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Regular Articles |
From the Department of Pathology and Laboratory
Medicine*
and Departments of Pediatrics and
Nutrition,
University of North Carolina School
of Medicine, Chapel Hill, North Carolina; and the Section of Molecular
Genetics and Microbiology and Institute for Cellular and Molecular
Biology,
University of Texas, Austin, Texas
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The influx of mononuclear inflammatory cells into infected host tissues has long been recognized as the hallmark of the host defense mechanism against viral infection.13,14 This cell-mediated immunity is the predominant mechanism for containment and recovery from primary viral infections. Consequently, patients who are immunocompromised, including those who are elderly or HIV-infected, are more susceptible to the extreme manifestations of viral infection.15 In general, the pathogenesis of lytic viral infections, such as influenza infections, can be divided into two phases, the cellular events that precede T cell invasion and those that follow it. The initial attack of influenza A on respiratory epithelial cells causes the extravasation of small numbers of blood-derived neutrophils, followed by larger numbers of blood monocytes/macrophages into the infected lung. During this period, viral replication continues in the epithelial cells and infection spreads to the collecting macrophages.16-18 These processes alone are not able to clear the virus from the lung. By day 7, CD8+ cytotoxic T cells (CTLs) from nearby mediastinal lymph nodes (MLNs) begin to accumulate in the infected lungs and the efficient process of T-cell mediated viral clearance begins.15,16,19 Although this cell-mediated mechanism is required for host protection and recovery, the overwhelming inflammatory cell accumulation can be harmful to the infected host by aggravating clinical symptoms and contributing to potentially lethal lung pathology.20,21 Although it is still unclear as to which inflammatory proteins mediate influenza A-induced leukocyte recruitment, virologists have recently begun to focus on the potential role of chemokines in this process.14,22
A number of in vitro studies have associated influenza A
infection with increased monocyte and epithelial cell expression of
several CC chemokines, including RANTES,17,23,24
MIP-1
,
and MCP-1,17,24,25
and the CXC chemokine,
IP-10.25
Additionally, MIP-1-deficient mice infected
with the influenza A virus displayed a marked reduction in
virus-induced pneumonitis, but a significant increase in viral titers
at days 6 and 7 postinfection.22
This work implicated
MIP-1 as a major T cell chemoattractant functioning in the lungs of
influenza-infected mice. Although it is known that macrophages are
responsible for the production of inflammatory mediators (including
MIP-1) that regulate the T cell response to influenza A virus
infection,26
it is not known which, if any, chemokines
promote the early activation and migration of these macrophages to
infected lungs.
In recent years insight into the pathogenesis of influenza A has been
gained through the use of murine transgenic and gene knockout animal
models.21,22,27-31
In this report, we have explored the
roles of two chemokine receptors, CCR5 and CCR2, in influenza A-induced
pulmonary macrophage recruitment. CCR2-deficient mice, in particular,
as well as MCP-1-deficient mice have been previously reported to have
significant defects in macrophage recruitment.32-35
In
these studies, we infected mice deficient in either CCR5, a receptor
for MIP-1, MIP-1ß, and RANTES, or CCR2, the primary receptor for
MCP-1, with a mouse-adapted influenza A virus (PR/8/34). We examined
the pathological manifestations of the virus as well as events
occurring simultaneously in the regional lymph nodes. We found that
CCR5 deficiency leads to a hyperacute inflammatory response that is
often fatal, whereas CCR2 deficiency leads to a milder inflammatory
response with reduced lung pathology and increased survival rates.
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Materials and Methods |
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CCR2-/-,
MIP-1-/-, and +/+ control mice were
generated and maintained as previously described22,32
and
were on mixed 129/Ola X C57BL/6J genetic background. Wild-type control
mice carry the CCR gene cluster derived from the 129/Ola strain.
CCR2-/- mice were bred with
MIP-1-/- mice to obtain mice that were
heterozygous for both mutations
(CCR2+/-/MIP-1+/-).
The double heterozygotes were then intercrossed to generate mice which
were doubly deficient
(CCR2-/-/MIP-1-/-).
CCR2 and MIP-1 genotypes were determined by methods that have been
described previously.21,32
CCR5-/-
mice were generated by targeted replacement of the entire CCR5
coding region with a neomycin-resistance gene in embryonic stem cells.
Correctly targeted embryonic stem cells were selected and male chimeras
were generated and mated to C57BL/6J females. F1 heterozygotes were
mated to obtain homozygous CCR5-/- mice (WA
Kuziel, TC Dawson, RL Reddick, and N Maeda, manuscript in
preparation). All experimental mice were bred and maintained in
pathogen-free conditions until viral inoculation. All animal
experiments were done in accordance with National Institutes of Health
guidelines and protocols approved by the Animal Care and Use Committee
at the University of North Carolina at Chapel Hill.
Virus Infection and Quantitation
Influenza A/Puerto Rico/8/34 (A/PR/8/34) virus grown in the allantoic cavity of 10-day-old embryonated hen eggs was a gift from Dr. John F. Sheridan, Ohio State University. At 6 to 10 weeks of age, mice were anesthetized with a ketamine-xylazine solution, and intranasally infected with 5 hemeagglutinating units (HAU) of the influenza A/PR/8/34 stock. Virus was isolated from the lungs of infected mice 5 days postinfection by first weighing, then grinding the tissue into minimal essential medium, followed by a series of freeze/thaw cycles and high-speed centrifugation of the ground tissue. The supernatant was collected and 10-fold serial dilutions of 0.1-ml aliquots were added in replicates of six to 96-well plates of confluent Madin Darby canine kidney cells. Plates were incubated for 24 hours at 37°C. Viral titers (expressed as TCID50) were determined as previously described.36 Individual group titers were compared to the control titers with unpaired Student’s t-tests.
Histological Analysis of the Lungs
At days 2, 3, 5, 16, and 25 postinfection, animals were sacrificed and one lung was resected, inflated, and embedded in freezing embedding medium (Tissue-Tek, Torrance, CA), then snap-frozen in isopentane cooled with liquid nitrogen. Frozen serial sections from all animals were stained with hematoxylin and eosin (H&E) and examined microscopically. Each specimen was assigned a score of 0 to 4+ based on the severity of lung pathology.22 Additional cryosections selected for immunohistochemical study were acetone-fixed and incubated with an antibody against one of several mouse leukocyte markers. Sections were incubated with a series of peroxidase-labeled secondary antibodies, developed with 9-amino-3-ethylene-carbazole, and counterstained with Mayer’s hematoxylin (Sigma Diagnostics, St. Louis, MO). Primary antibodies that were used included: a mouse macrophage/monocyte marker, MOMA-2 (Serotec, Oxford, UK); a mouse neutrophil marker, NIMPR40 (kindly received from Dr. Peter Heeringa, University of North Carolina, Chapel Hill); and lymphocyte markers CD8a, CD4, and CD3e (Pharmingen, San Diego, CA).
RNase Protection Assay
Total RNA from lungs and MLNs of uninfected mice (day 0) and infected mice at days 2 and 3 postinfection was prepared using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY). Chemokine mRNA levels were determined using the RiboQuant Multipurpose Ribonuclease Protection Assay (RPA) System with the mCK-5 probe set (Pharmingen, San Diego, CA). The dried gel was exposed to X-ray film and developed for 24 hours at -70°C. Bands were detected and densitometrically quantitated using RiboQuant software. All chemokine values were normalized to the housekeeping gene Gapdh. Each data point represents a sample of pooled RNA from two individual animals. Group values were compared independently to control values using unpaired Student’s t-tests.
Flow Cytometry and Cytospin Preparations
Cell suspensions from the MLNs and bronchoalveolar lavage (BAL) fluid of infected mice were obtained 5 days postinfection and pooled by genotype. Cells were stained with the following anti-mouse mAbs: PE anti-CD3, FITC anti-CD4, or FITC anti-CD8 (Pharmingen, San Diego, CA). After staining, cells were sorted and counted by FACS analysis on a FACScan machine using LYSYS II, Version 1.1 software (Becton Dickinson, San Jose, CA). Pooled BAL cells which were not used for FACS analysis were spun for 7 minutes at 1,000 rpm onto serum-coated slides using a cytocentrifuge (StatSpin; Shandon Scientific, Runcorn, UK). After Giemsa-Wright staining, leukocyte differentials were determined by averaging three counts of at least 250 cells each.
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Results |
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To asses the impact of CCR5 and CCR2 on susceptibility to
influenza A virus infection, age-matched control (+/+),
CCR5-/-, CCR2-/-, and
CCR2-/-/MIP-1-/-
(doubly-deficient) mice were inoculated intranasally with 5 HAU of
mouse-adapted influenza A/PR/8/34. As shown in Figure 1A
, at this dose, 30% of control mice
succumbed to the virus by day 10 postinfection. However, CCR5
deficiency resulted in a sharp increase in mortality early in the
course of infection; 42% of all CCR5-/- mice
died by day 6. CCR5-/- mice that survived this
early death recovered and lived well beyond day 16 postinfection. In
striking contrast to these results, CCR2-deficient mice were
substantially less susceptible to death from influenza A infection. As
a group, these animals exhibited only 10% mortality through day 16
postinfection. A strain of mice deficient in both CCR2 and MIP-1 was
the most protected, exhibiting no mortality during the observation
period for this experiment. Because all of the animals used are hybrids
between 129/Ola and C57BL/6 strains, we cannot rule out completely a
possibility that nonselected alleles have been sorted differently in
each group. However, considering that the loci unlinked to CCR5-CCR2
are equally randomized and that the wild-type controls carry CCR5-CCR2
loci from 129/Ola strain, it is unlikely that the random sorting of
alleles can account for the dramatic differences in pneumonitis
observed in these groups of animals.
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. Although
some of the CCR5-/- mice died prematurely,
there was no significant difference in the viral load harbored by the
surviving CCR5-/- mice when compared to the
control mice. In contrast, CCR2-deficient animals, which exhibited an
increased survival rate, had 10- to 100-fold higher viral titers than
the +/+ controls. Previous results obtained by Cook et
al,22
showed that deletion of the MIP-1 gene leads to a
similar increase in viral titers after influenza virus infection. Our
data using the
CCR2-/-/MIP-1-/-
double-knockout mice confirm this phenotype and seem to indicate that
the additional deletion of CCR2 exacerbates this effect by causing even
higher viral titers at this time point. These results suggest that the
absence of both CCR2 and MIP-1 may independently cause delays in the
migration of key leukocyte populations after influenza infection, which
may subsequently cause a delay in viral clearance. In addition, our
results along with those from Cook et al,22
show that
influenza-induced mortality does not directly correlate with increases
in virus replication. Altered Severity of Pulmonary Pathology in the Absence of CCR2 and CCR5
The above results confirm that the sometimes fatal, downstream
pathological events that occur after influenza infection are clearly
not because of an overwhelming viral burden in the lungs. This implies
that the harsh pulmonary pathology may be caused by the responding
inflammatory cells rather than by the cytopathic effects of the virus
itself. Indeed, the early increase in mortality seen in the surviving
CCR5-/- mice was associated with unusually
massive leukocyte accumulations and severe pulmonary tissue damage as
early as day 2 postinfection (Figure 2C)
,
compared to relatively mild inflammation in the control animals (Figure 2A)
. Likewise, the protective effect of the CCR2 deficiency as seen in
the survival study was confirmed by histological examination of the
intact lungs from infected mice. At days 3 and 5 postinfection,
CCR2-/- mice had a marked reduction in
pulmonary inflammatory cell infiltration (Figure 3, E and F)
, compared to control mice
(Figure 3, A and B)
. Additionally, the thickening of the septal walls,
disarray/disorganization of the bronchial epithelial cells, and the
areas of consolidation that were present in control lungs at day 5
postinfection (Figure 3B)
were noticeably absent in
CCR2-/- mice at day 5 postinfection (Figure 3F)
. However, an extended time course revealed that
CCR2-/- animals eventually developed some of
these histopathological characteristics. When histological sections
from all groups for five time points were scored 0 to 4+ based on the
amount of inflammation and the severity of tissue damage, the
histological scores of some of the CCR2-/- mice
at day 16 were as high as those of CCR5-/- mice
at day 2 (Figure 4)
. However, no deaths
occurred in the CCR2-/- animals, suggesting
that cell populations involved may be different in these two groups of
mice although the histological scores look similar. All surviving
animals were able to recover from the infection by day 25
postinfection. These results indicate that CCR5 deficiency accelerates
the onset of the pathological manifestations of influenza A virus,
whereas CCR2 deficiency causes a delay in the onset of the normal
pathological manifestations of influenza A virus.
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To determine which leukocyte subpopulation was prematurely
responding to the influenza A virus infection in
CCR5-/- mice, immunohistochemical analysis was
performed. As expected at day 2 postinfection, both control mice and
CCR2-/- mice had NIP/R40-positive neutrophils
present in the lungs (data not shown). However, macrophages were the
predominant cell type infiltrating at day 2 postinfection in
CCR5-/- lungs, as illustrated by an intense
positive MOMA-2 staining (Figure 2D)
compared to light staining in the
control lungs (Figure 2B)
. Neither CD4+ T cells, nor CD8+ T cells were
present at day 2 postinfection in either the +/+,
CCR2-/-, or CCR5-/-
mice (data not shown).
We and others have previously reported that mice deficient in either
MCP-1 or CCR2 have pronounced defects in macrophage function when
immunologically challenged.32-35
This also proved to be
true of CCR2-/- mice when challenged with the
influenza A virus. Although the acute neutrophilic response was
unaltered in the CCR2 knockout mice, these mice failed to mount the
expected mononuclear attack that begins with infiltration of
macrophages by day 3 postinfection in control mice. In fact, even at
day 5 postinfection, the few identifiable leukocytes that were present
in CCR2-/- lungs were still neutrophils (Figure 3G)
. This was confirmed by a cytological preparation of bronchoalveolar
lavage (BAL) cells 5 days postinfection, which clearly shows that
polymorphonuclear leukocytes (neutrophils) continue to be the
dominating cell type at this time point in CCR2-deficient mice (Figure 3H)
. When the BAL differentials were counted, only 10 ± 0.7% of
the BAL cells in CCR2-/- mice were
monocytes/macrophages, compared with 60 ± 1.5% in control mice
at the same time point (Figure 5)
. These
experiments illustrate that the delay in the pathogenesis of influenza
in the CCR2-/- mice is caused by their
inability to properly recruit macrophages during an inflammatory
attack.
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Although the defective macrophage recruitment in
CCR2-/- mice was not surprising, the hyperacute
macrophage accumulation in the CCR5-/- animals
was unexpected. To investigate the mechanism of this aberrant response,
we examined pulmonary chemokine expression levels of several relevant
CC chemokine genes after influenza virus infection. Image densitometry
of an RNase protection assay revealed a threefold increase in RANTES
RNA expression at day 2 postinfection in
CCR5-/- mice compared to control animals or
CCR2-deficient mice (Figure 6A)
. Excess
RANTES might attract additional macrophages through binding to its
alternate receptor, CCR1, on these cells. IP-10 expression was
increased in all animals with slightly more increase in the
CCR5-/- mice, which could also be contributing
to the macrophage response by their binding to other, intact receptors.
More likely, however, the excess macrophage accumulation in
CCR5-/- mice results from increased expression
of MCP-1, a key regulator of induced macrophage migration. Both the
CCR5-/- and CCR2-/-
animals exhibited a twofold increase in day 2 postinfection pulmonary
MCP-1 RNA levels relative to the levels in control mice (Figure 6A)
. As
would be expected, the overexpression of MCP-1 correlates with an
increased migration of macrophages in CCR5-/-
mice, but not in mice lacking CCR2, the major receptor for MCP-1. There
were no significant differences in eotaxin, MIP-1, MIP-1ß, MIP-2,
or TCA-3 levels in the CCR2-/- or
CCR5-/- mice when compared to controls.
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The cellularity of MLNs of mice infected with influenza A virus
increases rapidly, with maximal total cell counts reached by day 5
postinfection. All major lymphocyte subsets are involved in this
expansion, including CD4+ and CD8+ T
cells.37
At day 5 postinfection, ~23% of total MLN
cells in control mice were CD8+ (consistent with previous reports).
These numbers were modestly reduced in the MLNs of
CCR2-/- mice to ~19% CD8+ T cells (Table 1)
. Additionally, the percentage of total
(CD3+) T cells was substantially reduced in the
CCR2-/- MLNs (37% versus 53% in
+/+). As shown in Figure 6B
, this decrease in T cell numbers in MLNs of
CCR2-/- mice correlates with a decrease in MLN
expression of RANTES. These data imply that, relative to control MLNs,
MLNs of CCR2-/- mice are less activated, and
that fewer CTLs are homing to or dividing in the MLNs of these animals.
Subsequently, there will be fewer primed CD8+ T cells to localize to
the site of virus growth. In fact, FACS analysis did reveal a marked
reduction in the percentage of CD8+ T cells in the BAL fluid of
CCR2-/- mice compared to controls (data not
shown). This complements the results of immunohistochemical analyses
that indicated an absence of CD3+ or CD8+ T cells in the intact lungs
of CCR2-/- mice at day 5 postinfection (data
not shown). Altogether, these experiments demonstrate that proper
signaling through CCR2 is necessary for lymphocyte accumulation in
secondary lymphoid organs and for the subsequent trafficking to
influenza infected tissues.
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Discussion |
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. Third, the virus-infected
CCR2-/- mice have increased survival rates
compared to controls, in the presence of an early block in pulmonary
macrophage accumulation and an increased viral load. All of these
results highlight the importance of macrophages in generating an
appropriate immune response to influenza infection and in the
development of associated lung pathology. Macrophages may both directly
and indirectly contribute to influenza associated lung pathology. A
direct contribution of macrophages to influenza-induced lung pathology
is illustrated by their ability to express inducible nitric-oxide
synthase (NOS2) after becoming infected.38
This causes
macrophage secretion of potentially injurious inducible nitric-oxide
synthase-derived nitric oxide metabolites. Deletion of the inducible
nitric-oxide synthase gene confers protection against influenza-induced
lethal lung pathology, without altering CTL activity.28
Macrophages can also indirectly control the progression of influenza
pathogenesis by regulating T cell response. Just before the induction
of apoptotic cell death,17,39
infected macrophages secrete
cytokines such as TNF, IL-1, IL-6, and M-CSF, and chemokines such as
MIP-1, RANTES, and IP-10 all of which could potentially contribute
to lung pathology by inducing the activation and migration of CTLs to
the site of an influenza infection.17,21,26,40-42
Thus,
the macrophage response to the influenza virus leads to the rapid onset
of a proinflammatory signaling cascade and enhanced activation and
immigration of lymphocytes into the lung. Once present in the lung,
these lymphocytes, specifically CTLs, can recognize and directly lyse
virally infected cells, thereby eliminating the virus and initiating
recovery of the tissue.43 Inflammation in response to primary influenza virus infection not only involves macrophages and CTLs, but also involves chemokine-dependent trafficking of dendritic cells and natural killer cells in-between the site of infection, the circulation, and the draining lymph nodes. CC chemokine receptors CCR5 and CCR2 are expressed on all of these cell types,41,44 and several recent reports describe the production of various CCR5 and CCR2 ligands by mononuclear and epithelial cells after being infected by influenza A virus.17,23,24,45 Thus, CCR5 and/or CCR2 could potentially modulate both the afferent and efferent phases of the immune response to influenza virus infection.
In our studies, we show that CCR5 is not absolutely required for
macrophage recruitment into influenza-infected lungs or for control of
viral replication. CCR5 deficiency does, however, lead to a more
negative clinical outcome relative to controls, with approximately half
of all influenza-infected CCR5-deficient mice dying prematurely as a
result of early, overwhelming macrophage accumulation in the lungs.
Although not proven in our experiments, this accelerated macrophage
accumulation is likely to be linked to enhanced expression of MCP-1 and
RANTES (Figure 6A)
, and possibly of IP-10, and their binding to other
intact chemokine receptors on the CCR5-deficient macrophages. In other
experiments, we have also observed enhanced inflammatory cell
recruitment in the lungs of CCR5-/- mice after
primary infection with Cryptococcus
neoformans.46
The relationship between the
absence of CCR5 signaling and increased chemokine expression is not
clear at this time, but alterations in leukocyte development or
trafficking patterns directly related to CCR5 deficiency may be
involved.
The enhanced lung inflammation and pathology of
CCR5-/- mice stands in sharp contrast to the
reduced inflammation and pathology observed in mice deficient in
MIP-1, a CCR5 ligand.22
These results together suggest
that during influenza infections, MIP-1 primarily functions through
another of its known receptors (CCR1, CCR4), and that its interactions
with CCR5 have limited, if any, importance for the pulmonary
mononuclear response to influenza virus infection. These observations
underscore the complexity of the chemokine system and the difficulty of
relating the interplay of chemokines and their receptors in
vitro, with leukocyte function and biological consequences
in vivo.
It has been well established that the loss of functional CCR2 does not alter blood monocyte levels, but severely impairs the ability of blood monocytes/macrophages to respond to certain types of injurious stimuli or specific pathogenic agents.7,32-34 Our studies implicate influenza A virus as yet another such pathogen whereby mononuclear leukocyte activation and migration is mediated by a CCR2/MCP-1-dependent mechanism. Despite increased levels of MCP-1 RNA in the lungs of infected CCR2-/- mice, there is substantially reduced pulmonary macrophage accumulation in these animals. This correlates with reduced mortality even though the viral titers were 10- to 100-fold higher than in control or CCR5-/- mice at an early time point. These results suggest that the pathological manifestations of influenza infections are caused by the massive accumulation of inflammatory cells and not by the intrinsic cytopathic effects of the virus itself. Consistent with our previous experiments showing that the CCR2-deficient mice have enhanced early accumulation and delayed clearance of neutrophils in response to intraperitoneal thioglycollate injection,32 neutrophils persisted in the influenza virus infected lung of the CCR2-/- mice. The delay in macrophage response resulting in reduced phagocytosis is at least partly responsible for the prolonged presence of neutrophils in lungs of the CCR2-/- mice. It is also likely that the neutrophilic persistence seen at later time points in our flu experiments are because of the consistent migration of new neutrophils into the lung. No evidence of defective apoptosis of neutrophils has been demonstrated in CCR2-/- mice.
Our data with the CCR2-deficient mice further show that the delay in
the macrophage response leads to a subsequent delay in CTL development
and/or migration to the infected lungs. This delay in T cell response
is likely responsible for the increased viral load 5 days
postinfection. It has been shown that CCR2 expression is inducible on T
lymphocytes,47-48
and T lymphocytes can respond to
MCP-1 at high levels in vitro.49
Thus, the
apparent lack of T cell involvement in the lungs of
CCR2-/- mice could, at least in part, be
directly related to the absence of CCR2 from circulating T cells.
However, this is unlikely to account for the majority of the delays in
leukocyte accumulation in the CCR2-/- mice
because the delays are seen at very early time points, when monocytes,
not T cells, are widely regarded as the primary responders to induced
MCP-1 expression in vivo. It is also important to note that
whereas the CCR2 deficiency confers pathological protection at early
time points, CCR2-deficient mice eventually mount a pulmonary
mononuclear response that is sufficient to allow for tissue recovery
with no significant rise in mortality. Obviously, there is some
compensatory mechanism that might involve other chemokines and
chemokine receptors, such as MIP-1 or IP-10. We observed increased
expression of IP-10 (Figure 6A)
, and IP-10 expression is markedly
increased after influenza A virus infection of cultured human
monocytes.25
Like MCP-1, IP-10 promotes chemotaxis of
monocytes and T lymphocytes,50
but it functions through
CXCR3.4
Another intriguing possibility is the binding of
MCP-1 to receptors other than CCR2. High levels of local MCP-1,
resulting from the increased MCP-1 mRNA produced after infection, may
help facilitate the binding of MCP-1 to alternative chemokine
receptors.
Mice deficient in both CCR2 and MIP-1 show the best survival and the
highest viral titers among all groups used in this study. This is
consistent with the idea that MCP-1 and MIP-1 make distinct and
additive contributions to the pulmonary inflammation induced by
influenza virus infections. Absence of CCR2 clearly blocks the majority
of the early pulmonary macrophage accumulation, whereas others have
shown that MIP-1 is the major regulator of T-cell trafficking into
lymph nodes and specific tissues in response to a variety of antigenic
stimuli, including influenza A virus.22,51-52
Altogether, our data demonstrate that the cell-mediated mechanism normally used in influenza-infected host tissues includes initial macrophage activation and recruitment via MCP-1 expression, followed by a quick and precise CTL attack against the influenza A virus. We show that elimination of either CCR5 or CCR2 significantly affects the course immune mechanisms as well as the clinical outcome, although in profoundly different ways. Altering the homing or phenotypic characteristics of the alveolar macrophages can cause an overwhelming, sometimes fatal early leukocyte response, as in the CCR5-/- mice. In contrast, reducing the migratory capability of the peripheral blood monocytes/macrophages confers protection from severe influenza-induced tissue damage at early time points, as seen in the CCR2-/- mice. Although there is clear overlap in CC chemokine receptor expression patterns in this case (and many others), the availability of chemokines and chemokine receptors in vivo has been repeatedly shown to be a highly specific, finely controlled, and tightly regulated process, making therapeutic disruption a distinct possibility. Our data indicate that an efficacious therapeutic agent might be one that targets the beginning stages of the early leukocyte inflammatory cascade, or more particularly, one that can specifically alter normal macrophage function. However, decreasing macrophage function could cause an increased susceptibility to secondary bacterial infections.53 This possibility must be carefully considered during future investigations into the pathogenesis of the influenza A virus.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants HL42630 and HL54123.
Accepted for publication February 16, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  J. R. Aldridge Jr., C. E. Moseley, D. A. Boltz, N. J. Negovetich, C. Reynolds, J. Franks, S. A. Brown, P. C. Doherty, R. G. Webster, and P. G. Thomas From the Cover: TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection PNAS, March 31, 2009; 106(13): 5306 - 5311. [Abstract] [Full Text] [PDF]
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E. G. Pamer Tipping the balance in favor of protective immunity during influenza virus infection PNAS, March 31, 2009; 106(13): 4961 - 4962. [Full Text] [PDF]
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 J. Wang, R. Oberley-Deegan, S. Wang, M. Nikrad, C. J. Funk, K. L. Hartshorn, and R. J. Mason Differentiated Human Alveolar Type II Cells Secrete Antiviral IL-29 (IFN-{lambda}1) in Response to Influenza A Infection J. Immunol., February 1, 2009; 182(3): 1296 - 1304. [Abstract] [Full Text] [PDF]
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K. P. Y. Hui, S. M. Y. Lee, C.-y. Cheung, I. H. Y. Ng, L. L. M. Poon, Y. Guan, N. Y. Y. Ip, A. S. Y. Lau, and J. S. M. Peiris Induction of Proinflammatory Cytokines in Primary Human Macrophages by Influenza A Virus (H5N1) Is Selectively Regulated by IFN Regulatory Factor 3 and p38 MAPK J. Immunol., January 15, 2009; 182(2): 1088 - 1098. [Abstract] [Full Text] [PDF]
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C. L. Thio, J. Astemborski, R. Thomas, T. Mosbruger, M. D. Witt, J. J. Goedert, K. Hoots, C. Winkler, D. L. Thomas, and M. Carrington Interaction between RANTES Promoter Variant and CCR5{Delta}32 Favors Recovery from Hepatitis B J. Immunol., December 1, 2008; 181(11): 7944 - 7947. [Abstract] [Full Text] [PDF]
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 J. Santiago, J. L. Hernandez-Cruz, M. E. Manjarrez-Zavala, R. Montes-Vizuet, D. P. Rosete-Olvera, A. M. Tapia-Diaz, H. Zepeda-Peney, and L. M. Teran Role of monocyte chemotactic protein-3 and -4 in children with virus exacerbation of asthma Eur. Respir. J., November 1, 2008; 32(5): 1243 - 1942. [Abstract] [Full Text] [PDF]
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A. Mildner, M. Djukic, D. Garbe, A. Wellmer, W. A. Kuziel, M. Mack, R. Nau, and M. Prinz Ly-6G+CCR2- Myeloid Cells Rather Than Ly-6ChighCCR2+ Monocytes Are Required for the Control of Bacterial Infection in the Central Nervous System J. Immunol., August 15, 2008; 181(4): 2713 - 2722. [Abstract] [Full Text] [PDF]
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 M. E. Wildenberg, C. G. van Helden-Meeuwsen, J. P. van de Merwe, C. Moreno, H. A. Drexhage, and M. A. Versnel Lack of CCR5 on dendritic cells promotes a proinflammatory environment in submandibular glands of the NOD mouse J. Leukoc. Biol., May 1, 2008; 83(5): 1194 - 1200. [Abstract] [Full Text] [PDF]
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K. L. Lin, Y. Suzuki, H. Nakano, E. Ramsburg, and M. D. Gunn CCR2+ Monocyte-Derived Dendritic Cells and Exudate Macrophages Produce Influenza-Induced Pulmonary Immune Pathology and Mortality J. Immunol., February 15, 2008; 180(4): 2562 - 2572. [Abstract] [Full Text] [PDF]
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S. J. Turner, E. Olivas, A. Gutierrez, G. Diaz, and P. C. Doherty Disregulated Influenza A Virus-Specific CD8+ T Cell Homeostasis in the Absence of IFN-{gamma} Signaling J. Immunol., June 15, 2007; 178(12): 7616 - 7622. [Abstract] [Full Text] [PDF]
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S. A. Joosten, K. E. van Meijgaarden, N. D. L. Savage, T. de Boer, F. Triebel, A. van der Wal, E. de Heer, M. R. Klein, A. Geluk, and T. H. M. Ottenhoff Identification of a human CD8+ regulatory T cell subset that mediates suppression through the chemokine CC chemokine ligand 4 PNAS, May 8, 2007; 104(19): 8029 - 8034. [Abstract] [Full Text] [PDF]
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M. D. Wareing, A. Lyon, C. Inglis, F. Giannoni, I. Charo, and S. R. Sarawar Chemokine regulation of the inflammatory response to a low-dose influenza infection in CCR2-/- mice J. Leukoc. Biol., March 1, 2007; 81(3): 793 - 801. [Abstract] [Full Text] [PDF]
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 C. Moreno, C. Nicaise, T. Gustot, E. Quertinmont, N. Nagy, M. Parmentier, H. Louis, and J. Deviere Chemokine receptor CCR5 deficiency exacerbates cerulein-induced acute pancreatitis in mice Am J Physiol Gastrointest Liver Physiol, December 1, 2006; 291(6): G1089 - G1099. [Abstract] [Full Text] [PDF]
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M. N. Ajuebor, J. A. Carey, and M. G. Swain CCR5 in T Cell-Mediated Liver Diseases: What's Going On? J. Immunol., August 15, 2006; 177(4): 2039 - 2045. [Abstract] [Full Text] [PDF]
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S. Herold, W. von Wulffen, M. Steinmueller, S. Pleschka, W. A. Kuziel, M. Mack, M. Srivastava, W. Seeger, U. A. Maus, and J. Lohmeyer Alveolar Epithelial Cells Direct Monocyte Transepithelial Migration upon Influenza Virus Infection: Impact of Chemokines and Adhesion Molecules J. Immunol., August 1, 2006; 177(3): 1817 - 1824. [Abstract] [Full Text] [PDF]
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 M. R. Edwards, M. W. Johnson, and S. L. Johnston Combination Therapy: Synergistic Suppression of Virus-Induced Chemokines in Airway Epithelial Cells Am. J. Respir. Cell Mol. Biol., May 1, 2006; 34(5): 616 - 624. [Abstract] [Full Text] [PDF]
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 D. J. J. Carr, J. Ash, T. E. Lane, and W. A. Kuziel Abnormal immune response of CCR5-deficient mice to ocular infection with herpes simplex virus type 1. J. Gen. Virol., March 1, 2006; 87(Pt 3): 489 - 499. [Abstract] [Full Text] [PDF]
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 T Oshima, K-H Sonoda, C Tsutsumi-Miyahara, H Qiao, T Hisatomi, S Nakao, S Hamano, K Egashira, I F Charo, and T Ishibashi Analysis of corneal inflammation induced by cauterisation in CCR2 and MCP-1 knockout mice Br. J. Ophthalmol., February 1, 2006; 90(2): 218 - 222. [Abstract] [Full Text] [PDF]
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M. A. Carey, J. A. Bradbury, J. M. Seubert, R. Langenbach, D. C. Zeldin, and D. R. Germolec Contrasting Effects of Cyclooxygenase-1 (COX-1) and COX-2 Deficiency on the Host Response to Influenza A Viral Infection J. Immunol., November 15, 2005; 175(10): 6878 - 6884. [Abstract] [Full Text] [PDF]
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 C. A. Wysocki, Q. Jiang, A. Panoskaltsis-Mortari, P. A. Taylor, K. P. McKinnon, L. Su, B. R. Blazar, and J. S. Serody Critical role for CCR5 in the function of donor CD4+CD25+ regulatory T cells during acute graft-versus-host disease Blood, November 1, 2005; 106(9): 3300 - 3307. [Abstract] [Full Text] [PDF]
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N. Ank, K. Petersen, L. Malmgaard, S. C. Mogensen, and S. R. Paludan Age-Dependent Role for CCR5 in Antiviral Host Defense against Herpes Simplex Virus Type 2 J. Virol., August 1, 2005; 79(15): 9831 - 9841. [Abstract] [Full Text] [PDF]
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M. D. Wareing, A. B. Lyon, B. Lu, C. Gerard, and S. R. Sarawar Chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza A virus infection in mice J. Leukoc. Biol., October 1, 2004; 76(4): 886 - 895. [Abstract] [Full Text] [PDF]
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 B.-C. Chiu, C. M. Freeman, V. R. Stolberg, J. S. Hu, K. Zeibecoglou, B. Lu, C. Gerard, I. F. Charo, S. A. Lira, and S. W. Chensue Impaired Lung Dendritic Cell Activation in CCR2 Knockout Mice Am. J. Pathol., October 1, 2004; 165(4): 1199 - 1209. [Abstract] [Full Text] [PDF]
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H. M. Scott Algood and J. L. Flynn CCR5-Deficient Mice Control Mycobacterium tuberculosis Infection despite Increased Pulmonary Lymphocytic Infiltration J. Immunol., September 1, 2004; 173(5): 3287 - 3296. [Abstract] [Full Text] [PDF]
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S. Hawgood, C. Brown, J. Edmondson, A. Stumbaugh, L. Allen, J. Goerke, H. Clark, and F. Poulain Pulmonary Collectins Modulate Strain-Specific Influenza A Virus Infection and Host Responses J. Virol., August 15, 2004; 78(16): 8565 - 8572. [Abstract] [Full Text] [PDF]
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A. D. Schecter, A. B. Berman, L. Yi, H. Ma, C. M. Daly, K. Soejima, B. J. Rollins, I. F. Charo, and M. B. Taubman MCP-1-dependent signaling in CCR2-/- aortic smooth muscle cells J. Leukoc. Biol., June 1, 2004; 75(6): 1079 - 1085. [Abstract] [Full Text] [PDF]
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S. Fernandez, P. Jose, M. G. Avdiushko, A. M. Kaplan, and D. A. Cohen Inhibition of IL-10 Receptor Function in Alveolar Macrophages by Toll-Like Receptor Agonists J. Immunol., February 15, 2004; 172(4): 2613 - 2620. [Abstract] [Full Text] [PDF]
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J. Melchjorsen, L. N. Sorensen, and S. R. Paludan Expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function J. Leukoc. Biol., September 1, 2003; 74(3): 331 - 343. [Abstract] [Full Text] [PDF]
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C. Tsutsumi, K.-H. Sonoda, K. Egashira, H. Qiao, T. Hisatomi, S. Nakao, M. Ishibashi, I. F. Charo, T. Sakamoto, T. Murata, et al. The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization J. Leukoc. Biol., July 1, 2003; 74(1): 25 - 32. [Abstract] [Full Text] [PDF]
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 P Srivastava, P J Helms, D Stewart, M Main, and G Russell Association of CCR5{Delta}32 with reduced risk of childhood but not adult asthma Thorax, March 1, 2003; 58(3): 222 - 226. [Abstract] [Full Text] [PDF]
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W. G. Glass and T. E. Lane Functional Expression of Chemokine Receptor CCR5 on CD4+ T Cells during Virus-Induced Central Nervous System Disease J. Virol., December 6, 2002; 77(1): 191 - 198. [Abstract] [Full Text] [PDF]
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A. Nansen, J. P. Christensen, S. O. Andreasen, C. Bartholdy, J. E. Christensen, and A. R. Thomsen The role of CC chemokine receptor 5 in antiviral immunity Blood, February 15, 2002; 99(4): 1237 - 1245. [Abstract] [Full Text] [PDF]
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B. P. Chen, W. A. Kuziel, and T. E. Lane Lack of CCR2 Results in Increased Mortality and Impaired Leukocyte Activation and Trafficking Following Infection of the Central Nervous System with a Neurotropic Coronavirus J. Immunol., October 15, 2001; 167(8): 4585 - 4592. [Abstract] [Full Text] [PDF]
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 S. W. Chensue Molecular Machinations: Chemokine Signals in Host-Pathogen Interactions Clin. Microbiol. Rev., October 1, 2001; 14(4): 821 - 835. [Abstract] [Full Text] [PDF]
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Z. Dembic, J.-A. Rottingen, J. Dellacasagrande, K. Schenck, and B. Bogen Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level Blood, May 1, 2001; 97(9): 2808 - 2814. [Abstract] [Full Text] [PDF]
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M. Mack, J. Cihak, C. Simonis, B. Luckow, A. E. I. Proudfoot, H. Bruhl, M. Frink, H.-J. Anders, V. Vielhauer, J. Pfirstinger, et al. Expression and Characterization of the Chemokine Receptors CCR2 and CCR5 in Mice J. Immunol., April 1, 2001; 166(7): 4697 - 4704. [Abstract] [Full Text] [PDF]
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J. A. MacLean, G. T. De Sanctis, K. G. Ackerman, J. M. Drazen, A. Sauty, E. DeHaan, F. H. Y. Green, I. F. Charo, and A. D. Luster CC Chemokine Receptor-2 Is Not Essential for the Development of Antigen-Induced Pulmonary Eosinophilia and Airway Hyperresponsiveness J. Immunol., December 1, 2000; 165(11): 6568 - 6575. [Abstract] [Full Text] [PDF]
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),
CCR5-/-
(•),
CCR2-/-
(
), or
CCR2-/-/MIP-1
mice (n = 19 per
group) infected intranasally with 5 HAU of
influenza A (strain
A/PR/8/34) and monitored for 16 days. Data
represent two independent experiments combined. B: Influenza
A titers in +/+, CCR5-/-,
CCR2-/-, and
CCR2-/-/MIP-1
