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Originally published online as doi:10.1189/jlb.0506299 on December 19, 2006

Published online before print December 19, 2006
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(Journal of Leukocyte Biology. 2007;81:793-801.)
© 2007 by Society for Leukocyte Biology

Chemokine regulation of the inflammatory response to a low-dose influenza infection in CCR2–/– mice

Mark D. Wareing1, Ashley Lyon, Chandra Inglis, Francesca Giannoni, Israel Charo2 and Sally R. Sarawar

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Influenza virus infections induce chemokines and cytokines, which regulate the immune response. The chemokine receptor CCR2 plays an important role in macrophage recruitment and in the development of T1 immunity. In the present study, we addressed the role of CCR2 in influenza A virus infection. CCR2 knockout (–/–) mice are protected against influenza A virus infection, despite delayed recruitment of macrophages. We show that low-dose influenza infection of CCR2–/– mice leads to increased neutrophilia between Days 5 and 10 after infection and decreased monocyte/macrophage and CD4+ T cell recruitment to the lungs between Days 5 and 7 after infection. These changes in leukocyte recruitment did not result from or cause increased viral titers or delayed viral clearance. Neutrophilia in the lungs correlated with increased keratinocyte-derived chemokine (KC) and/or MIP-2 expression in CCR2–/– mice between Days 5 to 10 after infection, although the kinetics of neutrophil recruitment was not altered. MIP-2 mRNA and protein expression was increased three- to fivefold, and KC protein levels were increased two- to threefold in CCR2–/– compared with CCR2 wild-type mice at Day 5 after infection. This preceded the peak neutrophil influx, which occurred 7 days after infection. In vitro studies confirmed that MIP-2 and KC accounted for neutrophil chemotactic activity in the bronchoalveolar lavage. CCR2 deficiency also resulted in increased MIP-1{alpha}, MIP-1ß, MCP-1, and IFN-inducible protein 10 and decreased RANTES mRNA expression. Furthermore, IL-6 and TNF-{alpha} 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The initial response to primary influenza infection involves neutrophil, monocyte/macrophage, and NK cell recruitment to the lungs [1 , 2 ]. Macrophages are one of the most abundant immune cells present in the lungs during the early stages of an influenza infection [3 ]. Activated macrophages produce TNF-{alpha} 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{alpha}, MIP-1ß, RANTES, and IFN-inducible protein 10 (IP-10) [10 ]. MIP-1{alpha} 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
CCR2–/– mice were originally generated in the laboratory of Dr. I. Charo [27 ], and 6- to 8-week-old female mice were used in all experiments. CCR2–/– and CCR2 wild-type (+/+) breeding colonies were established under specific pathogen-free conditions at La Jolla Institute for Allergy and Immunology (La Jolla, CA, USA) and were on a mixed 129/Ola x C57BL/6J genetic background. The CCR2 genotype was determined as described previously [27 ].

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{alpha}, 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-{alpha} 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCR2–/– mice control low-dose influenza infected, despite defective monocyte/ macrophage recruitment
To determine whether CCR2 expression is necessary for a robust immune response capable of controlling and resolving a low-dose influenza infection, CCR2–/– and CCR2+/+ mice were i.n.-infected with 100 PFU/mL influenza PR/8. CCR2 deficiency had a minor impact on the course of infection, and similar virus titers and inflammatory cell numbers infiltrated the airways observed in –/– and +/+ mice (Fig. 1 ). Peak cellular infiltration of the airways occurred between Days 10 and 14 after infection, declining thereafter to remain significantly elevated (P=0.0013) by Day 30 in CCR2–/– mice compared with uninfected controls (Fig. 1) . The inflammatory infiltrate was associated with a significant increase in the number of monocyte/macrophages and lymphocytes between Days 7 and 14 after infection (Fig. 2b and 2c ). However, CCR2–/– mice experienced a significant delay in the recruitment of monocyte/macrophages in the BAL at Days 5 and 7 after infection compared with +/+ controls. Increased neutrophil infiltration of the airways of CCR2–/– mice had been observed previously at Day 5 after influenza infection [3 ]. In the present study, we found neutrophil infiltration of the airways occurred between Days 3 and 30 after infection in CCR2+/+ and –/– mice. Despite similar kinetics, neutrophil numbers were increased significantly in the airways of CCR2–/– mice between Days 5 and 10 after infection compared with +/+ mice (Fig. 2d) . The increase in neutrophils correlated with the period of peak viral replication in the lungs (Figs. 1a and 2d) .


Figure 1
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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.

 

Figure 2
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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.

 
As observed previously in C57BL6/J mice [10 ], peak lymphocyte numbers were observed in the airways of –/– and +/+ mice at Day 10 after influenza infection. Lymphocyte numbers and kinetics were unaffected by CCR2 deletion (Fig. 2c) . However, infiltrating eosinophils, despite being low in number, were reduced significantly in CCR2–/– mice at Days 10 and 14 after infection compared with +/+ mice (Fig. 2e) .

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{alpha}, 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{alpha}, 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{alpha}, 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) .


Figure 3
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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{alpha}, 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.

 

Figure 4
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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{alpha}, 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{alpha}, 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.

 
Similar mRNA expression levels of RANTES, a ligand for CCR5, was observed between CCR2–/– and +/+ mice, except at Day 7 p.i. when RANTES mRNA levels were significantly lower in –/– mice (Fig. 4b) . Expression of MIP-2 mRNA was also significantly higher in CCR2–/– mice at Day 10 p.i. compared with +/+ controls, which correlated with greater neutrophil numbers observed in the airways of –/– mice at this time-point. Neutrophil numbers returned to near-baseline levels from Day 14 p.i. onward in CCR2–/– and +/+ mice, as did MIP-2 mRNA expression, suggesting that MIP-2 was involved in neutrophil chemotaxis to the site of infection. KC, another polymorphonuclear cell chemoattractant, was constitutively expressed in the lungs of CCR2–/– mice and +/+ controls.

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 ].


Figure 5
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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).

 
To support the findings of increased neutrophil numbers and increased CXCR2 ligand expression in the inflammatory airways of CCR2–/– mice, chemotaxis assays were performed to demonstrate that MIP-2 and/or KC expression induced directed migration of neutrophils to the airways (Fig. 5b) . MIP-2 and KC were found to contribute to neutrophil chemotactic activity in the BAL. However, when anti-MIP-2 and anti-KC antibodies were combined, they induced a more significant reduction in chemotaxis when compared with the addition of either antibody to the BAL alone (Fig. 5b) .

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-{alpha}, in BAL fluid isolated from the airways. Similar kinetics of expression for IL-6 and TNF-{alpha} was observed in CCR2–/– and +/+ mice; however, CCR2–/– produced significantly higher levels of IL-6 at Days 5 and 7 p.i. and TNF-{alpha} 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.


Figure 6
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  		Figure 6. CCR2–/– mice produced higher levels of TNF-{alpha} and IL-6. BAL fluids were collected at various times after infection of CCR2–/– and +/+ mice with PR/8. Concentration of TNF-{alpha} 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).

 
CCR2 deficiency resulted in decreased eotaxin late in infection
To determine whether eotaxin and/or RANTES was in involved in eosinophil recruitment in +/+ mice at Days 10 and 14 after infection, we measured protein expression in BAL fluid of CCR2–/– and +/+ mice. Eotaxin, but not RANTES, was increased significantly (P=0.0379) in +/+ mice compared with CCR2-deficient mice (Fig. 7 ) at Day 10 after infection, which correlates with greater increases in eosinophil numbers in CCR2+/+ mice between Days 10 and 14 after infection (Fig. 2e) . By Day 14 after infection, eotaxin levels had reduced to near-background levels, and there was a corresponding decrease in eosinophil numbers between Days 14 and 30 after infection in CCR2+/+ and –/– mice. The expression of RANTES was comparable between CCR2–/– and +/+ mice.


Figure 7
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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).

 
CCR2–/– mice exhibit defective CD4+ T cell migration in response to low-dose influenza infection
CCR2 is only expressed on a small percentage of T cells [13 ]. Nevertheless, the altered mRNA expression profiles of inflammatory chemokines associated with CCR2 deletion (Fig. 4) may have an effect on the recruitment of lymphocyte subsets. Flow cytometric analysis showed that there was a delay in CD4+ T cell trafficking to the airways of CCR2–/– mice between Days 5 and 7 after infection, after which the percentage of CD4+ T cells returned to that observed in the airways of +/+ mice (Table 1 ). The percentages of CD4+ T cells in the BAL at these time-points were quite low: 15% and 7%, at Day 5 p.i. in +/+ and –/– mice, respectively, and ~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{alpha}, 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

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Pulmonary infection of CCR2-deficient mice leads to delayed trafficking of monocyte/macrophages to the site of inflammation [25 , 32 , 33 ]. Our studies support these findings and also concur with a previous study using an influenza infection model, which found that CCR2 deficiency did not result in increased morbidity [3 ]. However, in contrast to the present study, Dawson et al. [3 ] showed that defective monocyte/macrophage recruitment in influenza-infected CCR2–/– mice led to increased virus titers at Day 5 p.i. in CCR2–/– mice compared with +/+ controls. Similar results were observed in influenza-infected MIP-1{alpha}–/– 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-{alpha} in the lungs [38 , 39 ]. A previous study has also shown that increased TNF-{alpha} 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-{alpha} and IL-6 expression in CCR2–/– mice. Increased TNF-{alpha} production by alveolar macrophages may be stimulated by neutrophil-derived myeloperoxidase [41 ] and in vitro MCP-1- or MIP-1{alpha}-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{alpha}, probably contributed to enhance TNF-{alpha} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grant AI 44247 and the Infectious Disease Science Center.


   FOOTNOTES
 
2 Current address: Gladstone Institute, University of California, San Francisco, CA 94141, USA. Back

Received May 3, 2006; revised November 1, 2006; accepted November 2, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stein-Streilein, J., Guffee, J. (1986) In vivo treatment of mice and hamsters with antibodies to asialo GM1 increases morbidity and mortality to pulmonary influenza infection J. Immunol. 136,1435-1441[Abstract]
  2. Tsuru, S., Fujisawa, H., Taniguchi, M., Zinnaka, Y., Nomoto, K. (1987) Mechanism of protection during the early phase of a generalized viral infection. II. Contribution of polymorphonuclear leukocytes to protection against intravenous infection with influenza virus J. Gen. Virol. 68,419-424[Abstract/Free Full Text]
  3. Dawson, T. C., Beck, M. A., Kuziel, W. A., Henderson, F., Maeda, N. (2000) Contrasting effects of CCR5 and CCR2 deficiency in the pulmonary inflammatory response to influenza A virus Am. J. Pathol. 156,1951-1959[Abstract/Free Full Text]
  4. Bot, A., Reichlin, A., Isobe, H., Bot, S., Schulman, J., Yokoyama, W. M., Bona, C. A. (1996) Cellular mechanisms involved in protection and recovery from influenza virus infection in immunodeficient mice J. Virol. 70,5668-5672[Abstract/Free Full Text]
  5. Bender, B. S., Small, P. A., Jr (1992) Influenza: pathogenesis and host defense Semin. Respir. Infect. 7,38-45[Medline]
  6. Xu, L., Yoon, H., Zhao, M. Q., Liu, J., Ramana, C. V., Enelow, R. I. (2004) Cutting edge: pulmonary immunopathology mediated by antigen-specific expression of TNF-{alpha} by antiviral CD8+ T cells J. Immunol. 173,721-725[Abstract/Free Full Text]
  7. Hussell, T., Pennycook, A., Openshaw, P. J. (2001) Inhibition of tumor necrosis factor reduces the severity of virus-specific lung immunopathology Eur. J. Immunol. 31,2566-2573[CrossRef][Medline]
  8. Christen, U., McGavern, D. B., Luster, A. D., von Herrath, M. G., Oldstone, M. B. (2003) Among CXCR3 chemokines, IFN-{gamma}-inducible protein of 10 kDa (CXC chemokine ligand (CXCL) 10) but not monokine induced by IFN-{gamma} (CXCL9) imprints a pattern for the subsequent development of autoimmune disease J. Immunol. 171,6838-6845[Abstract/Free Full Text]
  9. Cerwenka, A., Morgan, T. M., Harmsen, A. G., Dutton, R. W. (1999) Migration kinetics and final destination of type 1 and type 2 CD8 effector cells predict protection against pulmonary virus infection J. Exp. Med. 189,423-434[Abstract/Free Full Text]
  10. Wareing, M. D., Lyon, A. B., Lu, B., Gerard, C., Sarawar, S. R. (2004) Chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza A virus infection in mice J. Leukoc. Biol. 76,886-895[Abstract/Free Full Text]
  11. Cook, D. N., Beck, M. A., Coffman, T. M., Kirby, S. L., Sheridan, J. F., Pragnell, I. B., Smithies, O. (1995) Requirement of MIP-1 {alpha} for an inflammatory response to viral infection Science 269,1583-1585[Abstract/Free Full Text]
  12. Jones, E., Price, D. A., Dahm-Vicker, M., Cerundolo, V., Klenerman, P., Gallimore, A. (2003) The influence of macrophage inflammatory protein-1{alpha} on protective immunity mediated by antiviral cytotoxic T cells Immunology 109,68-75[CrossRef][Medline]
  13. Mack, M., Cihak, J., Simonis, C., Luckow, B., Proudfoot, A. E., Plachy, J., Bruhl, H., Frink, M., Anders, H. J., Vielhauer, V., Pfirstinger, J., Stangassinger, M., Schlondorff, D. (2001) Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice J. Immunol. 166,4697-4704[Abstract/Free Full Text]
  14. Sato, N., Ahuja, S. K., Quinones, M., Kostecki, V., Reddick, R. L., Melby, P. C., Kuziel, W. A., Ahuja, S. S. (2000) CC chemokine receptor (CCR)2 is required for Langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, B cell outgrowth, and sustained neutrophilic inflammation J. Exp. Med. 192,205-218[Abstract/Free Full Text]
  15. Nansen, A., Marker, O., Bartholdy, C., Thomsen, A. R. (2000) CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection Eur. J. Immunol. 30,1797-1806[CrossRef][Medline]
  16. Schecter, A. D., Berman, A. B., Yi, L., Ma, H., Daly, C. M., Soejima, K., Rollins, B. J., Charo, I. F., Taubman, M. B. (2004) MCP-1-dependent signaling in CCR2(–/–) aortic smooth muscle cells J. Leukoc. Biol. 75,1079-1085[Abstract/Free Full Text]
  17. Blease, K., Lukacs, N. W., Hogaboam, C. M., Kunkel, S. L. (2000) Chemokines and their role in airway hyper-reactivity Respir. Res. 1,54-61[CrossRef][Medline]
  18. Traynor, T. R., Herring, A. C., Dorf, M. E., Kuziel, W. A., Toews, G. B., Huffnagle, G. B. (2002) Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity J. Immunol. 168,4659-4666[Abstract/Free Full Text]
  19. Scott, H. M., Flynn, J. L. (2002) Mycobacterium tuberculosis in chemokine receptor 2-deficient mice: influence of dose on disease progression Infect. Immun. 70,5946-5954[Abstract/Free Full Text]
  20. Chen, B. P., Kuziel, W. A., Lane, T. E. (2001) 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. 167,4585-4592[Abstract/Free Full Text]
  21. De Lema, G. P., Maier, H., Franz, T. J., Escribese, M., Chilla, S., Segerer, S., Camarasa, N., Schmid, H., Banas, B., Kalaydjiev, S., Busch, D. H., Pfeffer, K., Mampaso, F., Schlondorff, D., Luckow, B. (2005) Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice J. Am. Soc. Nephrol. 16,3592-3601[Abstract/Free Full Text]
  22. Moore, B. B., Paine, R., III, Christensen, P. J., Moore, T. A., Sitterding, S., Ngan, R., Wilke, C. A., Kuziel, W. A., Toews, G. B. (2001) Protection from pulmonary fibrosis in the absence of CCR2 signaling J. Immunol. 167,4368-4377[Abstract/Free Full Text]
  23. Moore, B. B., Kolodsick, J. E., Thannickal, V. J., Cooke, K., Moore, T. A., Hogaboam, C., Wilke, C. A., Toews, G. B. (2005) CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury Am. J. Pathol. 166,675-684[Abstract/Free Full Text]
  24. Boring, L., Gosling, J., Cleary, M., Charo, I. F. (1998) Decreased lesion formation in CCR2–/– mice reveals a role for chemokines in the initiation of atherosclerosis Nature 394,894-897[CrossRef][Medline]
  25. Hokeness, K. L., Kuziel, W. A., Biron, C. A., Salazar-Mather, T. P. (2005) Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-{alpha}/ß-induced inflammatory responses and antiviral defense in liver J. Immunol. 174,1549-1556[Abstract/Free Full Text]
  26. Peters, W., Scott, H. M., Chambers, H. F., Flynn, J. L., Charo, I. F., Ernst, J. D. (2001) Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis Proc. Natl. Acad. Sci. USA 98,7958-7963[Abstract/Free Full Text]
  27. Boring, L., Gosling, J., Chensue, S. W., Kunkel, S. L., Farese, R. V., Jr, Broxmeyer, H. E., Charo, I. F. (1997) Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice J. Clin. Invest. 100,2552-2561[Medline]
  28. Tannock, G. A., Paul, J. A., Barry, R. D. (1984) Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens Infect. Immun. 43,457-462[Abstract/Free Full Text]
  29. Sarawar, S. R., Lee, B. J., Anderson, M., Teng, Y. C., Zuberi, R., Von Gesjen, S. (2002) Chemokine induction and leukocyte trafficking to the lungs during murine {gamma}herpesvirus 68 (MHV-68) infection Virology 293,54-62[CrossRef][Medline]
  30. Bischoff, D. S., Zhu, J. H., Makhijani, N. S., Yamaguchi, D. T. (2005) KC chemokine expression by TGF-ß in C3H10T1/2 cells induced towards osteoblasts Biochem. Biophys. Res. Commun. 326,364-370[CrossRef][Medline]
  31. Biswas, R., Datta, S., Gupta, J. D., Novotny, M., Tebo, J., Hamilton, T. A. (2003) Regulation of chemokine mRNA stability by lipopolysaccharide and IL-10 J. Immunol. 170,6202-6208[Abstract/Free Full Text]
  32. Peters, W., Cyster, J. G., Mack, M., Schlondorff, D., Wolf, A. J., Ernst, J. D., Charo, I. F. (2004) CCR2-dependent trafficking of F4/80dim macrophages and CD11cdim/intermediate dendritic cells is crucial for T cell recruitment to lungs infected with Mycobacterium tuberculosis J. Immunol. 172,7647-7653[Abstract/Free Full Text]
  33. Cadillac, J. M., Sigler, R. E., Weinberg, J. B., Lutzke, M. L., Rochford, R. (2005) {gamma}Herpesvirus-induced lung pathology is altered in the absence of macrophages Lung 183,239-251[CrossRef][Medline]
  34. Sakai, S., Kawamata, H., Mantani, N., Kogure, T., Shimada, Y., Terasawa, K., Sakai, T., Imanishi, N., Ochiai, H. (2000) Therapeutic effect of anti-macrophage inflammatory protein 2 antibody on influenza virus-induced pneumonia in mice J. Virol. 74,2472-2476[Abstract/Free Full Text]
  35. Oliveira, S. H., Lira, S., Martinez, A. C., Wiekowski, M., Sullivan, L., Lukacs, N. W. (2002) Increased responsiveness of murine eosinophils to MIP-1ß (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8 J. Leukoc. Biol. 71,1019-1025[Abstract/Free Full Text]
  36. Lukacs, N. W., Standiford, T. J., Chensue, S. W., Kunkel, R. G., Strieter, R. M., Kunkel, S. L. (1996) C-C chemokine-induced eosinophil chemotaxis during allergic airway inflammation J. Leukoc. Biol. 60,573-578[Abstract]
  37. Goser, S., Andrassy, M., Buss, S. J., Leuschner, F., Volz, C. H., Ottl, R., Zittrich, S., Blaudeck, N., Hardt, S. E., Pfitzer, G., Rose, N. R., Katus, H. A., Kaya, Z. (2006) Cardiac troponin I but not cardiac troponin T induces severe autoimmune inflammation in the myocardium Circulation 114,1693-1702
  38. Zheng, L., He, M., Long, M., Blomgran, R., Stendahl, O. (2004) Pathogen-induced apoptotic neutrophils express heat shock proteins and elicit activation of human macrophages J. Immunol. 173,6319-6326[Abstract/Free Full Text]
  39. Thomassen, M. J., Divis, L. T., Fisher, C. J. (1996) Regulation of human alveolar macrophage inflammatory cytokine production by interleukin-10 Clin. Immunol. Immunopathol. 80,321-324[CrossRef][Medline]
  40. Robben, P. M., Laregina, M., Kuziel, W. A., Sibley, L. D. (2005) Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis J. Exp. Med. 201,1761-1769[Abstract/Free Full Text]
  41. Grattendick, K., Stuart, R., Roberts, E., Lincoln, J., Lefkowitz, S. S., Bollen, A., Moguilevsky, N., Friedman, H., Lefkowitz, D. L. (2002) Alveolar macrophage activation by myeloperoxidase: a model for exacerbation of lung inflammation Am. J. Respir. Cell Mol. Biol. 26,716-722[Abstract/Free Full Text]
  42. Speyer, C. L., Gao, H., Rancilio, N. J., Neff, T. A., Huffnagle, G. B., Sarma, J. V., Ward, P. A. (2004) Novel chemokine responsiveness and mobilization of neutrophils during sepsis Am. J. Pathol. 165,2187-2196[Abstract/Free Full Text]
  43. Traynor, T. R., Kuziel, W. A., Toews, G. B., Huffnagle, G. B. (2000) CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection J. Immunol. 164,2021-2027[Abstract/Free Full Text]
  44. Chiu, B. C., Freeman, C. M., Stolberg, V. R., Hu, J. S., Zeibecoglou, K., Lu, B., Gerard, C., Charo, I. F., Lira, S. A., Chensue, S. W. (2004) Impaired lung dendritic cell activation in CCR2 knockout mice Am. J. Pathol. 165,1199-1209[Abstract/Free Full Text]
  45. Graham, M. B., Braciale, T. J. (1997) Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice J. Exp. Med. 186,2063-2068[Abstract/Free Full Text]



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