Pathogenecity of the 1918 Virus: functionnal roles of alveolar macrophages and neutro

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Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice

Terrence M. Tumpey,¹^* Adolfo Garc?a-Sastre,² Jeffery K. Taubenberger,³ Peter Palese,² David E. Swayne,⁴ Mary J. Pantin-Jackwood,⁴ Stacey Schultz-Cherry,⁵ Alicia Sol?rzano,² Nico Van Rooijen,⁶ Jacqueline M. Katz,¹ and Christopher F. Basler² Influenza Branch, Mail Stop G-16, DVRD, NCID, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., Atlanta, Georgia,¹ Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029,² Department of Molecular Pathology, Armed Forces Institute of Pathology, Rockville, Maryland 20850,³ Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 934 College Station Road, Athens, Georgia 30606,⁴ Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706,⁵ Department of Molecular Cell Biology, Vrije University, Amsterdam, The Netherlands⁶
Received 25 July 2005/ Accepted 7 September 2005
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<table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> ABSTRACT </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The Spanish influenza pandemic of 1918 to 1919 swept the globeand resulted in the deaths of at least 20 million people. Thebasis of the pulmonary damage and high lethality caused by the1918 H1N1 influenza virus remains largely unknown. Recombinantinfluenza viruses bearing the 1918 influenza virus hemagglutinin(HA) and neuraminidase (NA) glycoproteins were rescued in thegenetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91)virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91virus was lethal for BALB/c mice without the prior adaptationthat is usually required for human influenza A H1N1 viruses.The increased mortality of 1918 HA/NA:Tx/91-infected mice wasaccompanied by (i) increased (>200-fold) viral replication,(ii) greater influx of neutrophils into the lung, (iii) increasednumbers of alveolar macrophages (AMs), and (iv) increased proteinexpression of cytokines and chemokines in lung tissues comparedwith the levels seen for control Tx/91 virus-infected mice.Because pathological changes in AMs and neutrophil migrationcorrelated with lung inflammation, we assessed the role of thesecells in the pathogenesis associated with 1918 HA/NA:Tx/91 virusinfection. Neutrophil and/or AM depletion initiated 3 or 5 daysafter infection did not have a significant effect on the diseaseoutcome following a lethal 1918 HA/NA:Tx/91 virus infection.By contrast, depletion of these cells before a sublethal infectionwith 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growthand mortality in mice. In addition, neutrophil and/or AM depletionwas associated with decreased expression of cytokines and chemokines.These results indicate that a human influenza H1N1 virus possessingthe 1918 HA and NA glycoproteins can induce severe lung inflammationconsisting of AMs and neutrophils, which play a role in controllingthe replication and spread of 1918 HA/NA:Tx/91 virus after intranasalinfection of mice.
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<table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> INTRODUCTION </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The influenza pandemic of 1918 was exceptional in that it resultedin an estimated 20 to 50 million deaths worldwide (9, 32, 50,59). Approximately 30% of the world's population was clinicallyinfected during the pandemic (15), and there was an unusuallyhigh lethality rate among healthy adults aged 15 to 34 years,a finding that has not been observed in subsequent pandemicsor epidemics caused by influenza A viruses (40). Histopathologicalanalysis of lung tissues from individuals who died from primaryinfluenza pneumonia in 1918 showed massive pulmonary edema withunique destruction of the alveolar architecture (35, 58, 72).The retrieval of viral RNA from archived autopsy materials andfrom the body of an Alaskan influenza victim buried in permafrosthas revealed the genetic code of this H1N1 subtype influenzavirus. Analyses of the complete coding sequences of the 1918hemagglutinin (HA), neuraminidase (NA), matrix, nonstructural,and nucleoprotein genes have not revealed the molecular basisfor its extreme virulence (4, 48-52, 58).
Plasmid-based reverse genetics has allowed for the generationof recombinant viruses containing one or more of the 1918 influenzavirus genes entirely from cloned cDNAs (4, 19, 30, 34, 64, 65).Using this approach, we previously generated recombinant virusespossessing the 1918 HA and/or NA segments rescued on the geneticbackground of the mouse-adapted A/WSN/33 (1918 HA/NA:WSN) virus(30, 64, 65). Although the full virulence of the 1918 pandemicvirus is most likely due to the constellation of virus genes,initial emphasis was placed on the HA and NA glycoproteins (30,34, 64, 65), since they are the major viral surface antigensand are important virulence factors in birds and mammals (22,25, 44, 74). Infection with the 1918 HA/NA:WSN virus resultedin severe morbidity and mortality in BALB/c mice, whereas arecombinant control virus possessing the HA and NA of the contemporaryA/New Caledonia/20/99 (H1N1) virus (N. Cal. HA/NA:WSN) inducedminimal lung pathology and was not lethal. Histological characterizationof 1918 HA/NA:WSN-infected mouse lungs demonstrated severe pulmonarylesions consisting of severe bronchitis and alveolitis as earlyas 72 h following an intranasal infection (30). More-recentstudies by Kobasa et al. (34) confirmed these results and alsoindicated that the 1918 HA alone confers increased replicationproperties and lethality in mice to human influenza viruses.Additional studies are needed to further characterize the pulmonaryinflammation and the functional roles of inflammatory cellsthat contribute to the unique pathogenic properties observedwith the 1918 HA and NA recombinant influenza viruses.
In its uncomplicated form, a human influenza A virus infectionof the H1 or H3 subtype results in viral replication in therespiratory epithelium and leads to the induction of an inflammatoryinfiltrate comprised mainly of mononuclear leukocytes, withlower levels of polymorphonuclear leukocytes (neutrophils) (1).Recovery from influenza infection is dependent on virus-specificT and B cells (10, 20, 74). However, neutrophils and alveolarmacrophages (AMs) can respond more quickly to virus infectionand play important roles in the pulmonary disease process (23,54, 56). AMs are the primary mononuclear phagocytes of the uninflamedlung alveoli and are considered the first cells of the immunesystem to encounter inhaled organisms and antigens. Therefore,they function as the major scavenger cells in the lower respiratorytract, and upon appropriate stimulation, these cells can releaseproinflammatory cytokines and chemokines (5, 39, 54). Neutrophilsmay also contribute to the release of cytokines, and althoughthey traditionally have been viewed as being important in fightingbacterial infections, these cells can be involved in virallyinduced pathology (46, 62, 67).
The basis for the exceptional virulence of the 1918 pandemicinfluenza virus remains elusive but most certainly involveshost responses to influenza infection. Because little is knownabout the mechanism(s) by which the 1918 influenza virus causedsevere lung pathology and death, this study was undertaken toinvestigate the pathogenesis in mice of a recombinant influenzavirus possessing the 1918 HA and NA glycoproteins. The geneticbackground of the remaining influenza genes used for the recombinant1918 virus was derived from a human influenza A H1N1 1991 strain,A/Texas/36/91 (1918 HA/NA:Tx/91) virus, which has been characterizedin humans under experimental settings (26) and is nonpathogenicin mice. Characterization of the 1918 HA/NA:Tx/91 virus revealeda high pathogenicity phenotype in mice in comparison to thatof the parental Tx/91 virus. An additional feature of the lethal1918 HA/NA:Tx/91 virus was its ability to induce elevated levelsof cytokines, AMs, and neutrophils in the infected mouse lung.While increased numbers of AMs and neutrophils in lungs contributeto the inhibition of virus replication as part of the host innateimmune response, they might also contribute to the enhancedimmunopathology and, therefore, be partially responsible forthe lethal phenotype of the 1918 HA/NA:Tx/91 virus. Using depletiontechniques, we found that these cells play a role in the initialstages of the innate immune response in limiting virus replication,virus spread, and lethality during 1918 HA/NA:Tx/91 virus infection.
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<table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> MATERIALS AND METHODS </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation of 1918 HA and NA cDNAs and recombinant viruses. The 1918 HA and NA cDNAs were constructed by PCR with overlappingdeoxyoligonucleotides corresponding to the published sequenceof the influenza A/South Carolina/1/18 (H1N1) virus HA (48)open reading frame or the influenza A/Brevig Mission/1/18 (H1N1)virus NA open reading frame (49). The noncoding regions of eachsegment are identical to those of the corresponding segmentof influenza A/WSN/33 (WSN) H1N1 virus. Recombinant viruseswere generated using the reverse genetics system of Fodor etal. (14) following the methods of Basler et al. (4). Virusespossessing 1918 HA and NA genes in a WSN (1918 HA/NA:WSN) or1918 HA/NA:Tx/91 virus background were generated under biosafetylevel 3 (BSL-3 with enhancements) containment (3) to ensurethe safety of laboratory workers, the environment, and the public.All subsequent laboratory and animal work with live virus alsowas performed under these high-stringency containment conditions.The control viruses included a whole parental Tx/91 virus rescued(rTx/91) in a similar manner and a wild-type Tx/91 stock providedby A. Klimov (Centers for Disease Control and Prevention, Atlanta,Georgia). The identity of the 1918 influenza virus genes inthe recombinant viruses was confirmed by reverse transcription-PCRand sequencing. Primer sequences and PCR conditions are availableupon request.
Infection of mice. Female BALB/c mice, 6 to 7 weeks old (Charles River Laboratories,Wilmington, Mass.), were anesthetized with an intraperitoneal(i.p.) injection of 0.2 ml of 2,2,2-tribromoethanal in tert-amylalcohol (Avertin; Aldrich Chemical Co., Milwaukee, Wis.), and50 ?l of infectious virus diluted in phosphate-bufferedsaline (PBS) was inoculated intranasally (i.n.). Fifty-percentlethal dose (LD₅₀) titers were determined by inoculating groupsof three mice i.n. with serial 10-fold dilutions of virus. LD₅₀titers were calculated by the method of Reed and Muench (47).For determination of lung virus titers, morbidity (measuredby weight loss), and mortality at multiple time points duringthe first week of infection, 24 additional mice were infectedi.n. with the highest inoculating dose (10⁶ PFU) of virus. Individualbody weights from eight mice were recorded for each group ondays 0, 2, 4, 6, 8, and 10 postinfection (p.i.) and monitoreddaily for disease signs and death for 14 days p.i. On days 2,4, 6, and 8 p.i., four mice from each group were euthanatized,and whole lungs, spleen, and brain tissues were removed asepticallyand homogenized in 1 ml of sterile PBS. After this process,homogenates were titrated for virus infectivity in 10-day-oldembryonated eggs (36). The statistical significance of virustiter data was determined by using analysis of variance (ANOVA).Fifty-percent egg infectious dose (EID₅₀/ml) titers were calculatedby the method of Reed and Muench (47).
Lung homogenates along with bronchoalveolar lavage (BAL) samples(described below) were also individually tested for bacterialinfection by routine bacteriological methods for the isolationand identification of bacteria. Briefly, serial 10-fold dilutionsof lung homogenates (day 4 p.i.) or BAL samples (days 0, 3,5, and 7 p.i.) were transferred to a tryptic soy agar plate,blood agar, chocolate agar, or a MacConkey agar plate (FisherScientific, Pittsburgh, PA). Five lung homogenates collectedon day 4 p.i. and 20 BAL samples collected on p.i. days 0, 3,5, and 7 per group were tested from both the mock control andinfected groups. Three individual bacteriological loops wereplated from each sample, and the number of CFU was determinedafter 72 h of incubation at 37?C.
Histopathology. Three mice from each group were euthanatized on day 5 p.i. A5-mm lung sample from the ventral end of each left lobe wascollected for histopathology, as were brain, lung, heart, spleen,liver, kidney, thymus, and bronchial lymph node samples. Duplicatesections were immunohistochemically stained to demonstrate influenzaA viral protein. The primary antibody was produced in chickensthat had been immunized twice with A/chicken/Wisconsin/68 (H5N9)virus and was used at a 1:100 dilution. This antibody recognizesconserved internal virus proteins of multiple subtypes. Thesecondary antibody was a goat anti-chicken immunoglobulin G(IgG) tagged with horseradish peroxidase (Southern Biotech,Birmingham, AL), and the chromogen diaminobenzidine (DAB Pluskit; Zymed, San Francisco, CA) was used for visualization. Forthe lung sections, the numbers of all viral antigen-positiverespiratory epithelial cells and macrophages in a minimum offive high-powered fields (HPF) were determined by counting,and averages were determined for individual mice and for treatmentgroups.
BAL sample and blood cell counts. BAL sample cells were obtained from euthanatized mice as previouslydescribed (31). Briefly, the BAL sample cells were washed fromthe lungs of anesthetized mice in 1 ml of PBS containing 1%bovine serum albumin (Sigma, St. Louis, MO). Absolute cell countswere performed by diluting cell suspensions 1:5 in Turks solution(2% acetic acid, 0.01% methylene blue). The cell numbers fromfive individual mice per group were determined in triplicateby counting in a hemocytometer. Portions were cytospun ontoglass microscope slides and stained with Hema 3 stain (FisherScientific), and the percentages of monocytes, polymorphonuclearneutrophils, and lymphocytes were determined. At least 100 cellswere counted for each slide; counts were performed in triplicateat a magnification of x1,000. For differential cell counts,peripheral blood samples (20 to 40 ?l) were obtained fromthe orbital plexus of two or three mice on days 1, 3, 5, and7 p.i., and smears were stained with Hema 3 stain. For eachsample, at least 100 cells were counted at a magnification ofx1,000. The percentage of leukocytes was determined followingtreatment with monoclonal antibody (MAb) RB6-8C5 or controlrat IgG antibody (described below).
Depletion of neutrophils and AMs. The hybridoma RB6-8C5, which produces a rat IgG2b anti-mousegranulocyte MAb, was a gift from R. Coffman, DNAX Research Institute(Palo Alto, CA). The antibody was purified from hybridoma culturemedium by affinity chromatography with protein G-Sepharose FastFlow (Pharmacia, LKB Biotechnology, Piscataway, NJ) and quantifiedby use of a radial immunodiffusion kit (ICN Immunobiologicals,Costa Mesa, Calif.). Each mouse received one i.p. injectionof 0.5 mg of purified antibody 5 h before infection and anotheron day 3 p.i. To maintain sufficient depletion of neutrophils,mice received 1 mg of antibody on days 5 and 7 p.i. Controlmice received rat IgG antibody (Sigma) in place of MAb RB6-8C5.The same treatment regimen also was followed for mice that receivedMAb RB6-8C5 treatment after infection, except that the firsti.p. injection of MAb RB6-8C5 was initiated on day 3 or 5 p.i.Thus, the first and second i.p. injections consisted of 0.5mg of MAb followed by 1 mg of antibody every other day. Fordepletion of AMs, liposome-encapsulated dichloromethylene diphosphonate(clodronate) (L-CL₂MDP) and liposomes containing PBS (L-PBS)were prepared as previously described (66). Dichloromethylenediphosphonate was a kind gift of Roche Diagnostics, Mannheim,Germany. BALB/c mice were anesthetized with Avertin before i.n.inoculation with L-CL₂MDP or L-PBS in a 50-?l volume ondays ?4 and ?2 before virus infection. In experimentswhere AMs were depleted after infection, mice received L-CL₂MDPor L-PBS on days 3 and 5 p.i or on days 5 and 7 p.i. This procedureresults in the selective depletion of AM so that other cellsremain unaffected after the elimination procedure (60, 69, 70).The toxicity of L-CL₂MDP was tested in five mice that were notlater infected with virus. In comparison to na?ve or L-PBS-treatedmice, no overt signs of clinical illness, including weight loss,were observed among animals that received L-CL₂MDP.
Cytokine and chemokine quantitation. To determine the in vivo levels of cytokine or chemokine proteins,five mice per group were exsanguinated from the axilla and euthanatized,and lung tissues were removed from na?ve and infected mice(day 5 p.i.). Individual whole-lung samples were immediatelyfrozen at ?70?C. On the day of analysis, tissues werethawed, homogenized in 1 ml of cold PBS, and centrifuged at150 x g for 5 min. The clarified cell lysates were assayed foralpha interferon (IFN-; assay sensitivity, 12.5 pg/ml), IFN-(assay sensitivity, 2 pg/ml), tumor necrosis factor alpha (TNF-;assay sensitivity, 5.1 pg/ml), macrophage inflammatory protein-1alpha (MIP-1; assay sensitivity, 1.5 pg/ml), and MIP-2 (assaysensitivity, 1.5 pg/ml) by use of enzyme-linked immunosorbentassay (ELISA) kits purchased from R & D Systems (Minneapolis,MN). Statistical significance of lung cytokine data was determinedby ANOVA.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction and characterization of recombinant viruses with 1918 HA and NA influenza virus genes. Genes encoding the 1918 pandemic influenza virus were reconstructedfrom deoxyoligonucleotides and corresponded to the reported1918 virus coding sequences (48, 49, 58). The HA and NA genesof the 1918 virus rescued in the genetic background of Tx/91(1918 HA/NA:Tx/91) or WSN (1918 HA/NA:WSN) virus had high infectivitytiters in MDCK cells and 10-day-old embryonated chicken eggs,similar to those of the wild-type Tx/91, rTx/91, and parentalrescued WSN (rWSN) viruses (Table 1). BALB/c mice were inoculatedi.n., and weight loss, virus replication, and LD₅₀ titers weredetermined. For comparison, groups of mice were infected withthe highly pathogenic 1918 HA/NA:WSN or rWSN virus, both ofwhich were previously shown to have high lethality in mice (65).Infection of mice with the 1918 HA/NA:Tx/91 recombinant virusresulted in lung virus titers that were approximately 250-foldhigher than those for rTx/91-infected mice on day 4 p.i. (Table1). The 1918 HA/NA:WSN and rWSN viruses were more lethal (LD₅₀10^2.75) than the 1918 HA/NA:Tx/91 virus (LD₅₀ of 10^4.75). Incontrast to the lethal outcomes of infection with the 1918 recombinantviruses, the rescued Tx/91 virus and its wild-type counterpartdid not kill mice and resulted in only minimal weight reduction.To better understand the differences in pathogenicity amongthe H1N1 recombinant viruses, we examined the kinetics of virusreplication in lung, brain, and spleen, the percent weight loss,and overall survival throughout the course of 1918 HA/NA:Tx/91virus infection. Mice infected i.n. with 10⁶ PFU of virus weresacrificed on days 2, 6, and 8 p.i. to determine virus titers(Fig. 1A). On days 2 and 6 p.i., virus titers recovered fromthe lungs of mice infected with the Tx/91 control viruses weresignificantly lower than those from the 1918 HA/NA:Tx/91-infectedmice. 1918 HA/NA:Tx/91-infected mice were unable to clear theinfection, showed signs of illness (e.g., ruffled fur and hunchedposture), and began to lose significant amounts of weight 4days after infection (Fig. 1B). Weight loss continued in 1918HA/NA:Tx/91-infected mice, and mortality reached 100% by day8, with a mean death time (MDT) of 7.3 days (Fig. 1C). In contrast,all Tx/91-infected mice survived the infection and displayedminimal weight reduction on day 2 p.i. only. We examined whetherthe 1918 HA/NA:Tx/91 virus replicated systemically in the mouseby removing brain, heart, and spleen tissues on day 6 p.i. (foursamples per tissue), when virus titers in these tissues aremaximal following infection with highly pathogenic influenzaviruses (36). All eight mice infected with 1918 HA/NA:Tx/91or Tx/91 control viruses had undetectable levels of virus (10^0.8EID₅₀/ml) in the brain, heart, and spleen tissues (not shown).
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<!-- null --> <nobr></nobr> <table border="1" width="100%"><tbody><tr><td><table cellpadding="0" cellspacing="10" width="100%"> <tbody><tr> <td rowspan="2" align="center" valign="middle">Virus^a</td> <td colspan="2" align="center" valign="bottom">Titer^b
<hr noshade="noshade" size="1"></td> <td rowspan="2" align="center" valign="middle">% Weight loss^c</td> <td rowspan="2" align="center" valign="middle">Lung titer (log₁₀ EID₅₀/ ml ? SE)^d</td> <td rowspan="2" align="center" valign="middle">LD₅₀^e</td> </tr> <tr> <td align="center" valign="bottom">log₁₀ EID₅₀/ml</td> <td align="center" valign="bottom">PFU/ml</td> </tr> <tr><td colspan="7"><hr></td></tr><tr> <td align="left" valign="top">Wild-type Tx/36/91</td> <td align="center" valign="top">8.2</td> <td align="center" valign="top">2.0 x 10⁷</td> <td align="center" valign="top">0.7</td> <td align="center" valign="top">3.3 ? 0.2</td> <td align="center" valign="top">>6</td> </tr> <tr> <td align="left" valign="top">Parental rTx/36/91</td> <td align="center" valign="top">8.7</td> <td align="center" valign="top">4.4 x 10⁷</td> <td align="center" valign="top">0.1</td> <td align="center" valign="top">3.6 ? 0.3</td> <td align="center" valign="top">>6</td> </tr> <tr> <td align="left" valign="top">Parental rWSN</td> <td align="center" valign="top">8.2</td> <td align="center" valign="top">2.2 x 10⁷</td> <td align="center" valign="top">21.8</td> <td align="center" valign="top">7.2 ? 0.2</td> <td align="center" valign="top">2.75</td> </tr> <tr> <td align="left" valign="top">1918 HA/NA:Tx/36/91</td> <td align="center" valign="top">8.7</td> <td align="center" valign="top">2.5 x 10⁷</td> <td align="center" valign="top">15.9</td> <td align="center" valign="top">6.0 ? 0.2</td> <td align="center" valign="top">4.75</td> </tr> <tr> <td align="left" valign="top">1918 HA/NA:WSN</td> <td align="center" valign="top">8.5</td> <td align="center" valign="top">2.1 x 10⁷</td> <td align="center" valign="top">21.7</td> <td align="center" valign="top">7.0 ? 0.3</td> <td align="center" valign="top">2.25</td> </tr> </tbody></table></td></tr></tbody></table>
<!-- tblfn -->^a All viral genomic segments derived from the WSN or Tx/91 virus unless otherwise indicated.
<!-- tblfn -->^b Calculated by the method of Reed and Muench (47) from titrations in eggs and MDCK cells.
<!-- tblfn -->^c Average percent weight loss on day 4 postinfection (five mice per group).
<!-- tblfn -->^d Average lung titers of four mice on day 4 postinfection.
<!-- tblfn -->^e Expressed as the log₁₀ PFU required to give 1 LD₅₀.




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</th> </tr> </tbody></table> <table style="width: 1px; height: 20px;" border="0" cellpadding="2" cellspacing="2"> <tbody><tr> <th bgcolor="#0033b4" nowrap="nowrap" width="50%">Clin. Vaccine Immunol.</th> <th bgcolor="#0033b4" nowrap="nowrap" width="50%">ALL ASM JOURNALS</th> </tr> </tbody></table>

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</nobr> </td><td align="left" valign="top"> TABLE 1. Properties of recombinant influenza viruses used in this study
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</nobr> </td><td align="left" valign="top"> FIG. 1. Comparison of lung virus titers (A), weight loss rates (B), and mortality rates (C) of BALB/c mice infected with 10⁶ PFU of 1918 HA/NA:Tx/91 (), rescued parental Tx/91 (?), and wild-type Tx/91 () virus. Four mice from each virus-infected group were euthanatized on the indicated days p.i., and titers of individual lungs were determined in eggs. The limit of virus detection was 10^1.2 EID₅₀/ml. The remaining eight mice from each group were observed for weight loss and mortality through a 14-day observation period. Asterisks indicate that the 1918 HA/NA:Tx/91 lung titers are significantly (P < 0.05) different than those of both Tx/91 controls, as determined by ANOVA.
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Mouse lung pathology. The presence of gross and histopathologic changes and virusantigen in the mouse lung was determined at 5 days p.i., a timethat immediately preceded the death of mice infected with thelethal 1918 HA/NA:Tx/91 virus. The three mice infected withthe control Tx/91 viruses had normal lungs in terms of color,texture, and size, with only occasional small dark red fociof pneumonia (Fig. 2a). Histologically, the lungs were normalor had minimal-to-mild bronchitis with lymphocytic-to-histiocyticperibronchitis and mild histiocytic alveolitis (Fig. 2b and c).By contrast, mice infected with the 1918 HA/NA:Tx/91 recombinantvirus generally had within the lungs large pneumonic areas primarilyaffecting the ventral portions of each of the lung lobes (Fig.2d). Histologically, a mild-to-severe necrotizing bronchitiswith associated moderate-to-severe alveolitis and moderate-to-severepulmonary edema was observed in these lungs (Fig. 2e). A strikingfeature of the 1918 HA/NA:Tx/91-infected lungs was the prominentnumber of neutrophils in the peribronchium and alveoli (Fig.2f). In comparing the two viruses, lungs from mice infectedwith rTx/91 and from those infected with wild-type Tx/91 virusgroups had 14.2 and 9.9 neutrophils/HPF of alveoli, respectively,while lungs from mice infected with 1918 HA/NA:Tx/91 had 68.3neutrophils/HPF of alveoli. To demonstrate that a similar pathologycould be induced with a second 1918 HA/NA recombinant virus,three mice were infected with the 1918 HA/NA:WSN virus, andhistopathologic changes were determined at day 5 p.i. The lungsfrom the 1918 HA/NA:WSN-infected mice also showed a severe necrotizingbronchitis with associated alveolitis (Fig. 2g). The moderate-to-severealveolitis was characterized by abundant AMs and neutrophils(Fig. 2h). Viral antigen commonly could be seen in necroticcellular debris within bronchial lumina and alveoli, and frequently,within AMs. The virus-positive respiratory epithelial cells(Fig. 2i and j) were infrequent in all groups, as evidencedby the presence of only 0.05, 0.28, and 0.3 infected epithelialcells/HPF in the rTx/91, wild-type Tx/91, and 1918 HA/NA:Tx/91virus-infected mouse lungs, respectively. The frequencies ofvirus-positive macrophages in the alveoli were also low in theTx/91 control virus groups (0 and 1.35/HPF) (Fig. 2k), but thefrequency was at least threefold higher in the lungs of 1918HA/NA:Tx/91 virus-infected mice (4.5/HPF). These histologicalresults indicate that neutrophils and AMs are prominent celltypes associated with and potentially mediating the severe lungpathology, especially in alveoli, following lethal 1918 HA/NArecombinant virus infection. <!-- null -->

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</nobr> </td><td align="left" valign="top"> FIG. 2. Pathologies of mice intranasally inoculated with 10⁶ PFU of rescued parental Tx/91, 1918 HA/NA:Tx/91, or 1918 HA/NA:WSN recombinant influenza viruses. Gross photographs (a and d) and photomicrographs (b, c, and e through h) of hematoxylin and eosin-stained tissue sections are shown. (a) Lungs of a mouse inoculated with Tx/91 virus; lungs at day 5 p.i. appear normal except for a small focus of pneumonia in left lung lobe. Bar = 5 mm. (b) Mild lymphocytic peribronchitis with mild alveolitis in a mouse inoculated with Tx/91; day 5 p.i. Bar = 200 ?m. (c) Predominant peribronchial histiocytic alveolitis with iatrogenic atelectasis in a mouse inoculated with Tx/91; day 5 p.i. Bar = 25 ?m. (d) Large areas of pneumonia affecting most of the left lung and cardiac lobe of the right lung. Smaller areas of pneumonia in apical and azygous lobes of the right lung of a mouse inoculated with 1918 HA/NA:Tx/91; day 5 p.i. Bar = 5 mm. (e) Diffuse severe alveolitis in a mouse inoculated with 1918 HA/NA:Tx/91; day 5 p.i. Bar = 200 ?m. (f) Severe neutrophilic-to-histiocytic alveolitis adjacent to bronchiole with necrotizing bronchitis. Furthermore, some alveoli have pulmonary edema evident as protein crescents in a mouse inoculated with 1918 HA/NA:Tx/91; day 5 p.i. Bar = 20 ?m. (g) Severe necrotizing bronchitis with severe alveolitis in a mouse inoculated with 1918 HA/NA:WSN; day 5 p.i. Bar = 100 ?m. (h) Higher magnification of panel g showing prominent necrotic debris in bronchial lumen (labeled "B" within panel) and abundant neutrophils and histiocytes in alveoli. Bar = 20 ?m. (j) Two virus-positive bronchial epithelia (arrowheads) in a mouse inoculated with 1918 HA/NA:Tx/91; day 5 p.i. Bar = 10 ?m. (k) Virus-positive AMs (arrowheads) in a mouse inoculated with 1918 HA/NA:Tx/91; day 5 p.i. Bar = 10 ?m.
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Because of the observed increases of lung neutrophils and theirdocumented association with secondary bacterial infections,it was of interest to determine whether the 1918 HA/NA:Tx/91virus-infected lungs harbored levels of bacteria greater thanthose in the Tx/91-infected or uninfected lungs. Twenty individualsamples from the BAL specimens collected on days 0, 3, 5, and7 p.i. (five per time period) and lung homogenates collectedon day 4 p.i (five mice per group) were evaluated for the presenceof bacteria. In general, there was no tendency for bacteriato be isolated more frequently or at higher levels from the1918 HA/NA:Tx/91-infected mouse lungs than from rTx/91-infectedor uninfected lungs (data not shown). For BAL specimens, 15%(3 of 20) of Tx/91-infected and 20% (4 of 20) of uninfectedmice were colonized with bacteria, whereas 5% (1 of 20) of BALsamples collected from the 1918 HA/NA:Tx/91-infected lungs demonstratedbacterial growth. Among the positive BAL samples, bacterialgrowth ranged from 10² to 10³ CFU/ml. The proportions of lunghomogenates that yielded growth of bacteria were also similarfor all three groups tested. In these samples, no viable bacteriawere present in either normal or 1918 HA/NA:Tx/91-infected lungs,and only one of five rTx/91-infected lung homogenates presentedlow bacterial growth, which ranged from <10¹ to 10² CFU/mlamong the triplicate samples tested. Taken together, these resultsindicated that the HA and NA genes of the 1918 virus enhancedthe virulence of the Tx/91 virus in mice and that this enhancementdoes not appear to be due to a higher incidence of coinfectionwith bacteria. Neutrophils and alveolar macrophages contribute to host defense after 1918 HA/NA:Tx/91 recombinant virus infection. To better characterize the cellular components in the lung followinga primary 1918 HA/NA:Tx/91 virus infection, total and differentialcell counts of BAL sample infiltrates were collected and analyzedfrom infected mice at 0.75 (18 h), 3, 5, and 7 days p.i. (Table2). The total BAL sample cells recovered from both the 1918HA/NA:Tx/91 virus-infected and control Tx/91-infected mice showedincreases of infiltrates as early as 18 h p.i., with cell numbersincreasing 1.5- to 6.7-fold by day 7 p.i. However, much largerincreases in total BAL sample cells were observed in 1918 HA/NA:Tx/91virus-infected lungs. During 1918 HA/NA:Tx/91 virus infection,peak cellular infiltration in the BAL samples on days 5 and7 p.i. was associated with a substantial increase in the numberof infiltrating neutrophils compared with that for control Tx/91-infectedmice. The lymphocyte and macrophage cell counts of the 1918HA/NA:Tx/91 virus-infected lungs generally followed patternssimilar to those of counts for control mice, except elevatedlevels of BAL sample macrophages were observed on days 3 and5 p.i. These results suggest that 1918 HA/NA:Tx/91 virus inducesa predominant neutrophil infiltrate and moderate macrophageincreases into the mouse lung that may be contributing to thepathogenesis of the enhanced disease associated with 1918 HA/NA:Tx/91virus infection.
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</nobr> </td><td align="left" valign="top"> TABLE 2. Total and differential counts in BAL specimens following infection with 1918 recombinant viruses
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The role of neutrophils and AMs in 1918 HA/NA recombinant virus-inducedlung pathology was investigated by eliminating such cells beforeand/or after infection with 1918 HA/NA:Tx/91 virus. Profoundneutrophil depletion could be achieved in BALB/c mice by MAbRB6-8C5 treatment. Blood samples from two or three animals werecollected 1 h before each i.p. injection in order to monitorthe neutrophil count. The depletion protocol produced and maintaineda depletion of blood neutrophils in a range from 96 to 99% withno apparent adverse affect on the percentages of lymphocytesand macrophages (Fig. 3A). The MAb RB6-8C5 protocol of depletingneutrophils was also efficient in the BAL samples without noticeablyaffecting the percentages of lymphocytes and macrophages (Table2). Moreover, RB6-8C5 MAb was unable to bind CD4⁺ or CD8⁺ Tcells, thymocytes, monocytes/macrophages, or B cells in flowcytometric experiments (38, 62; also data not shown), suggestingthat RB6-8C5 MAb has no detectable cross-reactivity with theseother cell types. Depletion of AM was performed by i.n. instillationof L-Cl₂MBP twice (days ?4 and ?1) before infection.This procedure resulted in an 82.7% reduction in AM numbersfor BAL samples compared with those for L-PBS-treated controlmice on day 2 p.i. (Fig. 3B) without adversely affecting theblood monocyte percentages (Fig. 3A) or neutrophil numbers (Table2). We initially investigated whether depleting neutrophilsand/or AMs after infection would reduce the pathogenesis ofthe 1918 recombinant virus. Groups of 12 mice were i.n. infectedwith a lethal dose (20 LD₅₀) of 1918 HA/NA:Tx/91 virus, andtreatment with MAb RB6-8C5 or L-Cl₂MBP was initiated on day3 p.i. Control mice received rat IgG in place of MAb RB6-8C5or L-PBS in place of L-Cl₂MBP. Virus replication, weight loss,and mortality rates among the neutrophil- and AM-depleted micewere compared with those among their IgG- and L-PBS-treatedcounterparts. Although neutrophils and AMs were barely detectablein the lungs of the treated mice, depletion of these two celltypes initiated after 1918 HA/NA:Tx/91 virus infection failedto reduce morbidity and mortality in comparison with those forcontrol animals (data not shown). Furthermore, the lethalitiesfor the neutrophil-depleted, AM-depleted, and control groupswere 100%, with MDTs of 7.4, 7.1, and 7.9, respectively. Theamounts of infectious virus recovered from the lungs of neutrophil-depletedand AM-depleted mice were significantly (P < 0.05) higher(7.1 ? 0.2 log₁₀ EID₅₀/ml and 7.4 ? 0.2 log₁₀EID₅₀/ml, respectively) in comparison to the amount from untreatedcontrol mice (6.0 ? 0.3 log₁₀ EID₅₀/ml), suggesting thatthese cells suppress influenza growth in the lung. In a secondindependent experiment, delaying neutrophil and/or AM depletionuntil day 5 p.i also resulted in levels of morbidity and mortalityfor treated animals that were comparable to those for controlanimals (data not shown). <!-- null -->

<center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top">
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</nobr> </td><td align="left" valign="top"> FIG. 3. Efficiency of neutrophil and AM depletion (depl) in vivo. (A) Effect of neutrophil and AM depletion on blood leukocyte counts. For neutrophil depletion, BALB/c mice received repeated i.p. injections of MAb RB6-8C5 or control rat IgG beginning 5 h before i.n. virus infection and on days 3, 5, and 7 after infection to maintain depletion. Neutrophil depletion was monitored 1 h before each MAb injection by performing differential cell counts on blood smears. The results at day 7 p.i. are shown and are expressed as the mean percentages of monocytes (monos), neutrophils (PMNs), and lymphocytes (lymphs). (B) For AM depletion, mice received 50-?l doses of L-Cl₂MBP i.n. on days ?4 and ?2 relative to the time of i.n. virus infection. Control mice received 50-?l doses of L-PBS (PBS-Lip). AM depletion was monitored on days 1, 3, 5, and 7 p.i. by collecting cells from lung BAL samples from infected mice, as described in Materials and Methods. The results at day 7 p.i. are shown and are expressed as the number of AMs in the BAL sample.
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We next examined what effect neutrophil and/or AM depletionwould have if depletion was initiated before a sublethal (0.2LD₅₀) 1918 HA/NA:Tx/91 virus infection. Accordingly, groupsof mice (20 per group) were depleted of neutrophils (by MAbRB6-8C5 treatment) and/or depleted of AMs. At various timesfollowing sublethal virus infections with 1918 HA/NA:Tx/91 virus,the lung and brain tissues of MAb RB6-8C5- and L-Cl₂MBP-treatedmice were collected for determination of infectious virus titers.Figure 4A shows that the mean virus titers in the lung weresignificantly higher at each time period examined in neutrophil-and/or AM-depleted animals compared with those of the controls.Infectious virus was also present in the brains of MAb RB6-8C5-treatedand/or L-Cl₂MBP-treated mice (Fig. 4B) but was not detectedin the control animals. At the sublethal infectious dose of1918 HA/NA:Tx/91 virus, symptoms of hunched posture, lethargy,and death were not observed in untreated control animals. However,we found that most neutrophil- and/or AM-depleted mice lostsignificant amounts of weight and were listless during the firstweek of infection; these symptoms were taken as signs of morbidity(data not shown). All AM-depleted mice succumbed to infectionby day 8, whereas 4 of 10 mice depleted of neutrophils died(Fig. 4C). All mice depleted of both AMs and neutrophils rapidlysuccumbed to virus infection, and death occurred slightly soonerthan it did in groups depleted of AM or neutrophils only. Incontrast, all L-PBS and rat IgG control mice survived the sublethalvirus infection. In a second independent experiment, both neutrophilsand AMs were depleted before a sublethal virus infection with0.2 LD₅₀ of 1918 HA/NA:Tx/91 virus or 10⁶ PFU of rTx/91 virus.All depleted mice challenged with 10⁴ PFU 1918 HA/NA:Tx/91 virusdied. In contrast, we did not observe any change in morbidityor mortality in mice challenged with 10⁶ PFU of rTx/91 virus.These results demonstrate that AMs and neutrophils play a significantrole in controlling the replication and spread of 1918 HA/NA:Tx/91virus after infection. <!-- null -->

<center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top">
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</nobr> </td><td align="left" valign="top"> FIG. 4. Depletion (depl.) of neutrophils (PMN) and AM results in increased lung virus titers (A), brain virus titers (B), and lethality (C). Neutrophils were depleted in BALB/c mice (20 mice/group) by repeated i.p. injections of MAb RB6-8C5 () or control rat IgG (?) 5 h before a 1918 HA/NA:Tx/91 virus infection and on days 3, 5, and 7 p.i to maintain depletion. For AM depletion, i.n. administration of 50 ?l of L-Cl₂MBP () on days ?4 and ?2 relative to the time of 1918 HA/NA:Tx/91 virus infection was performed. Control mice received 50-?l doses of L-PBS (). An additional group of mice was depleted of both AMs and neutrophils (). Mice were i.n. infected with sublethal doses of 0.2 LD₅₀ of 1918 HA/NA:Tx/91 virus. On the days indicated, mice (four per group) were sacrificed, and individual lung and brain tissues were homogenized in 1 ml of PBS. The titers of the lysates for virus infectivity in eggs were determined. The remaining eight mice were followed daily for survival. The key corresponds to panels A and B. Asterisks indicate that the control lung titers are significantly (P < 0.05) different from lung titers from depleted mice, as determined by ANOVA.
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Cytokine and chemokine expression in lungs of 1918 HA/NA-infected mice. Following primary infection of mice with influenza A viruses,many cytokines and chemokines are produced in the lung (7, 27,63) and are believed to contribute to the recruitment/activationof protective immune cells, thus facilitating the antiviraldefense against influenza infection. Individual lung tissueswere collected on day 5 p.i., a time point with maximal lungcytokine/chemokine levels following infection with highly pathogenicinfluenza strains (63). Tissues were homogenized, and lysateswere assayed for cytokines and chemokines by ELISA. Determinationof IFN-, IFN-, TNF-, MIP-1, and MIP-2 levels demonstrated thatall cytokines and chemokines were produced above their constitutivelevels 5 days after infection with both 1918 HA/NA:Tx/91 andrTx/91 virus (Fig. 5A). Although the lung IFN- levels were similarfor both virus-infected groups, IFN-, TNF-, and both MIP-1 andMIP-2 chemokines were detected at significantly higher levelsin 1918 HA/NA:Tx/91-infected mice than in Tx/91-infected mice.Concentrations of TNF- were low compared with concentrationsof the other cytokines measured, but protein levels of thiscytokine in 1918 HA/NA:Tx/91-infected mice were fourfold higherthan those in Tx/91-infected mice. <!-- null -->

<center><table cellpadding="0" cellspacing="0" width="95%"><tbody><tr bgcolor="#e1e1e1"><td><table cellpadding="2" cellspacing="2"> <tbody><tr bgcolor="#e1e1e1"><td align="center" bgcolor="#ffffff" valign="top">
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</nobr> </td><td align="left" valign="top"> FIG. 5. Lung cytokine/chemokine activity in 1918 HA/NA:Tx/91 virus-infected lungs (A) and AM- and/or neutrophil (PMN)-depleted (depl.) mice (B). At day 5 p.i., five individual lung tissues were removed from each treated group and frozen at ?70?C. Samples were thawed and homogenized in 1 ml of PBS, after which the clarified cell lysates were assayed by ELISA. The mean cytokine level ? standard deviation for each cytokine was determined. In addition, the constitutive cytokine level present in each tissue was determined by harvesting individual lung tissues from four uninfected BALB/c mice. The constitutive lung cytokine levels were 22, 27, and 35 pg/ml for IFN-, IFN-, and TNF-, respectively. The constitutive lung chemokine levels were 14 and 17 pg/ml for MIP-1 and MIP-2, respectively. (A) Asterisks indicate that the levels for the 1918 HA/NA:Tx/91-infected group were significantly (P < 0.05) different from those for the Tx/91-infected control group, as determined by ANOVA. (B) For AM depletion, mice received 50-?l doses of L-Cl₂MBP i.n. on days ?4 and ?2 relative to the time of 1918 HA/NA:Tx/91 virus infection. Control mice received 50-?l doses of L-PBS. For neutrophil depletion, mice received i.p. injections of MAb RB6-8C5 or control rat IgG beginning 5 h before 1918 HA/NA:Tx/91 virus infection and on day 3 after infection to maintain depletion until the day of tissue collection (day 5 p.i.). Asterisks indicate that the control lung cytokine/chemokine levels are significantly (P < 0.05) different than those detected in depleted mice, as determined by ANOVA.
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Since the previous experiments established that AM and neutrophilsplay a significant role in helping control the replication andspread of the 1918 HA/NA:Tx/91 virus after i.n. infection, wenext determined whether these cells influenced the expressionof cytokines/chemokines associated with virus infection. Wefound that neutrophil depletion was accompanied by a significantreduction of cytokine and chemokine expression in the infectedlungs (Fig. 5B). Compared with expression in rat IgG controlmice, IFN- and IFN- levels were decreased by 81% and 87%, respectively,and TNF- and MIP-2 levels fell by 24% and 37%, respectively.The level of the beta chemokine MIP-1 was also significantlylower (55%) in mice depleted of neutrophils than that in nondepletedcontrol mice. Treatment of na?ve mice with L-PBS or L-Cl₂MBPalone did not increase the production of detectable lung cytokinesor chemokines above the constitutive levels. AM-depleted micealso had decreased cytokine levels in the lungs following 1918HA/NA:Tx/91 virus infection. Marked decreases in IFN-, IFN-,TNF-, and MIP-1 levels were observed in AM-depleted mice, whereasthe level of MIP-2 was moderately reduced compared with cytokinelevels in the L-PBS control mice. These results suggest thatthe 1918 HA/NA glycoproteins possess molecular determinantsthat are capable of inducing high levels of cytokines/chemokinesin the mouse lung and that AMs and neutrophils are crucial fortheir optimal production. <!-- null -->
<table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> DISCUSSION </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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During the 1918 influenza pandemic, pathologists observed atautopsy severe destruction of lung tissue unlike that typicallyseen in cases of pneumonia. Histological observations of archivedfixed tissues from 1918 showed heavy infiltrations of whiteblood cells into influenza-infected lungs, including the alveoli,suggesting that such cells were involved in the pathogenesisof disease (35, 71, 72). The studies described herein were basedon a previous observation that the lethal 1918 HA/NA:WSN recombinantinfluenza virus and the nonlethal New Caledonia HA/NA:WSN controlvirus induced markedly different patterns of mouse lung inflammation(30). Although both viruses induced a necrotizing bronchitis,which is characteristic of uncomplicated influenza virus infections,histological samples of the lungs of 1918 HA/NA:WSN-infectedmice also displayed moderate-to-severe alveolitis characterizedby increases of AMs and neutrophils. Kobasa et al. (34) alsoobserved histologically a massive recruitment of neutrophilsinto the mouse lung following an intranasal infection of a 1918HA recombinant virus. In the current study, a recombinant viruspossessing the 1918 HA and NA genes on the genetic backgroundof a Tx/91 virus replicated efficiently in the mouse lung andwas lethal for this species, with an MDT of 7.3 to 7.9 days.Severe disease following 1918 HA/NA:Tx/91 virus infection wasdose dependent, with only high doses of virus (10⁵ PFU) causingsignificant morbidity and mortality. In contrast, the parentalTx/91 H1N1 virus did not cause mortality in mice at any virusdose and replicated to only low titers in mouse lungs. Thus,the 1918 HA/NA genes conferred upon the Tx/91 internal genecomplex the property to grow to high titers in the respiratorytract without the need for prior adaptation, which is generallyrequired for the replication of human H1N1 viruses in mice (28).In comparison to the Tx/91-infected control lungs, the 1918HA/NA:Tx/91-infected lungs displayed a heavy inflammatory infiltrationmarked by a predominance of neutrophils associated with an increaseof AMs and cytokines shortly before the death of these mice.To address the functional role of neutrophils and AMs during1918 HA/NA:Tx/91 virus infection, we depleted mice of thesecells and determined the severities of disease and degrees ofvirus clearance. In comparison to immunocompetent controls,AM- and neutrophil-depleted mice displayed higher virus titersin the lung tissue, which spread systemically to the brain afterinfection from an otherwise sublethal dose in nondepleted animals.In addition, depletion of neutrophils and/or AMs resulted insignificantly lower levels of cytokines and chemokines in infectedlung tissues. As a consequence, AM- and/or neutrophil-depletedmice displayed greater morbidity and mortality following sublethal1918 HA/NA:Tx/91 virus infection.
It is unclear why the lethal 1918 HA/NA:Tx/91 and parental Tx/91strains of influenza vary in their capacities to induce neutrophilmigration into the mouse lung, particularly the alveoli. Inthe recipients of the Tx/91 control virus infections, lung tissuesdisplayed a relatively modest inflammatory response accompaniedby clearance of infectious virus and viral antigen by day 8p.i. The inflammatory cells in these lungs appear restrictedto the level of the bronchial epithelium and parabronchial area.In contrast, 1918 HA/NA:Tx/91 virus infection induced an increasein neutrophils that could be detected as early as 18 h afterinfection, and by day 5 the neutrophil influx was evident bothin the peribronchium and alveoli. Furthermore, the pulmonaryneutrophilia did not appear to be due to a higher incidenceof coinfection with bacteria but was associated with highertiters of infectious virus and greater detected levels of viralantigen, including in the alveoli. However, the extent of viralreplication alone does not appear to account for the enhancedmouse lung pathology observed among 1918 recombinant viruses(30, 34). Further studies to elucidate the actual stimuli forneutrophil accumulation into the mouse lung are under way. Avariety of inflammatory mediators, including complement factors(29) and chemokines (7, 55), could cause neutrophil migration.Chemokine expression closely follows the level of influenzavirus replication and affects the type of inflammatory responseto infection (68). We focused on MIP-1 and MIP-2, since thesechemokines have been shown to activate and exert chemotacticeffects on neutrophils, although MIP-1 is also a potent chemoattractantfor monocytes and activated T cells (73, 75). It has been demonstratedthat influenza-infected mice carrying a disrupted MIP-1 (^?/?)gene have reduced inflammation and delayed clearance of thevirus when compared with infected wild-type (^+/+) mice (7);thus, MIP-1 may play an important role during influenza virusinfection. The greater expression of MIP-1 and MIP-2 chemokinesin the 1918 HA/NA:Tx/91-infected lungs relative to those inTx/91-control lungs correlates well with the increased neutrophilinflux observed on days 5 and 7 p.i.
Conceivably, excessive accumulation of neutrophils and AMs ina vital organ, such as the lung, could contribute to tissuedamage by causing vascular injury and destruction of the parenchymalcells (2, 37). The resulting loss of functional alveolar surfacearea would result in inadequate gas exchange, lower respiration,and, ultimately, death. In fact, deterioration of pulmonaryfunction has been observed in patients with prolonged neutropenia(2). Furthermore, the overproduction of cytokines, such as IFN-and TNF-, may be detrimental to the host and may be responsiblefor the enhanced morbidity and mortality by influenza virusinfection (8, 11, 57, 60). Increased expression of TNF- hasbeen associated with highly pathogenic influenza H5N1 virusesin vitro and has been noted in postmortem lung tissue of H5N1fatal cases (6, 42). TNF- can also function to enhance neutrophilicinfiltration into the lung tissue (33). We found that the expressionof TNF- was two- to threefold higher in the 1918 HA/NA:Tx/91-infectedlungs preceding the death of these mice (day 5 p.i.) than thatin to Tx/91-infected control lungs. Analysis of IFN- cytokinelevels in 1918 HA/NA:Tx/91-infected mouse lungs also demonstrateda significant increase over that in Tx/91-infected control lungs.Neutrophils elicit numerous responses in the presence of IFN-;these responses may include increased oxidative burst, inductionof antigen presentation, and chemokine production (12, 13).The neutrophils in the 1918 HA/NA:Tx/91-infected lungs may havebeen one source of the cytokines and chemokines that were observed,since neutrophil depletion resulted in significantly lower levelsof TNF-, IFN-, and both MIP chemokines. Migration of neutrophilsinto the lung is thought to occur in a cascading manner. Ina cycle that repeats itself, infiltrating neutrophils and AMsproduce more cytokines/chemokines, which in turn chemoattractadditional cells into the lung and thus increase lung inflammation(54, 77).
The notion of neutrophils and AMs driving the increased lungpathology and contributing to mortality following 1918 HA/NA:Tx/91virus infection was addressed in our initial depletion studies.However, we found that the depletion of neutrophils and/or AMsafter the initiation of lung inflammation had no effect on theoverall disease outcome following lethal challenge with the1918 HA/NA recombinant virus. Thus, despite effective and substantialdepletion of neutrophils and AMs in the lungs of treated animals,these mice displayed severe morbidity and had MDTs in the rangeof those of untreated control animals (7.1 to 7.4 days). However,it was observed in these initial experiments that delayed depletioncaused slightly greater viral burden in the infected lungs,suggesting that these cells may function either directly orindirectly to control virus replication in the lung. We subsequentlyfound that if mice were depleted of neutrophils and/or AMs beforea sublethal infection with 1918 HA/NA:Tx/91 virus, the animalsdisplayed significant weight loss and increased mortality. Furthermore,the virus spread systemically to the brain, a property not seenwith the immunocompetent control mice infected with the 1918HA/NA:Tx/91 virus. These results suggest that although neutrophilsare the predominant cell in the 1918 HA/NA:Tx/91-infected lungand may contribute to the pathology, they are essential forhost defense by controlling the replication and spread of virusafter a primary infection. This antiviral role of neutrophilsextends the findings of previous studies (16, 17, 41, 61), includingthose that identified antiviral substances with IFN-like activityagainst human and bovine herpes viruses (53). It remains unresolvedhow neutrophils exert their antiviral effect against influenza,as their defense capabilities are quite diverse. Neutrophilshave been shown to adhere to influenza virus-infected cellsand to phagocytose influenza virions in vitro (16, 46). It hasbecome clear that mature neutrophils, although limited in theircapacity to synthesize and secrete proteins, nevertheless producea variety of mediators, some of which may have antiviral activity.The production of immune mediators, such as myeloperoxidase(76), defensins (18), and reactive oxygen and/or nitrogen species(16, 56), by activated neutrophils may directly inhibit influenzagrowth in the lung. The production of IFN- or antiviral cytokines,such as TNF-, has antiviral activity against influenza (45,75).
Our results also show that the elimination of AM has a dramaticeffect on the host defense to 1918 HA/NA:Tx/91 virus infection.While only some of the neutrophil-depleted mice died followinga sublethal 1918 HA/NA:Tx/91 virus infection, 100% mortalitywas observed for AM-depleted mice. Similar to the response withneutrophil depletion, we found that in the absence of AMs, micewere unable to control 1918 HA/NA:Tx/91 virus infection or itsspread to the brain tissues. This does not appear to be dueto impaired neutrophil recruitment in AM-depleted lungs, becauseneutrophils are recruited in large numbers in 1918 HA/NA:Tx/91-infectedlungs but cannot functionally compensate for the antiviral functionsof the depleted AM. The exact mode of action of AMs in controlling1918 HA/NA:Tx/91 virus replication remains to be revealed. Mostlikely, AMs can phagocytose virus particles in the lung (54)and synthesize critical immune mediators, including nitric oxide(43) and cytokines (77). AMs can also regulate cell-mediatedimmune responses to influenza infection (60, 69). The significantreduction of cytokines and chemokines in AM-depleted mice wouldsuggest that AMs play an important role as a cytokine sourceduring the early stages of infection.
One explanation for the apparent alveolar tropism of the 1918recombinant viruses may be that it is partly due to the abilityof the 1918 HA to target certain cell types in the mouse lungalveoli. The HA surface glycoprotein possesses receptor-bindingproperties that determine the host range of influenza by bindingto cell surface glycoproteins containing terminal sialic acidresidues. The nature of the receptor sialic acid linkage toa neighboring galactose can occur in the alpha 2,3 linkage orthe alpha 2,6 linkage. Although both Tx/91 and 1918 HAs havea preference for binding 2,6-linked sialic acids (21), differencesin binding affinities or in binding specificities to differentglycosugars might be responsible for an expanded cell tropismof the 1918 recombinant viruses that results in increased replicationin the lungs. Alternatively, the 1918 HA and/or NA proteinsmight confer upon the virus an increased ability to evade theantiviral host response. In fact, influenza H1 and H3 subtypeshave been shown to deactivate neutrophil functions that appearto be mediated by HA binding to neutrophil surface sialic acid-bearingsites (24). Further studies are needed to determine the compositionof the sialic acid in the deeper airways of the alveoli andto determine whether the receptor affinity of the 1918 HA increasesthe cellular tropism for resident cells of the alveoli.
Arguably, the 1918 pandemic influenza virus was successful becauseof its ability to replicate efficiently in hosts with littleor no immunity to the virus of the H1N1 subtype. However, itsability to induce severe lung pathology, particularly in youngadults, suggests that host factors contributed to the high virulenceof this virus. In this study, a recombinant virus containingthe 1918 HA and NA glycoproteins prepared on the genetic backgroundof a nonlethal influenza H1N1 1991 strain gave the virus a lethalphenotype in mice. More striking still were the high titersof virus in the lungs of these mice, which were associated withan influx of substantial numbers of neutrophils and macrophagesinto the infected lung, particularly the alveoli. While theAMs and neutrophils may be contributing to the overall pathogenesis,due to their presence in the airways, their role is crucialin controlling the growth and promoting clearance of this highlyvirulent virus. These results may have important implicationsin defining the pathogenesis of severe influenza virus infectionscaused by highly virulent influenza strains.

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This work was partially supported by grants from the NIH toP.P., A.G.-S., C.F.B., and J.K.T. This work was partially supportedby USDA/ARS CRIS project number 6612-32000-039-00D and NIH grantPO1 AI058113-01. P.P. is a Senior Fellow of the Ellison MedicalFoundation. C.F.B. is a New Scholar of the Ellison Medical FoundationProgram in Global Infectious Diseases.
We thank Randy A. Albrecht and Claudia Chesley for criticalreviews of the manuscript.
The findings and conclusions in this report are those of theauthors and do not necessarily represent the views of the fundingagencies.
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<table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> FOOTNOTES </th></tr></tbody></table>
<!-- null --> * Corresponding author. Mailing address: Influenza Branch, Mail Stop G-16, DVRD, NCID, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., Atlanta, GA 30333. Phone: (404) 639-5444. Fax: (404) 639-2334. E-mail: tft9@cdc.gov<script type="text/javascript"><!-- var u = "tft9", d = "cdc.gov"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>.
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<table bgcolor="#e1e1e1" cellpadding="0" cellspacing="0" width="100%"> <tbody><tr><td align="left" bgcolor="#ffffff" valign="middle" width="5%"></td> <th align="left" valign="middle" width="95%"> REFERENCES </th></tr></tbody></table> <table align="right" border="1" cellpadding="5"><tbody><tr><th align="left"> Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
</th></tr></tbody></table>

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<hr> Journal of Virology, December 2005, p. 14933-14944, Vol. 79, No. 23
0022-538X/05/$08.00+0 doi:10.1128/JVI.79.23.14933-14944.2005
Copyright ? 2005, American Society for Microbiology. All Rights Reserved.