Published online before print December 19, 2006
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Torrey Pines Institute for Molecular Studies, San Diego, California, USA
1 Correspondence at current address: Center for Infectious Medicine, Karolinska Institute, Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden. E-mail: mark.wareing{at}ki.se
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, MIP-1ß, MCP-1, and IFN-inducible protein 10 and decreased RANTES mRNA expression. Furthermore, IL-6 and TNF- cytokine production were elevated after infection. These studies suggest that CCR2 plays a multifactorial role in the development of the immune response to influenza.
Key Words: neutrophils • virus • cell trafficking • lung
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and nitric oxide, which have antimicrobial properties; apoptotic neutrophils are also cleared from the site of infection by macrophages. The initial inflammatory infiltrate helps control viral replication until the virus is cleared by a viral-specific, CTL-mediated response [4
, 5
]. However, the inflammatory response to a respiratory infection may sometimes be detrimental to the host, causing augmented lung pathology [6
, 7
]. Identifying factors involved in the recruitment of proinflammatory leukocytes will further our understanding of the inflammatory response and immunopathology associated with respiratory viral infections.
It has been shown that the differential regulation of chemokines and chemokine receptors is functionally important in regulating leukocyte trafficking [8
, 9
]. Influenza infection of C57BL/6J mice induced chemokine gene expression for monocyte chemoattractants, MCP-1, MIP-1, MIP-1ß, RANTES, and IFN-inducible protein 10 (IP-10) [10
]. MIP-1 deficiency in influenza-infected mice resulted in reduced viral pneumonitis and increased viral titers [11
], although this could be a result of decreased CTL activity rather than impaired migration of CTL cells to the site of infection [12
]. RANTES knockout (–/–) and CXCR3–/– mice did not demonstrate increased susceptibility to influenza infection, although defective lymphocyte, neutrophil, and eosinophil recruitment to the airways was observed in CXCR3–/– mice [10
]. In contrast, influenza-infected CCR5–/– mice have been reported to exhibit increased mortality and severe lung pathology associated with hyperacute macrophage accumulation [3
].
CCR2 is expressed on monocytes/macrophages, immature dendritic cells (DC), and a small percentage (2–15%) of T cells in mice [13 14 15 ]. CCR2 is the only known functional receptor for MCP-1, although recent evidence suggests that an alternative receptor may be present in smooth muscle [16 ]. Infection models have shown that CCR2 deletion can result in the development of a Th2 response [14 , 17 , 18 ] or a decrease in Th1-associated factors [19 , 20 ]. Inflammatory disease models demonstrated that CCR2-deficient mice exhibited reduced pathological manifestations [21 22 23 24 ]. In the early stages of influenza infection, CCR2–/– mice have been shown to display defective macrophage recruitment, elevated virus titers, increased neutrophil accumulation, and elevated MCP-1 and IP-10 chemokine mRNA levels in the lungs [3 ]. The impaired ability to control infections in the absence of CCR2 has also been observed in Mycobacterium tuberculosis, Leishmania major, murine CMV, and mouse hepatitis virus infection models [14 , 20 , 25 , 26 ]. However, CCR2 deficiency had no effect on the ability to control a low-dose tuberculosis infection [19 ].
In the present study, we investigated the importance of CCR2 in the acute and late-phase immune response to a low-dose, nonlethal influenza infection. Our results suggest that CCR2 plays an important role in the recruitment of monocyte/macrophages and CD4+ T cells to the lungs during influenza infection. However, delayed recruitment of these cell types did not affect the control of viral replication or result in long-term lung damage. We also found that CCR2+ monocyte/macrophages, via a direct or indirect mechanism, influenced the numbers of neutrophils that are recruited to the lungs during influenza infection.
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Viral infection and sampling
Influenza A/Puerto Rico/8/34 (PR/8) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA; VR-1469), and stocks were grown in Madin Darby canine kidney (MDCK) cells (ATCC CCL34). Infectious virus yields were determined by plaque assay on monolayers of MDCK cells by the method of Tannock et al. [28
]. Mice were anaesthetized with Avertin (2,2,2-tribromoethanol) and infected intranasally (i.n.) with 30 µL virus at a concentration of 100 PFU/mL in PBS (
3 PFU/mouse), unless stated otherwise. Control mice were mock-infected with PBS alone. At various times after infection, mice were killed by terminal anesthesia with Avertin. The inflammatory cells infiltrating the airways were harvested by bronchoalveolar lavage (BAL) via the trachea, by performing three lavages with 1 mL PBS. Viable leukocyte counts were determined by Trypan blue exclusion. The lungs were removed, and a 10% (w/v) homogenate was prepared in PBS using a Tissue Tearor homogenizer (Fisher Scientific, Pittsburgh, PA, USA).
Cytospin preparations of BAL cells
BAL cells were cytocentrifuged onto microscope slides at 500 rpm for 7 min using 50 µL cell suspension containing 1.5 x 105 cells/mL in PBS 2% FBS. The cytocentrifuge preparations were fixed and stained using a Leukostat staining kit (Hema 3, Fisher Scientific).
Chemokine and chemokine receptor analysis
Chemokine and chemokine receptor expression was analyzed by RNase protection assay (RPA) as described previously [29
]. RNA from lungs of two or three mice from each experiment was analyzed individually. Chemokine or chemokine receptor mRNA levels were determined using a RiboQuant multiprobe template mCK-5 set (BD PharMingen, San Diego, CA, USA) or a modified mCR-5 probe set, which contained the additional probe templates for CCL21 (secondary lymphoid tissue chemokine) and CCR7. Custom murine probe templates for CXCR2, CXCR3, CXCR4, and CXCR5 or keratinocyte-derived chemokine (KC), MIP-2, MIP-3, and MIP-3ß were also used (BD PharMingen).
Levels of MIP-2, KC, RANTES, and eotaxin chemokines in BAL fluid were analyzed using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA).
Cytokine analysis
IL-6 and TNF- levels in BAL fluid were measured using commercial ELISA kits (R&D Systems).
Chemotaxis assays
Neutrophil chemotaxis assays were performed in 48-well, modified Boyden chambers (Neuro Probe, Gaithersburg, MD, USA) as described previously [27
].
Statistical analysis
Statistical analysis was performed using the Student’s t-test or the Mann-Whitney Rank Sum test, depending on whether the data were distributed normally.
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Figure 1. Lung virus titers and total BAL cell counts are similar in influenza PR/8-infected CCR2–/– (KO) and +/+ (WT) mice, which were infected i.n. with PR/8 or were mock-infected with PBS, and lungs and inflammatory cells were collected at various intervals. (a) Infectious virus yields in the lung. Virus titers were determined in 10% lung homogenates by plaque assay on MDCK cells. (b) Cell numbers in the BAL. Cells infiltrating the lung were collected by BAL via the trachea. Viable cell counts were determined by trypan blue exclusion. Data are means + SEM from three to five independent experiments.
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Figure 2. Kinetics of leukocyte recruitment to the airways following influenza infection. CCR2–/– mice and +/+ controls were infected i.n. with PR/8 or were mock-infected with PBS. Differential cell counts in the BAL population were determined using Hema-3 (Fisher Scientific)-stained cytospin preparations. (a) Percentage of granulocytes, (b) total macrophage/monocytes, (c) lymphocytes, (d) neutrophils, and (e) eosinophils in the BAL cell population were determined. Data shown are means + SEM from three to five independent experiments. Asterisks denote significant differences between cell populations in CCR2+/+ and –/– mice: ***, P < 0.001; **, P < 0.01; *, P < 0.05.
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Induction of chemokine mRNA in the lungs of low-dose, influenza-infected CCR2–/– mice
Deletion of CCR2 had a profound effect on the leukocyte kinetics and numbers of specific leukocyte subsets infiltrating the airways following influenza infection. To determine whether CCR2 deletion also led to a significant alteration in the chemokine profile, which could account for the altered leukocyte response, we used the RPA to measure chemokine mRNA expression in the lungs of influenza-infected CCR2–/– and +/+ mice. At Day 5 after infection, there were significantly higher levels of MIP-1, MIP-1ß, MCP-1, IP-10, and MIP-2 mRNA expression in the lungs of CCR2–/– mice compared with +/+ controls (Figs. 3
and 4
). Peak expression of most chemokines analyzed in +/+ mice occurred at Day 7 after infection. RANTES, KC, and MIP-3ß were constitutively expressed; only low or undetectable levels of mRNA expression for MIP-1, MIP-1ß, MCP-1, IP-10, and MIP-2 were observed in the lungs of uninfected CCR2+/+ or –/– mice. Peak expression at Day 7 after infection of MIP-3, the ligand for the chemokine receptor CCR6, which is expressed on memory T cells, B cells, and DC, was similar in CCR2–/– and +/+ mice; however, expression at Day 10 postinfection (p.i.) was significantly higher in CCR2–/– mice (Fig. 3b)
.
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Figure 3. RPA showing kinetics of chemokine mRNA induction in the lungs of PR/8-infected CCR2 +/+ and –/– mice. (a) RPA. Mice were infected with PR/8 or were mock-infected with PBS. At various time intervals after infection, the mice were killed and the lungs snap-frozen in liquid nitrogen. Total RNA was prepared from the lungs using Trizol reagent and hybridized with 32P-labeled antisense probes generated by in vitro transcription of templates for MIP-3, MIP-3ß, KC, and MIP-2 and housekeeping genes L32 and GAPDH (PharMingen). Protected probes were separated by PAGE and visualized using a PhosphorImager. A representative assay is shown. (b) Densitometric analysis. RPAs were performed as described in a. The resulting autoradiographs were analyzed by scanning densitometry of each lane using ImageQuant Software (Molecular Dynamics, Sunnyvale, CA, USA). Data are expressed as mean + SEM of arbitrary units normalized against values for the housekeeping gene, GAPDH, for three to four independent experiments at each time-point. Asterisks denote statistically significant differences in expression between CCR2+/+ and –/– mice: ***, P < 0.001; *, P < 0.05.
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Figure 4. CCR2–/– mice produce higher levels of chemokine mRNA early in influenza infection. (a) RPA. CCR2–/– or +/+ mice were infected i.n. with 100 PFU/mL PR/8, the lungs harvested, and RNA extracted as described in the legend to Figure 3
. RNA was hybridized with 32P-labeled antisense probes generated by in vitro transcription of templates for lymphotactin (Ltn), TCA-3, MCP-1, MIP-1, MIP-1ß, RANTES, eotaxin, MIP-2, IP-10, and housekeeping genes L32 and GAPDH (PharMingen). Protected probes were separated by PAGE and visualized using a PhosphorImager. A representative assay is shown. (b) Densitometric analysis. RPAs were performed as described in a. Data for MCP-1, MIP-1, MIP-1ß, RANTES, and IP-10 are expressed as mean + SEM of arbitrary units normalized against values for the housekeeping gene, GAPDH, for three independent experiments at each time-point. Asterisks denote statistically significant differences in expression between CCR2+/+ and –/– mice: ***, P < 0.001; **, P < 0.01; *, P < 0.05.
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Enhanced expression of neutrophil-attracting chemokines in the airways of influenza-infected CCR2–/– mice
To corroborate the increase in MIP-2 mRNA expression at Days 5 and 10 after influenza infection in CCR2–/– mice compared with +/+ controls, we measured MIP-2 protein levels in BAL fluid. Protein expression kinetics of MIP-2 mirrored the mRNA expression in –/– and +/+ mice, and peak levels were recorded at Day 5 and returning to near-baseline levels by Day 14 after infection (Fig. 5a
). However, MIP-2 protein expression in CCR2–/– mice was increased significantly at Days 5 and 10 p.i. compared with +/+ mice. Although no significant difference was observed in mRNA expression, KC protein expression was increased significantly in CCR2–/– mice at Days 5 and 7 p.i. compared with +/+ mice (Fig. 5a)
. This suggests that apart from differences in sensitivities between the two assays, there may be differential regulation of KC at the post-transcriptional level, possibly involving increased mRNA stability, at Days 5 and 7 p.i. [30
, 31
].
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Figure 5. MIP-2 and KC expression is up-regulated in CCR2–/– mice and correlates with neutrophil chemotaxis. (a) MIP-2 and KC expression was analyzed by ELISA in BAL fluid collected from PR/8-infected CCR2–/– and +/+ control mice at the times indicated. Data are expressed as mean protein concentration + SEM for three to five independent experiments (**, P<0.01; *, P<0.05, significantly different from PR/8-infected +/+ control). (b) Neutrophil chemotactic activity of BAL fluid collected at Day 5 after influenza infection. Cell-free BAL fluids were pooled from two to four mice, diluted 1/2, and their ability to induce neutrophil migration was analyzed in a microchemotaxis assay in the presence or absence of anti-MIP-2 and/or anti-KC or control antibodies (Ctrl Ig). Samples were assayed in duplicate, and 10 high-power fields (hpf; x400) were counted for each sample. Data are expressed as mean cell counts/10 hpf + SD for two independent experiments. Asterisks denote statistically significant differences in neutrophil chemotaxis relative to that for BAL fluid in the presence of a control antibody (***, P<0.001; **, P<0.01).
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Influenza infection of CCR2–/– mice leads to greater inflammatory cytokine expression
Increased numbers of neutrophils in the inflammatory airways of CCR2–/– mice compared with +/+ controls suggested that influenza infection may be associated with higher levels of inflammatory cytokines in the lungs of CCR2–/– mice. To address this, we measured the expression of the inflammatory cytokines, IL-6 and TNF-, in BAL fluid isolated from the airways. Similar kinetics of expression for IL-6 and TNF- was observed in CCR2–/– and +/+ mice; however, CCR2–/– produced significantly higher levels of IL-6 at Days 5 and 7 p.i. and TNF- at Day 5 p.i. (Fig. 6
). These results suggest that the level of inflammatory cytokines in the lungs of influenza-infected CCR2–/– mice was greater than that in +/+ mice. Nevertheless, histological analysis suggested that there was no long-term lung damage in these animals (data not shown). Thus, the increased inflammatory cytokine response elicited in CCR2-deficient mice was not sufficient to induce more pronounced pathology in these mice.
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Figure 6. CCR2–/– mice produced higher levels of TNF- and IL-6. BAL fluids were collected at various times after infection of CCR2–/– and +/+ mice with PR/8. Concentration of TNF- and IL-6 was determined by ELISA. Data shown are means + SEM from three to five independent experiments; *, P < 0.05 (significantly different from PR8 +/+ control).
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Figure 7. Eotaxin and RANTES expression in CCR2–/– mice. (a) Eotaxin and RANTES expression was analyzed by ELISA in BAL fluid collected from PR/8-infected CCR2–/– and +/+ control mice at the times indicated. Data are expressed as mean protein concentration + SD for three to four mice (*, P<0.05, significantly different from PR/8-infected –/– mice).
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6% and 4% at Day 7 p.i. in +/+ and –/–, respectively. The percentage of CD8+ T cells was similar between CCR2–/– and +/+ mice at all time-points examined. The lymphocyte subpopulations within the mediastinal lymph node were also equivalent between +/+ and –/– mice, suggesting that CCR2 deletion affected only the migration of CD4+ T cells to the site of infection. It is surprising that increased mRNA expression of MCP-1, MIP-1, MIP-1ß, and IP-10 at Day 5 after infection in –/– mice compared with +/+ controls did not appear to affect T cell or NK cell recruitment to the site of infection. |
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Table 1. Percentage of T Lymphocyte Subsets in BAL Populations
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–/– mice, which exhibited reduced pneumonitis and delayed viral clearance compared with controls [11
]. Differences in the infectious dose used may account for the disparity in the results observed between the current studies and those of Dawson et al. [3
]. Such a precedence has been reported, whereby differences in the immune response were observed between high- and low-dose M. tuberculosis infections of CCR2–/– mice [19
, 26
]. Significantly higher bacterial loads, severe pathology, and increased mortality were observed in high-dose M. tuberculosis i.v.-infected CCR2–/– mice [26
], whereas bacterial loads in low-dose-infected CCR2–/– mice did not exceed those detected in +/+ controls [19
]. Our results suggest that despite a temporal deficiency in monocyte/macrophage recruitment, the immune response to low-dose influenza infection is sufficiently robust to control and resolve the infection. Low-dose influenza infection of CCR2–/– mice resulted in increased neutrophilic and decreased eosinophilic infiltration to the lungs, an observation consistent, with respect to neutrophilia, with other infection models [3 , 26 ]. Nevertheless, contrary to the results of Dawson et al. [3 ], CCR2 deficiency did not augment neutrophil persistence in the infected lungs (Fig. 2) . In agreement with our data, Sakai et al. [34 ] showed that increased neutrophil counts in BAL fluid correlated with increased CXCR2 chemoattractant, MIP-2, and influenza virus replication in Institute of Cancer Research mice. CCR2 expression has been detected in eosinophils, but CCR2 deletion is unlikely to have had a direct effect on eosinophil chemotaxis, as eosinophils do not migrate toward MCP-1 [35 , 36 ]. Anti-MCP-1 gene therapy of mice was found to result in decreased eotaxin mRNA expression following induction of experimental autoimmune mycarditis [37 ]. This work suggests that monocyte activation and/or chemotaxis can influence eotaxin expression, which correlates with the results in our study.
Dawson et al. [3 ] reported that CCR2 deficiency led to increased MCP-1 and IP-10 mRNA expression in influenza-infected mice. In the present study, we showed that as well as MCP-1 and IP-10, several other chemokines were induced during airway inflammation. However, differences in the kinetics of chemokine expression were apparent, and Dawson et al. [3 ] reported peak induction of MCP-1 and IP-10 3–5 days earlier than observed in the present studies. The differences observed could be attributable to different infectious dose regimens used in the two studies.
Macrophages are an important source of TNF- in the lungs [38
, 39
]. A previous study has also shown that increased TNF- levels were induced in Toxoplasma gondii-infected CCR2–/– mice, despite a failure to recruit Gr-1+ monocytes [40
], which correlates with our observations of TNF- and IL-6 expression in CCR2–/– mice. Increased TNF- production by alveolar macrophages may be stimulated by neutrophil-derived myeloperoxidase [41
] and in vitro MCP-1- or MIP-1-stimulated polymorphonuclear cell culture IL-6 expression [42
]. Therefore, despite defective monocyte/macrophage recruitment, increased neutrophil numbers and the expression of chemokines, such as MIP-1, probably contributed to enhance TNF- and IL-6 production in the lungs of influenza-infected, CCR2-deficient mice.
Studies with CCR2–/– mice have indicated that this receptor promotes the development of T1-type immune responses in infection models [14 , 18 , 20 , 43 ]. The delayed recruitment of CD4+ T cells to the lungs is consistent with impaired trafficking of CD4+ and CD8+ T cells to the site of inflammation in corona virus-infected CCR2–/– mice [20 ]. Immature DC from CCR2–/– mice exhibit defective maturation with significantly decreased MHC Class II and CD40 expression [44 ]. This may potentially result in impaired activation of CD4+ T cells in CCR2–/– mice. Decreased expression of RANTES in CCR2-deficient mice may also have contributed to delayed recruitment of CD4+ T cells at Day 7 p.i. Nevertheless, the substantial reduction in CD4+ T cells trafficking to the lungs did not appear to influence virus replication and/or clearance, which suggests that there is some redundancy in the immune mechanisms involved in responding to a low-dose influenza infection. Studies by Graham and Braciale [45 ] with influenza infection of B cell-deficient mice illustrated that a more complex interaction of immune mediators is required to resolve high-dose viral infections compared with low-dose infections. Therefore, CCR2 expression may be necessary for controlling virus replication following high-dose infections, but it appears to be redundant in the response to low-dose influenza infections.
The studies presented here demonstrate that CCR2 plays an important role in the recruitment of monocyte/macrophages and CD4+ T cells to the lungs during influenza infection. Our data also suggest that CCR2 deficiency results in increased chemokine and inflammatory cytokine expression during influenza infection. The increase in inflammatory mediators produced during influenza infection also influenced the level, but not the kinetics, of the neutrophil and to a lesser degree, the eosinophil response in the inflamed lungs. Neutrophilia appeared to be associated directly with increased MIP-2 and KC expression, which in turn, correlated with viral replication. However, delayed monocyte/macrophage or CD4+ T cell recruitment did not affect viral clearance. Therefore, CCR2 deficieny may alter the development of the normal cell-mediated immune response against influenza but may not necessarily affect the outcome of infection.
Received May 3, 2006; revised November 1, 2006; accepted November 2, 2006.
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by antiviral CD8+ T cells J. Immunol. 173,721-725
-inducible protein of 10 kDa (CXC chemokine ligand (CXCL) 10) but not monokine induced by IFN- (CXCL9) imprints a pattern for the subsequent development of autoimmune disease J. Immunol. 171,6838-6845 for an inflammatory response to viral infection Science 269,1583-1585 on protective immunity mediated by antiviral cytotoxic T cells Immunology 109,68-75[CrossRef][Medline]/ß-induced inflammatory responses and antiviral defense in liver J. Immunol. 174,1549-1556herpesvirus 68 (MHV-68) infection Virology 293,54-62[CrossRef][Medline]Herpesvirus-induced lung pathology is altered in the absence of macrophages Lung 183,239-251[CrossRef][Medline]This article has been cited by other articles:
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