Introduction

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The collectin proteins (collagen-like lectins) are components of the multifaceted system that protects the mucosal surfaces of the respiratory tract from frequent exposure to pathogenic microorganisms and environmental allergens (1). In vitro, collectins inhibit infection by blocking pathogen-cell attachment (2), cause microbial aggregation (3), enhance phagocytosis and pathogen clearance (4, 5), enhance chemotaxis of macrophages (6), inhibit antigen- and mitogen-stimulated lymphocyte proliferation (7), regulate cytokine, chemokine, and radical production by macrophages, neutrophils, and eosinophils (10), and enhance antigen binding to immunocompetent cells (14, 15). Two members of the collectin family, surfactant protein (SP)-A and SP-D, are present in the lining fluid of the respiratory tract (16), in good position to provide immunologic surveillance and effector functions against both microbes and allergens.

Influenza A virus (IAV) is a common and serious human respiratory pathogen. The results of in vitro studies suggest that both SP-A and SP-D might contribute to the containment of IAV infection by direct inhibition of viral infectivity and/or promotion of viral uptake into phagocytic cells (17). Although both SP-A and SP-D attach to and neutralize certain strains of influenza virus, they appear to do so by different mechanisms (3). SP-A neutralizes IAV by directly occupying the sialic acid binding cleft in the viral surface glycoprotein hemaglutinin (HA) with the terminal sialic acid on the oligosaccharide located on the CRD of SP-A. Occupancy of this site blocks IAV-cell attachment (18). The binding of SP-A to influenza strains and SP-A-dependent neutralization in vitro are not influenced by the extent of HA glycosylation. In contrast, SP-D acts like a classic influenza -inhibitor by binding through the protein's carbohydrate-recognition domain to high-mannose oligosaccharides present at select sites on hemagglutinin (HA), a surface glycoprotein of IAV (3, 19). IAV strains lacking specific HA glycosylation sites are not as effectively neutralized by SP-D (19).

In addition to IAV neutralization in vitro, SP-A and SP-D agglutinate IAV, enhance neutrophil uptake, and potentiate influenza-induced neutrophil hydrogen peroxide responses (3, 20). SP-D is significantly more active than SP-A in these assays and SP-D appears to contribute most of the IAV-neutralizing activity in wild-type mouse bronchoalveolar lavage fluid (BALF), provided the IAV is a -sensitive strain (2). On the other hand, SP-A but not SP-D enhances rat alveolar macrophage uptake of IAV in vitro (21) and SP-A will neutralize IAV strains resistant to SP-D (3). At present, there is insufficient information to reliably translate the significance of these many in vitro studies to the physiologic situation. To isolate the role of SP-A during IAV infection in vivo, we have analyzed the response of mice deficient in SP-A but with normal amounts of SP-D to a strain of IAV lacking the major SP-D attachment site.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

SP-A-Deficient Mice

Mice deficient in SP-A were generated from 129 embryonic stem cells targeted with a replacement-type vector containing 1.1 kb and 8.9 kb homology regions of the murine SP-A gene. Exons 2, 3, and 4 of the SP-A gene, including the translation start site for and short segments of flanking intronic sequence, were replaced with Pgk-neo (1.8 kb) for positive selection (Poulain and colleagues, in preparation). Noncongenic SP-A+/+ and / littermates (F2 generation CD-1/129J) for this study were maintained in isolator cages in a barrier facility. Mice were 8-12 wk old when studied.

Virus Characterization

The influenza type A virus X-79 (H3N2) is a laboratory-derived, high yielding 6:2 reassortant of A/PR/8/34 (H1N1) and A/Phillipines/82 (H3N2) (22). The HA from A/Phillipines12/82, present on X-79, is potentially glycosylated at eight sites, including the critical asparagine at position 165 which determines -sensitivity (23). Parental X-79 replicates poorly in mice and causes almost no detectable illness (24). We obtained a mouse lung-adapted derivative of X-79 from Dr. Vaquelinokatz at the Centers for Disease Control (Atlanta, GA) that in preliminary experiments replicated well in wild-type mice and caused lethality. To determine if this virus had been selected for -resistance by its serial passage in mice, the HA1 domain of this derivative was amplified by RT-PCR and sequenced. For mouse inoculations, virus was grown in the allantoic cavity of 10-d embryonated hen's eggs, titered by standard hemagglutination and infectivity assays, and stored in aliquots at -80°C.

Mouse Model

To determine the mean lethal dose (LD₅₀), six mice of each genotype were lightly anesthetized with intraperitoneal ketamine and xylozine and inoculated intranasally with 50 µl allantoic fluid containing X-79167 serially diluted (10² to 10⁶) in endotoxin-free saline. The mice were weighed daily and observed for a total of 14 d. To further define the response to IAV infection, SP-A+/+ and SP-A/ mice were inoculated with a viral dose equal to 10 times the wild type LD₅₀ or with 50 µl saline alone. After 2 or 6 d, mice were killed by intraperitoneal phenobarbital. The lungs of four mice of each genotype at each time point were lavaged with 4 × 1 ml aliquots of 10 mM Tris, 100 mM NaCl, 0.2 mM EGTA, pH 7.4 for analysis of total protein, cell count and differential, and SP-A and SP-D levels. The unlavaged lungs of four mice of each genotype at each time point were frozen for RNA isolation, cytokine measurements, and determination of viral titers. Additional mice were killed for histology.

Cell Counts and Differentials

Bronchoalveolar lavage fluid (BALF) was centrifuged at 250 × g for 5 min at 4°C. The pellet was gently resuspended in 200 µl lavage buffer for cell counting. Cytospin slides were stained with Diff-Quik (Dade International, Miami, FL) for cell differential counts. A total of 20-25 high-power fields from four mice of each genotype at each time point were counted.

SP-A and SP-D Protein Measurements

The total protein content of the cell-free BALF was determined using bicinchoninic acid as a substrate. The protein concentration and volume of lavage were used to calculate the total lavage protein obtained from each mouse. Serial dilutions of cell-free BALF from mice of each genotype at 8 wk were analyzed for SP-A and SP-D content with a quantitative dot blot assay using monospecific polyclonal antibodies against recombinant mouse SP-A and SP-D, respectively. Standard curves using recombinant mouse SP-A and SP-D expressed in Chinese hamster ovary cells were used to determine the linear range of these assays and calculate absolute SP-A and SP-D BAL levels.

Cytokine mRNA and Protein Measurements

The mRNA levels of tumor necrosis factor (TNF)-, interleukin (IL)-6, interferon (IFN)-, macrophage inflammatory protein (MIP)-1, and MIP-2 were measured by RNase protection assay and normalized to L32 (RiboQuant; PharMingen, San Diego, CA). IL-6, IFN-, and MIP-2 levels were measured in tissue homogenates by sandwich ELISAs using standard protocols (Endogen, Woburn, MA).

Viral Titers

Two and six days after inoculation, lungs were homogenized in 1 ml Dulbecco's modified Eagle's medium. Viral titers were assayed by determining the tissue culture infectious dose in Madin- Darby canine kidney cells (TCID₅₀). The assays were performed in quadruplicate and the results presented as log (TCID₅₀) per gram of lung homogenate.

Microscopy

The lungs from two mice of each genotype at 2 and 6 d after inoculation were fixed intratracheally at 20 cm H₂O with 4% freshly prepared paraformaldehyde in 0.1M phosphate buffer and then prepared for paraffin sectioning using standard techniques. Midsagittal hematoxylin and eosin sections of the right lung were examined for morphologic changes.

Statistical Methods

All values are presented as mean ± standard deviation of the mean. Differences between groups were determined by two-sample t test assuming unequal variance. A P value of  0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Generation of SP-A/ Mice

The SP-A gene was inactivated in embryonic stem cells by standard homologous recombination techniques. Homozygous / mice had no detectable SP-A mRNA or protein (data not shown). SP-A null homozygous and heterozygous mice maintained in a barrier facility were indistinguishable by appearance, weight, and activity from wild-type littermates. Infection was not detected in unchallenged mice maintained in isolator cages in a barrier facility. A full description of the gene-targeting and unchallenged phenotype will be described elsewhere (Poulain and colleagues, in preparation).

Sequence of IAV HA

The entire HA1 domain of the mouse adapted X-79 strain was amplified by RT-PCR and sequenced. A point mutation in codon 167 resulted in an amino acid change from threonine to isoleucine and the loss of the consensus sequence for glycosylation of the critical -type collectin binding site at position 165.

Dose-Dependent Lethality of X-79167

Unlike the parental X-79 strain (24), X-79167 caused a lethal illness in both +/+ and / mice. The LD₅₀ was significantly less in / mice (10⁴) compared with their +/+ littermates (10^5.6) (Figure 1). SP-A/ mice also lost significantly more weight (average of 21%) than their +/+ littermates (average of 12%) after inoculation with a dose of virus equivalent to 10 times the +/+ LD₅₀ (n = 6, P < 0.05).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Kaplan-Maier survival curves after intranasal inoculation of X-79167 virus. On Day 0, mice 8-12 wk of age (6 mice of each genotype at each dose) were inoculated with 50 µl saline containing 10-fold dilutions (102 to 106) of a stock of X-79167 virus. The calculated LD50 of +/+ mice (104 dilution) was significantly greater than the LD50 of SP-A/ mice (105.6). Top panel: SP-A+/+ mice; bottom panel: SP-A/ mice. Open squares, 102; filled diamonds, 103; filled circles, 104; open triangles, 105; and filled squares, 106. Note the data points for 103 and 104 dilutions overlap with the points for 102 dilution from Days 11 and 12, respectively in the lower panel.

Viral Replication

The mean viral titers in the lungs of / mice trended higher on both Days 2 and 6 compared with +/+, but the differences were not significant on either day (P = 0.09 on Day 2 and 0.3 on Day 6). Virus was also cleared poorly from the lungs of both groups (Figure 2). As expected, virus was not detected in controls given saline.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Viral titers in the lung after infection with IAV. SP-A+/+ mice (open bars) and SP-A/ mice (closed bars) were inoculated intranasally with 50 µl saline or a 10× WT LD50 dose of X-79167 on Day 0. On Days 2 and 6, groups of four mice per genotype were killed. There was no significant difference between WT and SP-A/ mice in the viral load (mean ± SD) on either Day 2 or Day 6.

Total BAL Protein, Cell Counts, and Differentials

Cell-free BAL protein was not significantly different between saline-treated / or +/+ mice on either Day 2 or Day 6. Cell-free BAL protein after inoculation with X-79167 was unchanged on Day 2 but was significantly elevated with IAV infection from saline-treated controls by Day 6 in both +/+ and SP-A/ mice. The total BAL cell counts followed a similar pattern with no significant elevation on Day 2 with infection but a significant similar increase in total BAL cells on Day 6 after infection in both genotypes. There was a significant increase in the absolute number (Table 1) and percentage of neutrophils in the infected SP-A/ mice compared with infected +/+ mice on Day 2 (19 ± 8% in / compared with 8 ± 5%, n = 4, P < 0.05). The neutrophil counts (absolute counts or percentage of total cells) were not significantly different on Day 6. Saline-treated animals of either genotype had less than 1% neutrophils at both time points.

Cytokines

TNF-, IL-6, MIP-2, and MIP-1 mRNA levels were not significantly different in saline-treated +/+ and / mice on Day 2 or Day 6. On Day 2 after IAV inoculation, TNF- and MIP-2 mRNA levels were significantly but similarly increased compared with saline-treated controls in both +/+ and / infected mice. IL-6 and MIP-1 mRNA levels were significantly increased in / mice but not in infected +/+ mice on Day 2. IFN mRNA levels were unchanged in both genotypes on Day 2. By Day 6, all cytokine mRNA levels were significantly increased in IAV-infected mice compared with saline-treated mice. No differences between infected +/+ and / mice were seen for TNF-, MIP-1, or MIP-2 mRNA levels, but IL-6 and IFN- mRNA levels were significantly higher in infected +/+ compared with infected / mice (Table 2).

IL-6, IFN-, and MIP-2 protein levels were measured in duplicate in lung homogenates. Because no significant differences were seen between genotypes given saline only, the uninfected controls were pooled for the data presented in Figures 4 and 5. MIP-2 was not detectable in the lung homogenates of mice given saline only at any time point. On both Day 2 and Day 6 after infection, MIP-2 was significantly more elevated in / compared +/+ (Figure 3). There were no differences in IL-6 or IFN- levels between infected and uninfected mice or between +/+ and / mice on Day 2. On Day 6, both IL-6 and IFN- were significantly more elevated in +/+ than / mice (Figure 4). TNF- and MIP-1 protein levels were not measured in this set of experiments.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Lung homogenate IL-6 and IFN- levels on Day 6. On Day 6, IL-6 and IFN- protein levels were significantly elevated in lung homogenates of IAV-infected compared with saline-treated mice of both genotypes. IL-6 and IFN- protein were significantly higher in SP-A+/+ infected mice (open bars) compared with SP-A/ infected mice (closed bars) on Day 6 (*P < 0.05).

View larger version (70K): [in this window] [in a new window]   Figure 5.   Histology of saline and IAV inoculated mice on Day 6. (A) SP-A+/+ mouse inoculated with saline. SP-A/ mice inoculated with saline are similar to the +/+ (not shown). (B) SP-A+/+ mice infected with X-79167. (C) SP-A/ mouse infected with X-79167. Bar: 100 µm.

View larger version (12K): [in this window] [in a new window]   Figure 3.   Lung homogenate MIP-2 levels. MIP-2 levels (mean ± SD) were measured in lung homogenates on Days 2 and 6 in SP-A+/+ (open bars) and SP-A/ mice ( filled bars). No MIP-2 was detected in saline-treated mice of either genotype on either day. MIP-2 levels were significantly higher in SP-A/ mice infected with X-79167 on both Day 2 and Day 6 (*P < 0.05).

Histology

On Day 2 after infection, there was very little change in distal airway or alveolar histology. Scattered areas of airway epithelial injury were seen in both / and +/+ mice. By Day 6, airway epithelial injury increased in / mice compared with +/+ mice. Uninfected +/+ and / mice had intact airway epithelium without evidence of inflammation. Infected +/+ mice had numerous inflammatory cells within and overlying a generally intact airway epithelium, whereas the airways of infected / mice were largely missing an intact epithelial layer (Figure 5). The alveolar parenchyma of infected / mice was qualitatively more edematous and congested (and had a greater number of inflammatory cells in the airspace lumen than infected +/+ mice on Day 6.

SP-A and SP-D Levels

As expected, SP-A was not detected in BALF from / mice. In +/+ mice, SP-A levels were not significantly different after infection on Day 2 or Day 6 compared with saline controls. In contrast, in +/+ mice BAL SP-D levels increased 1.7-fold on Day 2 and 6.8-fold on Day 6 (P < 0.05 on both days) (Figure 6). In / infected mice, the changes were less dramatic although in the same direction (1.1-fold increase on Day 2 and 4.4-fold increase on Day 6, P < 0.05 on Day 6).

View larger version (13K): [in this window] [in a new window]   Figure 6.   SP-A and SP-D levels in SP-A+/+ mice after infection with X-79167 virus. The results (mean of four mice per time point) are expressed as a fold change from the saline treated mice. SP-D (open bars) was significantly (*P < 0.05) increased in IAV-infected mice while there was no significant change in SP-A levels ( filled bars) on either day.

	Discussion

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In this study, mice with a targeted disruption of the SP-A gene succumbed to IAV infection more readily than wild-type littermates. Despite having a viral burden comparable to that of +/+ mice, / mice showed exaggerated early inflammation with MIP-2 protein levels and a greater influx of neutrophils. An increased inflammatory response to infection of SP-A-deficient mice has been previously reported following group B streptococcal, pseudomonas, respiratory syncytial virus infection, and adenoviral infection (25). Although our results are broadly in line with these studies which all reported an increased inflammatory response to pathogen challenge, the specific nature and magnitude of the response varies considerably between studies. Factors that probably contribute to this variability include the genetic background of the mice, the pathogen, dose, route of infection, and time of sampling. For our studies we used outbred mice, a well-defined mouse-adapted virus, intranasal infection and sampled on Days 2 and 6 after inoculation with a lethal dose of virus. Day 2 was chosen to look at the inflammatory response at the time of expected maximal viral replication, and Day 6 was chosen as the day before the onset of mortality with the dose used. The cytokine response to IAV infection is very dynamic (29) and we would expect significantly different profiles had we sampled on other days after infection.

In the previous reports of pathogen challenge in SP-A/ mice, pathogen replication was greater and clearance was delayed, making it difficult to distinguish between a dysregulated host response and an appropriately upregulated response to a greater pathogen load (25). Our results suggest a dysregulated host response to -resistant IAV infection in the absence of SP-A as the viral load was similar in SP-A/ and +/+ mice. We do not know whether this pattern of response is specific to IAV or is limited to the dose and particular viral strain used in this study. Our findings are similar to the enhanced mortality and pathology with IAV infection after exposure to UV radiation without any change in viral burden or early TNF- response (30). SP-A/ mice also display a dysregulated host response to a strain of endotoxin that does not bind SP-A in vitro (10).

Although the mice used for this study were not congenic, littermate controls were used to standardize as much as possible potential genetic modifiers of the host response. The unchallenged phenotype of the / mice will be described in detail elsewhere, but relevant to this report, we found no differences between SP-A/ mice and their +/+ littermates in lung histology, surfactant lipid pools, surfactant activity, or BAL cell analysis (data not shown). These findings are generally consistent with the reported description of an independently derived SP-A deficient line in the 129J background (31).

Both SP-A and SP-D have IAV-neutralizing activity in vitro (3). The available in vitro data suggest SP-D binding to -sensitive IAV strains is of significantly higher avidity and contributes most, if not all, the viral neutralizing activity in human BALF (2). We determined that SP-D levels were comparable between uninfected / and +/+ mice and rose significantly in both genotypes in response to IAV infection. SP-D binds and neutralizes IAV in a strain-specific fashion. To address the role of SP-A during IAV infection independent of the effects of SP-D, we used an IAV strain that was sensitive to SP-A-neutralizing activity but resistant to SP-D. It has been previously reported that poorly glycosylated influenza strains or mouse- and bovine-adapted strains with a mutation that prevents N165 glycosylation bind SP-D weakly and are not neutralized effectively by SP-D (19). We confirmed that X-79167, the strain used in our studies, had lost the consensus sequence required for the N-linked glycosylation of the asparagine at position 165 that is important for SP-D but not SP-A- neutralizing activity. Despite the inability of SP-D to bind X-79167, we detected a significant increase in SP-D levels in BALF on both Days 2 and 6 in IAV-infected mice. This result is consistent with elevated SP-D levels in mice infected with -sensitive strains of IAV (19). In our studies, there was no corresponding increase in SP-A levels, suggesting the two collectins are differentially regulated during IAV infection. Although SP-D levels increased, we would not expect a major role for SP-D in X-79167 infection as SP-D does not bind or neutralize this IAV strain effectively in vitro. It is possible that SP-D could have influenced the host immune response to X-79167 by weakly interacting with the virus or by interacting in a nonopsonic fashion with immune-competent cells contributing to the IAV response. Mice deficient in both SP-A and SP-D will be needed to address this possibility.

Further studies are needed to explain the shift in LD₅₀ in the SP-A/ mice. Infection with IAV causes severe hypophagia and decreased locomotion in mice with food and water intake dropping to almost nothing within 72 h of infection with virulent virus (32). This "sickness behavior" leads to severe weight loss and certainly contributes to mortality after IAV infection. Although increased cytokine release, specifically TNF-, IL-1, and IL-6 in some studies, may contribute to sickness behavior, cytokine inhibitors did not prevent wasting in at least one mouse model of IAV infection (32). Nevertheless, an altered early response to IAV in SP-A/ mice might contribute to the exaggerated weight loss and increased lethality we observed. We have not identified the actual mediators or mechanisms involved. Direct lung injury might also contribute to the increased mortality. Qualitative histology suggested significantly more airway damage with extensive loss of epithelium in the / mice by Day 6. Surprisingly, the increase in IFN- and IL-6 levels in lung homogenates (both mRNA and protein) in response to IAV were blunted in / compared with +/+ mice. Although several cell types might contribute to the late rise in IFN- in mice infected with IAV, IAV-specific cytotoxic T cells are probably the most important source (33). These cells are thought to be critical for the clearance of IAV during an established infection (33). Further studies will be needed to determine if the IAV-specific T cell response is blunted in SP-A/ mice or whether other elements of the host response, such as dysregulated reactive oxygen or nitrogen species generation (34), contribute to the increased lethality.