In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1α (MIP-1α). Here we examine the role of MIP-1α in the pathogenesis of this disease using mice deficient in MIP-1α or its receptor, CCR1. The inflammatory response to PVM in MIP-1α-deficient mice was minimal, with ∼10–60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1α-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1α/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.

The aim of this study is to delineate the specific mechanisms underlying the pathogenesis of respiratory viral infection with a focus on the role of the eosinophilic leukocyte (1). Respiratory syncytial virus (RSV),3 a member of the pneumovirus subfamily of the family Paramyxoviridae, is a frequent cause of respiratory infection in both young children and the elderly and is the major etiologic agent responsible for hospitalization of children <1 year of age with respiratory infection worldwide (2, 3). Several recent findings link eosinophils to the pathophysiology of RSV infection: several groups have demonstrated that eosinophils are recruited to and degranulate into the respiratory tract in association with RSV infection in infants (4, 5, 6, 7) and that respiratory epithelial cells infected with RSV express potent eosinophil chemoattractants (7, 8, 9, 10, 11, 12). Whereas most of this research has focused on the harmful role of eosinophils in promoting tissue damage and bronchospasm, recent work from our laboratory suggests that eosinophils may also play a beneficial role in this disease. Specifically, we have found that human eosinophils can block RSV infection of target cells in vitro and that the antiviral effect is mediated at least in part by the unique eosinophil secretory RNases, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (13, 14, 15, 16).

To study the antiviral effects of eosinophils and their secretory RNases in vivo, we have extended our studies to investigate the inflammatory responses of mice to infection with pneumonia virus of mice (PVM). PVM is a paramyxovirus, subfamily pneumovirus, that naturally targets a rodent host (17, 18, 19) and replicates many of the acute inflammatory responses described for RSV in humans (20, 21). Easton and colleagues (22, 23, 24, 25) have characterized the genome of this virus, documenting similarities in gene order and among specific primary sequences and demonstrating that PVM is the phylogenetically nearest known relative of RSV. They have also demonstrated that BALB/c mice are readily infected by intranasal inoculation with PVM strain J3666 (20). We have characterized the inflammatory response to this virus in mice, which includes prominent pulmonary eosinophilia and neutrophilia. We also noted elevated local production of the chemokine, macrophage inflammatory protein-1α (MIP-1α), which suggested a molecular mechanism for the observed cellular response (21).

MIP-1α is a member of the CC subfamily of chemokines and is a chemoattractant for neutrophils and eosinophils in vitro as well as in several mouse models of disease (26, 27, 28, 29). MIP-1α has been detected in response to RSV infection both in tissue culture (7, 11) and in human subjects (11, 30, 31). Cook et al. (32, 33) have generated a MIP-1α-deficient gene-deleted mouse strain that displays reduced inflammatory response to infection with influenza virus. Similarly, Salazar-Mather et al. (34) have described reduced inflammatory and protective responses in livers of MIP-1α-deficient mice infected with murine cytomegalovirus. The major receptor for MIP-1α on neutrophils and eosinophils is CCR1 (35, 36, 37, 38, 39, 40); Gao et al. (41) have generated a CCR1-deficient gene-deleted mouse strain that responded to infection with the fungus, Asperigillus fumigatus, with accelerated mortality. Gerard et al. (42) described a second CCR1-deficient mouse strain that also demonstrated a role for this receptor in systemic inflammatory responses.

In this work, we examine the role of MIP-1α and its receptor CCR1 in promoting and sustaining the inflammation observed in mice in response to infection with PVM. In experiments performed with either the MIP-1α or CCR1 gene-deleted mouse strains, we found that both elements were crucial for recruiting the vast majority of the neutrophils and all of the eosinophils that accumulated in response to infection with PVM. Furthermore, deficiency of either MIP-1α or CCR1 resulted in recovery higher levels of infectious virus during the course of active infection compared with fully competent mice, and that deficiency of CCR1 correlated with reduced survival of PVM-infected mice. These data suggest that the antiviral inflammatory response protects mice from the lethal sequelae of viral infection.

Male and female mice (6–20 wk old) were used in all experiments. C57BL/6 mice with a targeted disruption of the gene encoding MIP-1α−/− described by Cook et al., (32) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice with a targeted disruption of the gene encoding CCR1 (CCR1−/−) and littermate controls (CCR1+/+) described by Gao et al. (41) were backcrossed to the C57BL/6 background for six generations and are maintained at the National Institutes of Health. Stocks of PVM (strain J3666) at 3 × 105 PFU/ml were stored in liquid nitrogen. All procedures were reviewed and approved by the National Institutes of Health Animal Welfare Review Board (protocol LHD8E).

To facilitate infection, mice were placed under anesthesia for ∼1 min by inhalation of 15% halothane (Ayerst Laboratories, Philadelphia, PA) diluted in mineral oil. Mice infected with PVM received a single intranasal dose of 80 μl containing varying amounts of virus (10- 200 PFU) diluted from concentrated stocks in IMDM (Life Technologies, Gaithersburg, MD). Mice described as “uninfected” were inoculated with 80 μl IMDM; mice contributing to “day 0” points were not inoculated.

Lungs to be sectioned for microscopic analysis were inflated transbronchially with ∼0.3 ml 10% formalin before removal from the body cavity. Inflated lungs were fixed in 10% formalin before sectioning, slide preparation, and staining with standard hematoxylin and eosin stains (American Histolabs, Gaithersburg, MD). Lungs to be processed for ELISA and viral titer determination were removed from the body cavity under aseptic conditions and immersed in prechilled tissue culture medium (IMDM). Lung tissue was homogenized (Tissumizer, Tekmar, Cincinnati, OH), and cellular debris was removed by low speed centrifugation. Clarified supernatants were flash frozen in a dry ice-ethanol bath and stored at −80°C. Viral titers were determined by standard plaque assay using the BS-C-1 epithelial cell line (American Type Tissue Collection, Manassas, VA). MIP-1α concentrations in homogenized lung tissue were determined by ELISA (Quantikine, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Total protein was determined by the Bradford colorimetric assay.

After dissection to expose the trachea and lungs, the lungs were inflated transbronchially with 0.8 ml PBS containing 0.25% BSA. The fluid was collected and the process was repeated, with a recovery of 1.2–1.5 ml bronchial washings per mouse. Total cells were determined by either hemocytometer counts or, in the cases in which very few cells were recovered, from direct counts from cytospin preparations. Differential leukocyte counts were assessed by visual inspection of Diff-Quik-stained cytospin preparations. CD8+ lymphocyte counts were assessed by fluorescent microscopic inspection of cells stained with polyclonal rabbit anti-CD8 (H-160, Santa Cruz Biotechnology, Santa Cruz, CA) followed by TRITC-tagged goat anti-rabbit IgG (Pierce, Rockford, IL).

All data points represent the average ± SEM of samples from 2–3 mice unless otherwise indicated. Statistical significance was determined by paired and unpaired Student t tests.

In previous work, we described the infection of 6- to 8-wk-old BALB/c mice with PVM strain J3666, and identified pulmonary eosinophilia as an early response to PVM infection. This inflammatory response was accompanied by local production of the leukocyte chemoattractant MIP-1α (21). We show here that wild-type C57BL/6 mice responded to this virus in a similar fashion. The data in Table I demonstrate that lethal infection in C57BL/6 mice was established with as few as 20 PFU of virus. Furthermore, the time elapsed between inoculation and the onset of death (ultimately reaching 100% mortality in all cases shown) was directly related to the viral titer in the inoculum in a manner similar to that shown for BALB/c mice. Likewise, the lower respiratory tract infection established in C57BL/6 mice was accompanied by eosinophil and neutrophil recruitment (see below) and by local production of MIP-1α (Table II).

Table I.

Average time elapsed until deatha

PFU/MouseTime Elapsed (days ± SEM)
200 6.0 ± 0.32 
100 6.6 ± 0.24 
40 7.4 ± 0.24∗ 
20 8.2 ± 0.37∗∗ 
PFU/MouseTime Elapsed (days ± SEM)
200 6.0 ± 0.32 
100 6.6 ± 0.24 
40 7.4 ± 0.24∗ 
20 8.2 ± 0.37∗∗ 
a

Eight-week-old C57BL/6 mice (n = 5 mice/group) were inoculated on day 0 with PVM. Statistical significance: ∗, p < 0.005, ∗∗, p < 0.0005 in comparison with 200 PFU/mouse.

Table II.

Detection of MIP-1α in lung homogenates from wild-type C57BL/6 mice infected with 200 PFU PVMa

DayMIP-1α (pg/ml/mg protein)
C57BL/6BALB/c (60 PFU inoculated)a
InfectedUninfectedInfectedUninfected
6.7 ± 6.7 32 ± 16 
73 ± 11 133 ± 67 6.6 ± 4.1 
120 ± 21 103 ± 35 1.9 ± 1.9 
DayMIP-1α (pg/ml/mg protein)
C57BL/6BALB/c (60 PFU inoculated)a
InfectedUninfectedInfectedUninfected
6.7 ± 6.7 32 ± 16 
73 ± 11 133 ± 67 6.6 ± 4.1 
120 ± 21 103 ± 35 1.9 ± 1.9 
a

Taken from Ref. 21 for comparison purposes.

Levels of MIP-1α were measured in lung homogenates of MIP-1α+/+ and MIP-1α−/− mice that were inoculated intranasally with PVM (200 PFU). No MIP-1α was detected in lung homogenates of either +/+ or −/− mice before inoculation (day 0; Table III). At days 4 and 5 postinoculation, MIP-1α was detected in lung homogenates from all +/+, but not from any of the −/− mice.

Table III.

Detection of MIP-1α (pg/ml/mg protein) in lung homogenates of MIP-1α+/+ and MIP-1α−/− micea

DayGenotype
MIP-1α+/+MIP-1α−/−
37 ± 2.3 
55 ± 3.1 
DayGenotype
MIP-1α+/+MIP-1α−/−
37 ± 2.3 
55 ± 3.1 
a

Each mouse was inoculated with 200 PFU PVM on day 0.

Tissue sections of lung from MIP-1α+/+ and MIP-1α−/− mice harvested on day 3 after inoculation with 200 PFU PVM are shown in Fig. 1. As early as day 3 after inoculation, we observed numerous inflammatory foci in the lungs of the MIP-1α+/+ wild-type mice (Fig. 1,A). A higher power view of this field is shown in Fig. 1,B; two of the many eosinophilic leukocytes recruited to this inflammatory focus are indicated by arrows. In contrast, no inflammatory foci were observed in sections of lung tissue prepared from infected MIP-1α−/− mice (Fig. 1 C).

FIGURE 1.

Microscopic pathology. Sections of lung tissue obtained 3 days after inoculation of MIP1α+/+ mice (A, B) or MIP-1α−/− mice (C) with 200 PFU PVM. A and C, ×400; B, ×1000. Arrows in B point to the eosinophilic leukocytes that are recruited as part of the inflammatory response to this virus.

FIGURE 1.

Microscopic pathology. Sections of lung tissue obtained 3 days after inoculation of MIP1α+/+ mice (A, B) or MIP-1α−/− mice (C) with 200 PFU PVM. A and C, ×400; B, ×1000. Arrows in B point to the eosinophilic leukocytes that are recruited as part of the inflammatory response to this virus.

Close modal

An analysis of total leukocytes and leukocyte subpopulations recruited to the lungs of MIP-1α+/+ and MIP-1α−/− mice in response to infection with PVM is shown in Table IV. The inflammatory response in the MIP-1α+/+ mice was rapid and sustained, with >106 leukocytes/ml detected in BAL fluid harvested on day 3, remaining elevated through day 6. The predominant cell type at all time points tested is the neutrophil (polymorphonuclear leukocyte), although lymphocytes are present in significant numbers at days 3 and 6 postinoculation. From 10 to 15% of the lymphocytes detected at day 3 in wild-type mice were CD8+. Interestingly, the absolute eosinophil count varied substantially during the course of infection. The number of eosinophils present in the lungs rises from virtually 0 to 1.4 × 105/ml between days 2 and 3 postinoculation (at which point they represent ∼10% of the total leukocyte count), and then declines rapidly 10-fold (to ∼104/ml) by day 6 (p < 0.05). In contrast, only a minimal inflammatory response was observed in the PVM-infected MIP-1α−/− mice. Only ∼50 total leukocytes were detected in 1 ml BAL fluid harvested from MIP-1α−/− mice on day 3 postinoculation, a number that likewise remained stable thereafter. Virtually all the cells accumulating in response to infection in the MIP-1α−/− mice were neutrophils; few lymphocytes and no eosinophils were detected in any of the samples evaluated. Interestingly, eosinophils can be elicited in normal numbers in response to i.p. thioglycolate treatment of MIP-1α−/− mice (data not shown).

Table IV.

Total and differential counts of cells in BAL fluid obtained from MIP-1α+/+ and MIP-1α−/− micea

DayGenotype: MIP-1α+/+Genotype: MIP-1α−/−
Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)
<1       
<1    .011 ± 0.005 .008 ± 0.006 0.003 ± 0.002 
1660 ± 210 143 ± 31 1140 ± 200 380 ± 140 .049 ± 0.006 .049 ± 0.006 
860 ± 58 88 ± 20 770 ± 78 <1 .062 ± 0.032 .059 ± 0.033 0.001 ± 0.001 
690 ± 120 46 ± 27 640 ± 190 <1 .050 ± 0.014 .045 ± 0.011 0.003 ± 0.003 
850 ± 93 11 ± 7b 590 ± 69 305 ± 70 .022 ± 0.015 .020 ± 0.018 0.002 ± 0.001 
DayGenotype: MIP-1α+/+Genotype: MIP-1α−/−
Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)
<1       
<1    .011 ± 0.005 .008 ± 0.006 0.003 ± 0.002 
1660 ± 210 143 ± 31 1140 ± 200 380 ± 140 .049 ± 0.006 .049 ± 0.006 
860 ± 58 88 ± 20 770 ± 78 <1 .062 ± 0.032 .059 ± 0.033 0.001 ± 0.001 
690 ± 120 46 ± 27 640 ± 190 <1 .050 ± 0.014 .045 ± 0.011 0.003 ± 0.003 
850 ± 93 11 ± 7b 590 ± 69 305 ± 70 .022 ± 0.015 .020 ± 0.018 0.002 ± 0.001 
a

Each mouse was inoculated with 200 PFU PVM on day 0. <1, beneath limits of detection. Eos, eosinophils; PMNs, neutrophils; lymphs, lymphocytes.

b

Total eosinophils detected at day 6 differ from total eosinophils at detected at day 3, p < 0.05.

Viral titers (PFU PVM × 106/g) recovered from lungs harvested from MIP-1α+/+ and MIP-1α−/− mice on days 3, 4, and 5 after inoculation with 200 PFU PVM are shown in Fig. 2. Statistically significant differences were observed at day 5 after inoculation, with recoveries calculated at 1.4 ± 0.08 × 106 PFU/g from MIP-1α+/+ mice and 11 ± 2 × 106 PFU/g from MIP-1α−/− mice (p < 0.05).

FIGURE 2.

Viral recovery from mouse lung homogenates. Viral titers (PFU × 106/g) were determined by standard plaque assay from lung homogenates generated on days 3, 4, and 5 after inoculation with 200 PFU PVM. The comparison shown is between MIP-1α+/+ (•) and MIP-1α−/− (▪) mice. Statistical significance between the groups of +/+ and −/− mice is observed at day 5 postinoculation. ∗, p < 0.05.

FIGURE 2.

Viral recovery from mouse lung homogenates. Viral titers (PFU × 106/g) were determined by standard plaque assay from lung homogenates generated on days 3, 4, and 5 after inoculation with 200 PFU PVM. The comparison shown is between MIP-1α+/+ (•) and MIP-1α−/− (▪) mice. Statistical significance between the groups of +/+ and −/− mice is observed at day 5 postinoculation. ∗, p < 0.05.

Close modal

As shown in Table V, the CCR1 ligand MIP-1α was present in lung homogenates from CCR1+/+ and CCR1−/− mice infected with 200 PFU PVM. MIP-1α expression was somewhat delayed in the CCR1−/− mice, although the peak response observed exceeded that of their CCR1+/+ counterparts. Although the significance of this observation remains unclear, we can conclude that both CCR1+/+ and CCR1−/− mice are able to generate this proinflammatory mediator in response to viral infection.

Table V.

Detection of MIP-1α (pg/ml/mg protein) in lung homogenates from CCR1+/+ and CCR1−/− micea

DayGenotype
CCR1+/+CCR1−/−
62.5 ± 4.1 
161 ± 6.5 319 ± 83 
DayGenotype
CCR1+/+CCR1−/−
62.5 ± 4.1 
161 ± 6.5 319 ± 83 
a

Each mouse was infected with 200 PFU PVM on day 0.

An analysis of total leukocytes and leukocyte subpopulations recruited to the lungs in CCR1+/+ and CCR1−/− mice in response to infection with PVM is shown in Table VI. As anticipated from earlier results (Table IV), the peak leukocyte count in CCR1+/+ mice (5 × 105/ml) was sustained through day 5 and was composed of predominantly neutrophils, with eosinophils peaking (∼10%) at day 3, and ∼10% of the lymphocytes as CD8+. Similar to the observations made with the MIP-1α−/− mice, the peak leukocyte count in the CCR1−/− mice was dramatically reduced (>103-fold to ∼150 cells/ml total) and eosinophils were not detected.

Table VI.

Total and differential counts of cells in BAL fluid obtained from CCR1+/+ and CCR1−/− micea

DayGenotype: CCR1+/+Genotype: CCR1−/−
Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)
<1       
493 ± 54 42 ± 12 330 ± 3 120 ± 11 0.148 ± 0.059 0.140 ± 0.057 0.0083 ± 0.0017 
313 ± 20 22 ± 12 290 ± 14 <1 0.113 ± 0.033 0.107 ± 0.030 0.0067 ± 0.0033 
333 ± 40 24 ± 11 300 ± 18 <1 0.220 ± 0.017 0.212 ± 0.022 0.0034 ± 0 
DayGenotype: CCR1+/+Genotype: CCR1−/−
Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)Total (×103/ml)Eos (×103/ml)PMNs (×103/ml)Lymphs (×103/ml)
<1       
493 ± 54 42 ± 12 330 ± 3 120 ± 11 0.148 ± 0.059 0.140 ± 0.057 0.0083 ± 0.0017 
313 ± 20 22 ± 12 290 ± 14 <1 0.113 ± 0.033 0.107 ± 0.030 0.0067 ± 0.0033 
333 ± 40 24 ± 11 300 ± 18 <1 0.220 ± 0.017 0.212 ± 0.022 0.0034 ± 0 
a

Each mouse was infected with 200 PFU PVM on day 0. <1, beneath limits of detection. Abbreviations as per the legend to Table 4.

Viral titers (PFU PVM × 107/g) recovered from lungs harvested from CCR1+/+ and CCR1−/− mice on days 3, 4, and 5 after inoculation with 200 PFU PVM are shown in Fig. 3. Statistically significant differences were observed at two of the three time points, with recoveries calculated at 0.8 ± 0.12 × 107 and 5.2 ± 1.6 × 107 PFU/g from CCR1+/+ mice and 19 ± 5 × 107 and 41 ± 14 × 107 PFU/g from CCR1−/− mice on days 4 and 5 postinoculation, respectively (p < 0.05).

FIGURE 3.

Viral recovery from mouse lung homogenates. Viral titers (PFU × 107/g) were determined by standard plaque assay of lung homogenates generated on days 3, 4, and 5 after inoculation with 200 PFU PVM. The comparison shown is between CCR1+/+ (•) and CCR1−/− (▪) mice. Statistical significance between the groups (+/+ vs −/−) is observed at days 4 and 5 after inoculation. ∗, p < 0.05.

FIGURE 3.

Viral recovery from mouse lung homogenates. Viral titers (PFU × 107/g) were determined by standard plaque assay of lung homogenates generated on days 3, 4, and 5 after inoculation with 200 PFU PVM. The comparison shown is between CCR1+/+ (•) and CCR1−/− (▪) mice. Statistical significance between the groups (+/+ vs −/−) is observed at days 4 and 5 after inoculation. ∗, p < 0.05.

Close modal

When CCR1−/− and age-matched CCR1+/+ littermates were each infected with 10 PFU PVM, the first death of the CCR1−/− group occurred on day 6, with an average time until death calculated to be 7.8 ± 0.48 days after inoculation (Fig. 4). The first death of the CCR1+/+ group occurred on day 8, with an average time to death calculated at 9.2 ± 0.42 days (p < 0.001). These results suggest a protective function for CCR1 in antiviral host defense, most likely due to its role in recruiting eosinophils and neutrophils to the lung during the earliest stages of acute viral infection.

FIGURE 4.

Percentage of CCR1+/+ vs CCR1−/− mice surviving after inoculation with 10 PFU PVM. Average time until death for CCR1−/− mice given this inoculum on day 0 was 7.8 ± 0.48 days; for CCR1+/+ age-matched littermates, it was 9.2 ± 0.42 days (p < 0.001).

FIGURE 4.

Percentage of CCR1+/+ vs CCR1−/− mice surviving after inoculation with 10 PFU PVM. Average time until death for CCR1−/− mice given this inoculum on day 0 was 7.8 ± 0.48 days; for CCR1+/+ age-matched littermates, it was 9.2 ± 0.42 days (p < 0.001).

Close modal

In this study, we have shown that MIP-1α is a major determinant of the inflammatory response to PVM in mice, apparently acting via its receptor CCR1 on eosinophils and neutrophils. The presence of MIP-1α demonstrates a striking parallel between PVM infection in mice and acute RSV infection in humans, in that several groups have detected MIP-1α in supernatants of RSV-infected cultured epithelial cells (7, 11), and MIP-1α has been detected in both upper (30, 31) and lower respiratory tract secretions (7, 31) of infected children.

The inflammatory response to paramyxovirus infection is generally considered to be an unnecessary and unwanted physiologic response serving solely to exacerbate the disease process. We show here that MIP-1α-induced inflammation may be a protective response, as the absence of MIP-1α was associated with increased yields of infectious virus from lung tissue compared with those obtained from MIP-1α-competent mice. Specifically, on days 4 and 5 after inoculation with 200 PFU PVM, lung homogenates from the MIP-1α−/− mice contained ∼10 times as many infectious virions as did those of their fully competent counterparts. This observation suggests that the inflammatory response promoted by MIP-1α may have a role in blunting viral replication the earliest stages of this infection.

The studies focusing on CCR1, the major receptor for MIP-1α expressed in both eosinophils and neutrophils (36, 37, 41), support the notion of a protective role for the inflammatory response to PVM. Similar to what we observed with the MIP-1α−/− mice, BAL fluid samples from infected CCR1−/− mice contained ∼103 fold fewer neutrophils than did those from their infected CCR1+/+ littermates, and they were devoid of eosinophils. Interestingly, Gao et al. (41) reported that eosinophil recruitment proceeds normally in CCR1−/− mice with schistosome-induced granulomata. Taken together, these results demonstrate the existence of distinct CCR1-dependent and CCR1-independent pathways for eliciting tissue eosinophilia in vivo, analogous to those described for CCR3 in vitro (43). Other ligands recognized by CCR1 include RANTES and MCP-3. Several groups have demonstrated that human epithelial cells synthesize and secrete RANTES in response to RSV infection (7, 8, 9, 10, 11), and RANTES has been detected in response to RSV infection in upper and lower respiratory tract samples in several clinical studies (7, 30, 31). However, we have shown previously that RANTES is synthesized constitutively in mouse lung, with no modulation seen in response to PVM infection (21). In contrast, Becker et al. (10) reported that no detectable MCP-3 was produced in response to RSV infection in primary human bronchial epithelial cells; to the best of our knowledge, MCP-3 has not been associated with this infection either in vitro or in vivo.

Similar to our findings with MIP-1α−/− mice, deficiency of CCR1 and the concomitant reduction in pulmonary inflammation are also associated with an increased yield of virus from lung tissue. Furthermore, we have shown that the presence of CCR1 and the CCR1-mediated inflammatory response correlate with prolonged survival of PVM-infected mice. Specifically, the average time to death of CCR1−/− mice inoculated with 10 PFU PVM on day 0 was found to be 7.8 ± 0.48 days; the average time to death of their CCR1+/+ littermates was 9.2 ± 0.42 (p < 0.001). Although we cannot be certain that there is a direct causal relationship, these results indicate a strong correlation between prolonged survival and the ability to mount an acute inflammatory response to infection with PVM. This result is most interesting in light of the recent findings of Uzel et al. (44) documenting the enhanced risk and severity of RSV infection in individuals with genetic defects in cellular inflammatory responses.

Although antiviral activities of CD8+ cells (24, 45) and neutrophils and their components (44, 46, 47, 48, 49) have been discussed in the literature, we are particularly interested in the contribution of eosinophils to host defense against respiratory viral pathogens. We have shown in our earlier work that human eosinophils can reduce the infectivity of respiratory syncytial virus in vitro, an activity that is directly dependent on the activity of their unique secretory RNases, EDN and eosinophil cationic protein (13, 14, 15, 16). Consistent with this observation, Adamko et al. (50) reported impaired recovery of virions of the paramyxovirus, parainfluenza virus type 3, from the eosinophil-enriched lungs of allergen-sensitized guinea pigs. Given these findings, it is interesting to focus on the dramatic differences in eosinophil number and percentage when comparing the inflammatory responses of both sets of +/+ vs −/− mice in our study. Specifically, the highest eosinophil counts for both sets of +/+ mice occurred on day 3 after inoculation, peaking at ∼10% (1.4 × 105/ml and 0.42 × 105/ml; Tables IV and VI, respectively). In contrast, no eosinophils (0%, 0 cells/ml) were detected in any BAL fluid samples from either the MIP-1α−/− or the CCR1−/− mice. Although the large reduction in the number of neutrophils precludes any strong conclusions on the role of eosinophils in antiviral host defense, it remains possible that some (if not all) of the enhanced recovery of infectious virions and the earlier demise of the CCR1−/− mice may be attributable to the absence of these eosinophils. To the best of our knowledge, this is also the first study to provide genetic evidence linking MIP-1α and CCR1 to eosinophilic inflammation in vivo.

In conclusion, our results indicate that MIP-1α and its specific receptor CCR1 are host factors contributing to the inflammatory response to paramyxovirus infection in the lung as well as to control of viral replication and to the rate of disease progression. Moreover, our results suggest that leukocyte recruitment to the lung is beneficial in that it controls both the viral burden and clinical outcome. Additional work will be needed to define specific roles of neutrophils and eosinophils in the pathogenesis of pulmonary infection by paramyxoviruses.

We thank Howard Adams and the Building 7 animal facility staff for their care of the animals used in these experiments; and Dr. Harry L. Malech, Dr. John I. Gallin, and Dr. Leonard B. Weiner for their ongoing support of the work in progress in our laboratories.

3

Abbreviations used in this paper: RSV, respiratory syncytial virus; EDN, eosinophil-derived neurotoxin; PVM, pneumonia virus of mice; MIP-1α, macrophage inflammatory protein-1α; BAL, bronchoalveolar lavage; MCP-3, monocyte chemoattractant protein-3.

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