-characterized apoptosis molecules bax and
caspase 3 in status epilepticus (14). Second, tachykinins
are known to be involved in the recruitment of inflammatory cells
(2); this could be a highly effective strategy for the
control of MHV-68 infection. Finally, tachykinins could be involved in
the effective function of recruited inflammatory cells (e.g., secretion
of IFN- or tumor necrosis factor alpha). In each instance, the
greater and more rapid recruitment of inflammatory cells to the site of infection seen in the PPT-A/ mice could
occur to compensate for the deficit.
In spite of the unusually low level of infectious virus in CD1 mice,
MHV-68 caused splenomegaly and established latency with an efficiency
comparable to that seen in other strains, such as BALB/c and C57BL/6.
The factors controlling the number of latently infected cells in the
spleens of mice have not been fully delineated. However, while the host
response early during infection in the lungs is largely innate
(5), a major contributory factor to the response in the
spleen is known to be adaptive, i.e., virus antigen-specific
CD8+ T cells (7, 28). The delay in
the clearance of latently infected cells in
PPT-A/ mice relative to wt mice
suggests that tachykinins could be involved in this response.
Although other studies have shown that tachykinins are produced in
response to virus infection (19, 26), this study is the
first to show directly that tachykinins play a role in the host
response to control a virus infection. However, these peptides could
function in a number of ways and at a number of sites during infection.
Since the completion of this study, tachyknin-deficient mice in a
129Sv/Ev background have become available. Since productive infection
in 129 mice is more akin to the pattern seen in other strains, these
mice may be more amenable for use in future studies of tachykinin
function. The delineation of the precise function of these molecules in
this context therefore awaits the results of detailed immunological
studies using these mice along with NK-1 receptor-deficient transgenic strains.
| |
ACKNOWLEDGMENTS |
|---|
James P. Stewart and John P. Quinn made equal contributions to the manuscript.
This work was supported by The Royal Society (London), the Biotechnology and Biological Sciences Research Council, and the Medical Research Council.
We thank Alan Basbaum, University of California, San Francisco, for the
generous gift of the PPT-A/ mice. J.P.S.
is a Royal Society university research fellow.
| |
FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall, Edinburgh EH9 1QH, United Kingdom. Phone: 44 131 650 7939. Fax: 44 131 650 6511. E-mail: james.stewart{at}ed.ac.uk.
Present address: Department of Pathology, The University of
Edinburgh, Edinburgh EH8 9AG, United Kingdom.
Present address: Physiological Laboratory and Department of Human
Anatomy and Cell Biology, University of Liverpool, Liverpool L69 3BX,
United Kingdom.
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Journal of Virology, November 2001, p. 10467-10471, Vol. 75, No. 21
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.21.10467-10471.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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