ORIGINAL ARTICLE

Influenza viruses differ in ability to infect macrophagesand to induce a local inflammatory response followingintraperitoneal injection of micePatrick C Reading1,2, Paul G Whitney1, Danielle L Pickett1, Michelle D Tate1 and Andrew G Brooks1

Strains of influenza A virus show marked differences in their ability to infect murine macrophages (MU) such that strainA/PR/8/34 (PR8; H1N1) infects MU poorly while strain BJx109 (H3N2) infects MU to high levels. Given the central role of MUin initiating and regulating inflammatory responses, we hypothesized that virus strains that infect MU poorly may also be poor atinitiating inflammatory responses. Studies to compare the inflammatory response of mice after intranasal inoculation with eitherBJx109 or PR8 were confounded by the rapid growth of the PR8 virus in lung tissues. Consequently, we have characterized thecellular inflammatory response following inoculation into the peritoneal cavity, as influenza viruses do not replicate at this site.Herein, we report marked differences in the local inflammatory response to BJx109 or PR8 in the peritoneal cavity with strainPR8 being particularly poor in its ability to recruit and activate peritoneal leukocytes, including NK cells and MU. In vitroBJx109, but not PR8, stimulated release of high levels of type I IFNs and TNF-a from PEC MU, and treatment of mice withneutralizing antibodies to either cytokine inhibited the ability of BJx109 to recruit and activate NK cells and MUs in theperitoneal cavity. Together, these data suggest that the ability of influenza virus strains to infect MU and stimulate cytokinerelease is an important factor governing the nature of the acute inflammatory response.Immunology and Cell Biology advance online publication, 9 February 2010; doi:10.1038/icb.2010.11

Keywords: inflammation; influenza; macrophage

Macrophages (MF) are a heterogeneous group of cells with roles inphagocytosis, antigen presentation and modulation of the immuneresponse to infection. During influenza infection, pulmonary MFsmay contribute to early host defence in a number of ways, acting as asite of abortive infection, removing infectious virions via phagocytosisas well as secreting innate cytokines such as IFN-a/b and IL-12.Evidence for each of these roles has been found in vitro. For example,infection of MF by influenza virus leads to the expression of viralproteins, but replication is incomplete and no infectious progeny areproduced.1,2 MF can also ingest influenza virus-infected cells3,4 andantibody-opsonized influenza virus particles.5 Furthermore, influenzavirus-infected MF undergo apoptosis, but before death produce type IIFNs and pro-inflammatory cytokines and chemokines.6,7 Critically,the selective elimination of alveolar MF using liposomes containingdichloromethylene diphosphonate enhanced virus replication in thelungs of influenza-infected mice,8 highlighting the role of MF indefense against influenza infection in vivo. Together, these findingssuggest that early interactions between MF and influenza virus may becritical in initiating a protective inflammatory response and thereforedetermining the outcome of infection.

We have previously reported a marked difference in the ability ofcertain strains of influenza virus to infect murine MF in vitro. Thesestudies identified the MF mannose receptor (MR) as a majorendocytic receptor mediating infection of MF by influenza virus.9

As such, viruses bearing few N-linked glycans (for example, A/PR/8/34, H1N1) interact weakly with this receptor and are inefficient intheir ability to infect murine MF. In contrast, virus strains bearingheavily glycosylated surface glycoproteins (for example, BJx109; areassortant virus bearing internal proteins derived from PR8 andsurface glycoproteins from strain A/Beijing/353/89, H3N2) bindefficiently to the MR and infect MF to high levels. In this study, weaimed to determine if the poor ability of strain PR8 to infect MFimpacted on the magnitude and quality of the inflammatory responsein vivo.Studies to compare the inflammatory response of mice after

intranasal (i.n.) inoculation with a standard dose of either BJx109or PR8 were confounded by the rapid growth of the PR8 virus in lungtissues relative to BJx109. Consequently we have characterized thecellular inflammatory response in the peritoneal cavity, as influenzaviruses do not replicate at this site. Intraperitoneal (i.p.) inoculation

Received 5 October 2009; revised 17 January 2010; accepted 18 January 2010

1Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia and 2WHO Collaborating Centre for Reference and Researchon Influenza, North Melbourne, Victoria, AustraliaCorrespondence: Dr PC Reading, Department of Microbiology and Immunology, The University of Melbourne, Royal Parade, Parkville 3010, Victoria, Australia.E-mail: preading@unimelb.edu.au

Immunology and Cell Biology (2010), 1–10& 2010 Australasian Society for Immunology Inc. All rights reserved 0818-9641/10 $32.00

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allowed for a direct comparison of the inflammatory potential ofBJx109 and PR8 at an equivalent dose of each virus. Herein, we showmarked differences in the local inflammatory response to BJx109 orPR8 in the peritoneal cavity, with strain PR8 being particularly poor inits ability to recruit and activate peritoneal leukocytes, including NKcells and MF. In vitro, BJx109, but not PR8, stimulated release of highlevels of type I IFNs and TNF-a from PEC MF, and treatment of micewith neutralizing antibodies to either cytokine inhibited the ability ofBJx109 to recruit and activate NK cells and MFs in the peritonealcavity. Together, these data suggest that the ability of influenza virusstrains to infect and stimulate cytokine release from murine MF maydetermine its ability to recruit and activate leukocytes at a site of localinflammation.

RESULTSBJx109 and PR8 differ in their ability to infect and induce cytokinesfrom murine MUIn previous studies, we have shown that influenza viruses differmarkedly in their ability to infect murine MF in vitro as assessed byimmunofluorescence microscopy at 8–10h post-infection.9 Herein, weshow that at a multiplicity of infection of 10PFU per cell, strainBJx109 infected murine MF to high levels, whereas an equivalent doseof PR8 infected MF poorly (Figure 1a). This hierarchy was a commonfeature of murine MF, including PEC MF and MF from BAL ofC57BL/6 mice and the MH-S MF cell line. Exposure of PEC MF toBJx109 resulted in 490% cytopathic effect (CPE, rounding ofadherent cells, cytoplasmic granularity and cell lysis) whereas exposureto PR8 resulted in o10% CPE, indicating that PR8 did not haveadverse effects on MF viability. In contrast, Madin–Darby caninekidney (MDCK) epithelial cells were equally sensitive to infection byBJx109 and PR8 viruses and both viruses induced 490% CPE onthese cells.To examine infection of resident peritoneal MF in situ, mice were

inoculated i.p. with 107 PFU of BJx109 or PR8, and 6 h later PECswere collected and intracellular NP was detected by flow cytometry.BJx109 infected F4/80high PECs with greater efficiency than PR8(33.9±5.8% of F4/80high cells from BJx109-inoculated mice comparedwith o5% from PR8-inoculated mice) (Figure 1b), showing that thedifference in infectivity for peritoneal MF observed in vitro could bereproduced in situ.The ability of BJx109 and PR8 to induce secretion of IFN-a/b and

TNF-a from peritoneal MF paralleled their ability to infect these cellsin vitro. Thus, peritoneal MF that had been purified by adherence andinfected in suspension with influenza virus produced high levels ofIFN-a/b (Figure 1c) and TNF-a (Figure 1d) in response to BJx109,but not to PR8. Induction of either cytokine required infectious virusas BJx109 that had been UV-irradiated for 30min to destroy virusinfectivity failed to induce either cytokine (data not shown). Together,these studies indicate that the BJx109 and PR8 differ markedly in theirability to infect and induce cytokine production from murine MF.

Viral replication following i.n. or i.p. inoculation with BJx109or PR8We hypothesized that the poor ability of PR8 to infect murine MFin vitro may have important consequences with respect to the timing,magnitude and nature of the inflammatory responses induced by eachvirus in vivo. Initial studies to compare lung inflammation followingintranasal infection of mice with 105 PFU of BJx109 or PR8 wereconfounded by the high pathogenicity of the mouse-adapted PR8virus. As seen in Figure 2a, 4100-fold more virus could be recoveredfrom the lungs of PR8-infected mice by 12h post-infection (6.1±0.2

vs 3.3±0.3 log10PFU/lung, for PR8 and BJx109-infected mice, respec-tively). High virus titres were maintained throughout PR8 infectionand mice were euthanized at day 4 post-infection due to the severity ofdisease. In contrast, virus could not be detected in lung homogenatesfrom BJx109-infected mice by day 4 post-infection and no evidence ofclinical disease was observed. Marked differences in pathogenicity

Figure 1 Ability of influenza viruses to infect and induce cytokines frommurine MF. (a) Infection of primary MF, the MH-S MF cell line and MDCKepithelial cells in vitro by 106PFU of influenza virus strains BJx109 (blackcolumns) and PR8 (striped columns). Monolayers were infected in chamber-slides and stained by immunofluorescence for expression of influenza virusNP at 8–10h post-infection. Data represent the mean±s.d. from 4–5independent fields and are representative of 43 independent experiments.(b) Infection of peritoneal MF in situ by influenza viruses. Mice wereinoculated i.p. with 107PFU of BJx109 or PR8 or with an equivalentvolume of diluent (PBS) and 6h later PECs were collected and stained forcell-surface expression of MF marker F4/80 and for intracellular expressionof influenza virus NP (white histograms) or mouse IgG as a control (greyhistograms). Shown are representative histograms from one mouse in eachgroup of 4–5 mice with a marker to indicate the percent of F4/80+ PEC thatstain for intracellular expression of influenza NP. (c and d) BJx109 and PR8differ in their ability to induce IFN-a/b (c) or TNF-a (d) from peritoneal MF.Adherence-purified MF were infected with influenza viruses at 10PFU percell and culture supernatants were collected 24h post-infection. Datarepresent the mean±1 s.d. from three independent experiments.

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were also seen following intranasal infection of C57BL/6 or CBAmouse strains with either BJx109 or PR8 (data not shown). Clearly,comparison of the inflammatory responses elicited in the airwaysfollowing infection with BJx109 or PR8 would be confounded by themarked differences in viral load observed in the lungs. Therefore, wehypothesized that the peritoneum may represent a convenient site tocompare the local inflammatory responses induced by BJx109 andPR8 as neither virus would be likely to replicate in the peritonealcavity. BALB/c mice were inoculated via the i.p. route with 106 PFU ofeither BJx109 or PR8 and at various times post-infection, peritonealwashouts were performed and levels of infectious virus in cell-freesupernatants were determined. As seen in Figure 2b, BJx109 and PR8did not replicate to significant levels in the peritoneal cavity andinfectious virus could not be detected after 1 day post-inoculation.

Thus, the peritoneal cavity serves as a convenient site to examineinflammatory responses to comparable doses of BJx109 andPR8 viruses.

Cellular inflammatory responses after i.p. inoculation of micewith BJx109 or PR8BALB/c mice were inoculated via the i.p. route with 106 PFU of BJx109or PR8 viruses and leukocytes infiltrating the peritoneal cavity wererecovered by lavage and differentially stained. As seen in Figure 3a,injection of BJx109 led to the progressive accumulation of leukocytesin the peritoneum, such that by day 5 the total number of PECrecovered was approximately 3-fold higher than the number recoveredfrom naive mice (t!0). At day 5 post-inoculation, the local inflam-matory response to BJx109 was characterized by infiltrationof MF (Figure 3b) and lymphocytes (Figure 3c) into the peritoneum.At an equivalent time, exudates from PR8-inoculated mice displayedonly a modest increase in numbers of total cells (Figure 3a) andnumbers of MF (Figure 3b) and lymphocytes (Figure 3c) were similarto those of naive mice. Neutrophils comprised o5% of PECs fromBJx109- or PR8-inoculated mice at day 5 post-infection (data notshown). The differential ability of BJx109 and PR8 to induce inflam-matory responses in the peritoneal cavity was a feature of immuno-competent (BALB/c, C57BL/6 and CBA) and immunodeficient(B6.RAG"/", SCID, BALB/c nu/nu) mouse strains, indicative of aninnate immune response.

Activation of NK cells and MU in the peritoneumby influenza virusNK cells are rapidly induced and activated in response to viralinfections10 and have a protective role during influenza virus infectionof mice.11 We compared the ability of BJx109 and PR8 to recruit andactivate NK cells in the peritoneal cavity following i.p. inoculation. NKcells were identified by flow cytometry as DX5+, CD3e" (for BALB/cmice) or NK1.1+, CD3e" (for B6 mice) cells in the lymphocyte gate.Inoculation with BJx109 led to rapid accumulation of NK cells withinthe peritoneal cavity and NK cell numbers had increased approxi-mately 10-fold by day 1 and remained high through to day 5(Figure 4a). A similar pattern of NK cell recruitment was seenfollowing inoculation of BALB/c mice with BJx109 (data notshown). Peritoneal NK cells recovered following inoculation withBJx109 were activated as indicated by intracellular IFN-g production(Figure 4b) and the ability of PEC to lyse NK-sensitive YAC-1 targetcells (Figure 4c). Peak numbers of IFN-g+ NK cells were detected inthe peritoneal cavity 1 day after inoculation and declined rapidlythereafter whereas only modest NK cell cytotoxicity was detected atday 1, with a clear peak observed 3 days after injection of BJx109.Although NK cell numbers remained high 5 days after BJx109injection (Figure 4a), few NK cells in the peritoneum producedIFN-g (Figure 4b) or mediated cytotoxic activity against YAC-1 targets(Figure 4c) at this time. Exudate cells recovered from B6 mice(Figure 4) or BALB/c mice (data not shown) inoculated with106 PFU of PR8 were characterized by lower NK cell numbers(Figure 4a), poor IFN-g production by NK cells (Figure 4b) andPECs displayed weak cytotoxic activity against YAC-1 targets(Figure 4c).The induction of activated or ‘cytotoxic’ MF capable of lysing

virus-infected cells in the absence of an antibody has been reportedfollowing i.p. or i.n. inoculation of mice with influenza virus.12

BJx109, but not PR8, induced the recruitment and activation of MFin the peritoneal cavity as indicated by (i) the tumouricidal activity ofday 5 PECs from BJx109-injected mice against the MF-sensitive P815

Figure 2 Virus titres in (a) lung homogenates and (b) peritoneal washoutsfollowing inoculation of BALB/c mice with influenza virus strains BJx109and PR8 by i.n. or i.p. routes, respectively. Groups of 3–4 mice wereinoculated i.n. with 105PFU/mouse or i.p. with 106PFU/mouse of BJx109(squares) or PR8 (circles) and, at various times after inoculation, titres ofinfectious virus in lung homogenates and peritoneal washouts weredetermined by plaque assay on MDCK cell monolayers. The dashed linerepresents the lower limit of detection of virus in each sample. Data indicatemean virus titres (±1 s.d.) at 4 h, 12h, 1, 2, 3, 4 and 5 days afterinoculation.

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cell line (Figure 5a), (ii) the ability of day 5 PECs from BJx109-inoculated mice to produce high levels of nitrite (as a measure of NOproduction) following overnight culture (Figure 5b), and (iii)

enhanced expression of Class II MHC and co-stimulatory moleculesCD80 and CD86 (Figure 5c) on F4/80+ MF from BJx109-inoculatedmice. In each assay, PEC MF taken 5 days after i.p. inoculation withan equivalent dose of PR8 displayed a phenotype more similar to thatof PEC MF from mock-infected mice, which received an i.p. injectionof diluent alone.

NK cells and IFN-c are essential for activation of PEC MUin response to i.p. inoculation with influenza virusT-cell independent, IFN-g-mediated MF activation has beendescribed as part of the early inflammatory response to intracellularpathogens13 and to viruses such as HSV-1 and MCMV.14 NeutralizingmAbs specific for murine IFN-g (or rat IgG as a control) wereadministered to mice, followed by i.p. inoculation with 106 PFU ofBJx109. Five days after inoculation, PECs were harvested and counted,and the degree of MF activation was determined. Neutralization ofendogenous IFN-g led to reduced numbers of PEC at day 5 post-inoculation (Figure 6ai). Furthermore, MF activation was markedlyreduced with low levels of nitrite detected in PEC supernatants fromIFN-g-depleted mice inoculated with BJx109 (8±3mM) comparedwith virus-inoculated mice that received either control IgG (44±5mM)or no treatment (52±7mM) mice (Figure 6aii). The absolute require-ment for IFN-g was confirmed using mice in which the IFN-g genewas disrupted by homologous recombination. Compared with B6controls, inoculation of B6.IFN-g"/" mice with 106 PFU of BJx109 didnot induce MF recruitment (Figure 6biii) and levels of nitriteproduction by PEC from virus-inoculated B6.IFN-g"/" mice werebelow detection (o5mM; Figure 6biv), indicating poor MF activation.MF activation was found to proceed in response to i.p. inoculation

with 106 PFU of BJx109 in BALB/c nu/nu mice (BALB/c!56±4mM,BALB/c nu/nu!48±6mM; n!3–5 mice/group; data not shown) andB6.RAG1"/" mice (B6!51±9mM, B6.RAG1"/"!57±7mM; n!4mice/group; data not shown), both of which lack functional T cells,suggesting that NK cells are likely to be an important source of IFN-gproduction. Treatment of C57BL/6 mice with anti-NK1.1 mAb wasused to deplete endogenous NK cells and 1 day later mice wereinoculated i.p. with 106 PFU of BJx109. NK cell depletion wasconfirmed to be 490% 3 days after inoculation (data not shown)and 5 days after inoculation we found both the number (Figure 6cv)and activation (Figure 6cvi) of PEC MF to be markedly reduced.Finally, B6.RAG"/" (lacking T and B cells) and B6.RAG2"/".gC"/"

(lacking T cells, B cells and NK cells) mice were inoculated i.p. with106 PFU and 5 days later PECs were harvested, counted and char-acterized. MFs accumulated in the peritoneal cavity of B6.RAG"/"

and, to a lesser extent, B6.RAG2"/".gC"/" mice (Figure 6dvii).Moreover, only MF from B6.RAG"/" mice were highly activated asdetermined by high levels of nitrite in PEC supernatants (Figure6dviii; 52±3mM for B6.RAG1"/" mice compared with (o5mM forB6.RAG2"/".gC"/" mice). Together, these data indicate that activatedNK cells recruited to the peritoneum following i.p. inoculation withBJx109 activate MF via the secretion of IFN-g.

TNF-a and IFN-a/b mediate leukocyte recruitment and activationin the peritoneal cavity in response to BJx109 virusBJx109 is highly efficient in its ability to infect (Figures 1a/b) andinduce secretion of TNF-a (Figure 1c) and IFN-a/b (Figure 1d) fromPEC MF, whereas strain PR8 is not. To assess the contribution ofTNF-a and IFN-a/b to the cellular inflammatory response in theperitoneum, neutralizing antibodies were used to deplete mice of eachof these cytokines before i.p. inoculation with 106 PFU of BJx109.Three days after virus inoculation, PECs were harvested from half of

Figure 3 Characterization of leukocytes recovered from peritoneal washoutsafter i.p. inoculation with BJx109 or PR8. Groups of 4–5 BALB/c mice wereinoculated i.p. with 106PFU of influenza virus BJx109 or PR8, andperitoneal cells were recovered and characterized at various times afterinoculation. (a) Total cell counts were performed by microscopic examinationof cell suspensions in the presence of trypan blue. Shown are mean (±s.d.)PEC counts from mice inoculated with BJx109 (squares) or PR8 (circles) at4, 12h, 1d, 2d, 3d, 4d and 5d after inoculation. (b and c) Numbers of (b)MF and (c) lymphocytes in peritoneal washouts 5 days after i.p. inoculationwith 106PFU of BJx109 or PR8. Differential leukocyte counts wereperformed and cell types were identified by morphological evaluation underlight microscopy. Cell counts from mice inoculated 5 days previously withdiluent alone (‘Mock’) are included for comparison. Data represent the meannumber (±1 s.d.) of macrophages or lymphocytes from groups of 4–5 mice.**Cell numbers from PR8 inoculated mice were significantly different tothose from BJx109-inoculated mice (Po0.01, one way ANOVA followed byTukey’s multiple comparison test) but not to mock-inoculated mice.

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the mice for determination of NK cell numbers and cytotoxicityagainst NK cell-sensitive YAC-1 targets. Five days after inoculation,PECs were removed from the remaining mice, counted and culturedovernight to allow for the determination of nitrate levels in culturesupernatants. Neutralization of either TNF-a (Figure 7a) or IFN-a/b(Figure 7b) led to a reduction in NK cell number (Figures 7a and bi)and cytotoxic activity (Figures 7a and bii), as well as inhibiting MFnumbers (Figures 7a and biii) and activation of PEC MF (Figures 7aand biv) in response to i.p. inoculation with BJx109. Together, thesedata show a role for both TNF-a and IFN-a/b in the recruitment andactivation of NK cells in the peritoneum after i.p. inoculation withinfluenza virus. Moreover, recruitment and activation of NK cellsappears to be critical for the subsequent activation of MF in thelocal inflammatory response to influenza virus.

DISCUSSIONWe have previously reported marked differences in the ability ofinfluenza virus strains to infect murine MF in vitro.15 These studiesidentified the MR as a major endocytic receptor mediating infection ofmurine MF by influenza virus and showed that poorly glycosylatedviruses such as PR8 were inefficient in their ability to infect MFwhereas virus strains bearing heavily glycosylated HA moleculesinfected MF to high levels. Here, we extend these findings andshow that virus strains capable of infecting MF were markedlymore efficient in their ability to recruit and activate leukocytes inthe peritoneal cavity in response to i.p. inoculation. This, in turn,correlated with enhanced ability of the virus to induce high levels ofIFN-a/b and TNF-a from purified PEC MF in vitro and depletion ofendogenous IFN-a/b or TNF-a in vivo inhibited the ability of BJx109virus to recruit and activate NK cells and MF in the peritoneum.Together, these data suggest an important link between the ability of aparticular virus strain to infect and stimulate cytokine release frommurine MF and its ability to recruit and activate leukocytes at a site oflocal inflammation.The poor ability of PR8 to infect murine MF correlated with poor

production of the inflammatory mediators TNF-a and type I IFNs(Figures 1c and d). In contrast, we have found that murine epithelialcells exposed to BJx109 or PR8 produce similar levels of type I IFNs inresponse to either virus strain (unpublished observations). Of interest,exposure of PEC MF to BJx109 virus that had been UV-irradiated todestroy virus infectivity failed to induce detectable levels of eithercytokine. While evidence suggests that MR engagement by micro-organisms, particles or specific antibodies is associated with signaltransduction pathway/s that lead to modulation of cell surfacereceptors and cytokine production (reviewed by Stahl and Ezekowitz(1998)) we can infer that in our studies the engagement of MR (and/or other cell surface receptors) by influenza viruses does not appearsufficient to induce secretion of type I IFNs or TNF-a. UV-inactivatedvirus retained full receptor-binding activity (as assessed by thehemagglutination assay) and would be expected to be bound andinternalized into the endosomal compartment by MR. These findingssuggest that the MR may facilitate receptor-mediated endocytosis todeliver the virus into endocytic vesicles, but that subsequent steps in

Figure 4 Recruitment and activation of NK cells in the peritoneum of B6mice following i.p. inoculation with BJx109 or PR8 viruses. (a) Number ofNK cells in PECs. At days 1, 3 and 5 after inoculation with 106PFU ofeither BJx109 (black columns), PR8 (striped columns), PECs wererecovered and stained with mAbs to NK1.1 and CD3e. NK cells wereidentified as NK1.1+, CD3e" cells in the lymphocyte gate. Data show themean±1 s.d. from groups of 4–5 mice. Numbers from uninfected controlmice (Un; white bars) are included for comparison. (b) Number of IFN-g+

NK cells in the peritoneal cavity. At 1, 3 and 5 days after inoculation with106PFU of either BJx109 (black columns) or PR8 (striped columns), PECswere isolated and incubated for 4 h with Brefeldin A to retain cytokines intheir cytoplasm. Cells were stained with FITC-labeled anti-NK1.1, APC-labeled anti-CD3e, and after permeabilization using saponin, with PE-labeled anti-IFN-g before analysis by three-color flow cytometry. Data areshown from groups of 3–5 mice and are representative of two independentexperiments. Numbers of IFN-g+ NK cells from uninfected control mice (Un;white bars) are included for comparison. (c) Cytotoxic activity of PECsagainst NK cell-sensitive target cells. PECs from 3–4 mice were pooled andassayed for cytotoxicity against the YAC-1 cell line using a standard 51Crrelease assay. Data represent the percent lysis from triplicate cultures at aneffector/target (E/T) ratio of 50:1. s.e.m. of all cultures were less than 15%.The dashed line represents the percent lysis of YAC-1 cells by PECs fromnaive mice at an E/T of 50:1.

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the infective cycle (including fusion of virus with the endosomalmembrane, release of viral genome and transport to the nucleus tocommence replication of the viral genome) are required to elicit thesecytokine responses from MF.Pattern recognition receptors of the toll-like receptor (TLR) family

such as TLR317 and TLR7,18 and additional nucleic acid recognitionproteins such as retinoic acid-inducible gene I (RIG-I)19,20 are presentin internal cellular compartments and have been implicated inmediating intracellular activation and cytokine production followinginfluenza infection. Our findings indicate that initiation of viralreplication and/or production of intermediate viral RNAs werenecessary to stimulate cytokine production by MF and epithelialcells. Furthermore, given that BJx109 carries internal components ofthe PR8 virus, the poor ability of PR8 to elicit inflammatory mediatorsfrom MF is likely to reflect an inability of the surface glycoproteins ofthe PR8 strain to interact with cell surface ‘delivery receptors’ rather

Figure 5 Activation of peritoneal MF following i.p. inoculation with influenzavirus. MF activation was measured 5 days after i.p. inoculation of BALB/cmice with 106PFU of BJx109 or PR8, or with diluent alone (‘Mock’).(a) Lysis of P815 cells by PECs. PECs from groups of 3–5 mice werepooled, counted and used as effectors in a standard 51Cr release assayagainst the MF-sensitive P815 cell line. Data shown represent the percentlysis as determined by 51Cr release from triplicate cultures. s.e.m. from allcultures were less than 15%. (b) Production of NO. PECs from 3–5 micewere pooled, counted and 1#106 cells/0.5ml were cultured for 24h beforethe levels of nitrites in the culture supernatants were determined. Resultsrepresent the mean±1 s.d. of triplicate cultures. The dashed line representsthe limit of detection of the assay at 5mM. *nitrite levels from PR8inoculated mice were significantly different to those from BJx109-inoculatedmice (Po0.01, one way ANOVA followed by Tukey’s multiple comparisontest) but not to mock-inoculated mice. (c) Expression of activation markerson the cell surface of peritoneal MF 5 days after i.p. inoculation with106PFU of either BJx109 (white histograms) or PR8 (black histograms), orwith diluent alone (grey histograms). F4/80+ PEC MF from 3–5 mice werepooled and examined by flow cytometry for binding of mAbs to murine I-Ad(Class II MHC), CD80 or CD86. Histograms shown are representative of twoor more independent experiments.

Figure 6 IFN-g and NK cells are critical for activation of peritoneal MF inresponse to i.p. BJx109. Groups of 3–5 mice were inoculated i.p. with106PFU of BJx109 and, 5 days later, PECs were recovered for analysis. Insome experiments, mice were treated with antibodies to deplete IFN-g or NKcells, or with control IgG, before i.p. inoculation with BJx109 virus. (Panelsi, iii, v and vii) MF numbers were determined following cytospin analysisand data shown represent the mean (±1 s.d.) cell number per mouse.(Panels ii, iv, vi and viii) Aliquots of 1#106 PECs were cultured for 24h andnitrite levels in cell-free supernatants were determined. Data shown are themean (±1 s.d.) from triplicate cultures. The dashed line represents the limitof detection of the assay at 5mM. (a) B6 mice were treated i.p. with 2mg ofanti-IFN-g mAb R4-6A2 (‘IFNg’) or control rat IgG (‘IgG’), or with anequivalent volume of PBS ("), 24h before i.p. inoculation with 106PFU ofBJx109. Control animals were not treated with antibodies and wereinoculated with virus diluent alone. (b) B6 or B6.IFNg"/" (‘IFNg"/’) micewere inoculated i.p. with 106PFU of BJx109 or with an equivalent volumeof virus diluent alone. (c) B6 mice were treated with 1mg of either mAbNK1.1 or control mouse IgG, 24h before i.p. inoculation with 106PFU ofBJx109. Control animals were not treated with antibodies, and wereinoculated with virus diluent alone. (d) RAG1"/" mice (‘RAG"/’) orRAG2"/".gC"/" mice (‘gC"/"’) were inoculated i.p. with 106PFU of BJx109or with an equivalent volume of virus diluent and PECs were characterized 5days later. Experiments shown in a, b, c and d were performed at least twicewith similar results. For statistical analysis, ***Po0.001, **Po0.01 and*Po0.05 one way ANOVA and Tukey’s multiple comparison test). In panelsi, ii, v and vi, asterisks indicate that antibody-treated mice were statisticallydifferent to both mice treated with control IgG (‘IgG’) or with PBS (‘"’)before i.p. inoculation with BJx109.

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that an intrinsic deficit in its ability to initiate cytokine production perse. Indeed, once the virus is internalized and commences replication,the production of cytokines would be expected to be similar: ourfindings that BJx109 and PR8 induce equivalent levels of type I IFNsfrom epithelial cells is consistent with this notion. It is noteworthy thatthe ability to infect PEC MF efficiently in vitro and to induce a localinflammatory response following i.p. injection of mice is not specificto the BJx109 virus strain. Other H3N2 virus strains, including HKx31(H3N2) and Phil/82-X (H3N2), which are high-yielding reassortantsof A/Aichi/2/63 (H3N2) and A/Philippines/2/82 (H3N2), respectively,with PR8 were also found to infect PEC MF efficiently and to induce alocal inflammatory response following i.p. injection of mice. Inaddition, wild-type viruses (that is, not reassortants with PR8),including A/Guandong/25/93 (H3N2), A/Beijing/32/92 (H3N2) andA/Brazil/11/78 (H1N1) also infected PEC MF and induced a stronginflammatory response in the peritoneum (Reading PC, unpublishedobservations).Infection of MF by BJx109 led to the rapid production of IFN-a/b

and TNF-a, which in turn played a role in the early recruitment andactivation of NK cells, and subsequently MF, in the peritoneumfollowing i.p. inoculation with BJx109. The failure of PR8 to inducethese cytokines from PEC MF in vitro or to recruit and activate

leukocytes in the peritoneum is striking. A number of studies havedescribed the ability of innate cytokines, including type I IFNs, TNF-a, IL-12, IL-15 and IL-18 to modulate NK cell responses during viralinfections (reviewed by Biron et al., 1999). Type I IFNs promote NKcell activation during infection with vaccinia virus21 and enhance NKcell proliferation and cytotoxicity in MCMV-infected mice.22 Further-more, IFN-a secreted from influenza virus-infected MF has beenshown to stimulate NK cell production of IFN-g23 and to promoteinduction of IL-15,24 an essential mediator of NK cell proliferationand homeostasis. The role of TNF-a in regulating NK cell responses isless clear; TNF-a blockade was associated with reduced NK cellrecruitment to the liver following infection with recombinant adeno-virus;25 however, accumulation of NK cells in the liver during MCMVinfection occurs independently of TNF-a.26 Following peritonealinoculation of influenza virus, both TNF-a and IFN-a/b have criticalroles in modulating NK cell recruitment and cytotoxicity, and pre-sumably also in potentiating IFN-g production from NK cells, giventhe poor activation of MF that is observed following depletion ofeither cytokine.NK cells respond to viral infection to mediate cytotoxicity and

produce IFN-g, as well as to proliferate and accumulate at sites ofinfection. Innate cytokines induced early after infection have a critical

Figure 7 Effect of in vivo depletion of TNF-a or IFN-a/b on activation of NK cells and MF in response to BJx109 virus. Groups of 3–5 mice were treatedwith antibodies or with diluent 24h before i.p. inoculation with 106PFU of BJx109. Control animals were pretreated with PBS, and 24h later inoculated i.p.with virus diluent alone (‘No virus’ or ). Three days after virus inoculation, (i) PECs from individual mice were counted and numbers of NK cells determinedby flow cytometry. Data show the mean NK cell number (±1 s.d.) per mouse. (ii) PECs were pooled and assayed in triplicate for cytotoxicity against the NKsensitive YAC-1 cell line. Results are expressed as percent lysis as described in Methods. s.e.m. of all triplicate cultures were o15%. Five days after virusinoculation, (iii) PECs from individual mice were counted and MF numbers determined after cytospin analysis. Data show the mean MF cell number (±1s.d.) per mouse. (iv) PECs were pooled and aliquots of 1#106 PECs were cultured for 24h and the nitrite levels from triplicate cultures were determined.Data shown are the mean (±1 s.d.) from triplicate cultures. The dashed line represents the limit of detection of the assay at 5mM. (a) Mice were treated with2mg of anti-TNF-a mAb MP6XT-22 (‘TNF’ or ) or control rat IgG (‘IgG’ or $), or with an equivalent volume of diluent (‘PBS’ or ’), 24h before i.p.inoculation with 106PFU of BJx109. (b) Mice were treated with 1000 units of anti-IFN-a/b rabbit antiserum (‘IFN’ or ) or an equivalent volume of normalrabbit serum (‘NRS’ or $) or PBS (‘PBS’ or ’) as described in Methods, and then inoculated i.p. with 106PFU of BJx109. Experiments were repeated withsimilar results. For statistical analysis, asterisks indicate that antibody-treated mice were statistically different to both mice treated with control IgG (‘IgG’) orwith PBS (‘PBS’) before i.p. inoculation with BJx109; ***Po0.001, **Po0.01, one way ANOVA and Tukey’s multiple comparison test).

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role in modulating particular NK cell functions. In the case of MCMV,production of IFN-a/b stimulated NK cell cytotoxicity in a STAT1-dependent manner, whereas IL-12 promoted STAT4-mediated IFN-gproduction by NK cells.22,27 Production of IFN-a/b during MCMVinfection also promoted expression of IL-15 and therefore NK cellproliferation.22 Following intraperitoneal inoculation with influenzavirus strain BJx109, NK cell numbers were elevated in the peritoneumat days 1, 3 and 5, yet IFN-g production peaked at day 1 and declinedrapidly thereafter. In contrast, cytotoxic activity was low at day 1,peaked at day 3 and declined thereafter. These findings suggest that inthe influenza model of peritoneal inflammation, particular innatecytokines might also modulate distinct NK cell effector functions andtherefore the composition of the first cytokine responses are likely tobe critical in determining a coordinated and appropriate NK cellresponse. Experiments to monitor the timing and the levels ofIFN-a/b, TNF-a and other innate cytokines, including IL-12 andIL-15, in response to intraperitoneal inoculation with influenza virusmay help to resolve this issue.Activation and differentiation of MF following MCMV

infection is an IFN-g-dependent response also regulated by innatecytokines. Intraperitoneal infection of immunocompetent or immu-nodeficient SCID mice with MCMV induces an inflammatory infil-trate consisting largely of activated MF;14 however, the high levels ofIFN-a/b induced by MCMV were found to impair MF responsivenessto IFN-g leading to reduced expression of Class II MHC.28 In thecontext of MCMV, neutralization of TNF-a has been linked toinhibition of infiltration and activation of MF in the peritoneum,14

consistent with our findings following i.p. injection of BJx109(Figure 6b). Production of TNF-a by MF is known to modulateexpression of adhesion molecules on endothelial cell walls,29 andthis in turn may influence NK cell and MF infiltration into theperitoneal cavity. Additional cytokines, including IL-12 and IL-18,might also contribute to NK cell and/or MF activation in vivo,although neither cytokine could be detected in the supernatants24h after infection of adherence purified PEC MF with BJx09(PC Reading, unpublished observations).This study has examined the early interactions of MF in the

peritoneum of mice with influenza virus to gain insight into factorsthat may initiate an appropriate and protective inflammatory responsein the lung and therefore determine the outcome of respiratoryinfection with different virus strains. The use of the i.p. route ofinfection was critical to allow for assessment of MF responses to viruswithout the confounding effects of concomitant viral replication inepithelial cells of the lung. Although it is well established that MFfrom different sites of the body can vary considerably with respect tobiochemistry and function,30,31 a number of observations suggest thatthe innate responses to BJx109 and PR8 in the peritoneum may wellrecapitulate the response of airway MF to each of these virus strains.First alveolar MF showed the same differential pattern of infectionwith BJx109 and PR8 (Figure 1a). Furthermore, IFN-a/b and TNF-acan be detected in supernatants from alveolar MF exposed to BJx109,but not PR8 (PC Reading, unpublished observations). In addition, theearly response to intranasal infection of mice with 105 PFU of BJx109,which replicates to only a low level in the lung (Figure 2a), was similarto that in the peritoneum with respect to activation of airway MFthrough a NK cell- and IFN-g-dependent mechanism (PC Reading,unpublished observations). Finally, in an independent study we haveshown that depletion of airway MF by clodronate-loaded liposomesled to the development of severe viral pneumonia in BJx109-infectedmice, but did not modulate disease severity in PR8-infected mice(MD Tate and PC Reading, unpublished observations).

A virulent strain of influenza virus such as PR8 is, by definition,able to escape at least some components of innate host defense.There is growing evidence to suggest that carbohydrate on the surfaceglycoproteins of influenza virus serve as a target for recognition bylectin-like receptors of the innate immune system, including solublecollectins in serum and lung fluids and the MR on MF and dendriticcells. The PR8 strain, which carries no glycosylation on the globularhead of its HA molecule,32 avoids neutralization and opsonization bySP-D and related collectins9,33 as well as the ‘dead-end’ route of uptakeand destruction by MF.15 Thus, following inhalation, PR8 avoidsthese innate defenses and can infect epithelial cells of the respiratorytract allowing for rapid virus growth and subsequent tissue damage inthe lungs.

METHODSViruses and cell linesThe influenza type A viruses used in this study were the Mt. Sinai strain of A/PR/8/34 (H1N1) (PR8) and BJx109 (H3N2), which is a laboratory-derived,high-yielding reassortant of PR8 and A/Beijing/353/89 known to bear the HAand NA genes of the A/Beijing/353/89 virus strain.15 Viruses were grown in theallantoic cavity of 10-day embryonated hens’ eggs by standard procedures andinfectivity titres of allantoic fluids were determined by plaquing on MDCK cellsin the presence of trypsin.34 The murine MH-S MF cell line was maintained ina-minimal essential medium (Gibco Grand Island, NY, USA) supplementedwith 2mM glutamine, 2mM pyruvate, 30mgml–1 of gentamicin and 10% FCS.MDCK (canine kidney epithelial cell line) were passaged in RPMI 1640supplemented with 2mM glutamine, 2mM pyruvate, 30mgml–1 of gentamicinand 10% FCS.

Infection and treatment of miceC57BL/6 (B6), BALB/c, CBA, B6.RAG1"/", B6.RAG2"/".gC"/" and C.B-17severe combined immunodeficiency (SCID) mice were bred and housed inspecific pathogen-free conditions at the Department of Microbiology andImmunology, University of Melbourne, Australia. Athymic BALB/c nu/nu micewere obtained from the animal facility at the Walter and Eliza Hall Institute ofMedical Research, Victoria, Australia. B6.IFN-g"/" mice were obtained fromProfessor Mark Smyth, Peter MacCallum Cancer Centre, Victoria, Australia.Male 6- to 10-week-old mice were used in all experiments.

Mice were lightly anesthetized with Penthrane (Abbott Australasia, Botany,NSW, Australia) and inoculated via the i.n. route with 105 PFU of influenzavirus in phosphate-buffered saline (PBS). For i.p. inoculation, a 26-G needlewas used to inoculate 100ml of influenza virus diluted in PBS into theperitoneal cavity. To deplete IFN-g or TNF-a, mice were injected with 2mgof purified mAb R4-6A2 or mAb MP6XT-22. As a negative control, micereceived an equivalent dose of rat IgG. To deplete NK cells, B6 mice wereinjected i.p. with 1mgml–1 purified mAb PK136 (anti-NK1.1) or 1mg ofcontrol mouse IgG (Sigma-Aldrich, St Louis, MO, USA) in sterile PBS, 24hbefore inoculation with virus. To deplete type I interferon (IFN), 200ml ofrabbit antiserum raised to murine IFN-a/b (Lee BioMolecular ResearchLaboratories Inc., San Diego, CA, USA) was injected into the peritoneal cavityof mice 24 h before inoculation with 106PFU of BJx109. Control animals wereinjected with an equivalent volume of normal rabbit serum (NRS).

Titration of virus in tissue samplesAt various times after i.p. inoculation with virus, mice were euthanized and theperitoneal cavity was washed with 2ml of ice-cold RPMI 1640 mediumsupplemented with 30mg gentamicin per ml. Mice inoculated via the i.n. routewere killed and the lungs removed, rinsed and disrupted in 1.5ml of Hanks’balanced salt solution containing 100Uml–1 of penicillin and 100mgml–1 ofstreptomycin by being pressed through a sieve with the plunger of a disposable2ml syringe. Lung extracts and peritoneal washes were clarified by centrifuga-tion, and the supernatants were frozen at"70 1C. Titres of infectious virus weredetermined by plaquing on MDCK cell monolayers in the presence of trypsin.34

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Infection of murine MU by influenza virusResident BAL or peritoneal MF, murine MF cell lines, or the MDCK epithelialcell line were prepared and infected with influenza virus in 8-well chamberslides as previously described.15 Slides were stained with a monoclonal antibody(mAb A-3, a gift from Nancy Cox, Influenza Branch, Centers for DiseaseControl and Prevention, Atlanta, USA) specific for the nucleoprotein of type Ainfluenza viruses, followed by FITC-conjugated sheep anti-mouse immunoglo-bulin (Silenus, Melbourne, Victoria, Australia) and viewed under #128magnification. The percentage of fluorescent cells was determined in a mini-mum of four random fields (4200 MF).

Cytokine production and assayPeritoneal MF were purified from PECs following overnight adherence toplastic flasks. The adherent cell population was detached by incubation withHBSS containing 5mM EDTA for 20min on ice and resuspended in serum-freeDMEM/F-12 medium (Gibco BRL). Cells (2#106) were incubated in sterileteflon pots (Savillex, Minnetonka, MN, USA) with 2#107 PFU of influenzavirus for 60min at 37 1C, washed three times to remove free virus and culturedfor 24h in sterile 24-well flat-bottomed plastic plates (Nunc). Culture super-natants were collected, absorbed twice with 5% packed volume of chickenerythrocytes for 30min on ice to remove residual virus and stored at "20 1C.

TNF-a activity in culture supernatants was assayed by inhibition ofproliferation of the WEHI 164 cell line as described.35 Optical density at540/690 nm was measured and titres were standardized by comparison withrecombinant mouse TNF-a (Genzyme Diagnostics, Cambridge, MA, USA).IFN-a/b was assayed by cytopathic reduction assay of Semliki Forest virus(SFV) infection in L929 cells. Duplicate dilutions of samples were added toL929 cell monolayers in 96-well flat-bottomed (Nunc) and incubated for 24hat 37 1C in 5% CO2. Samples were removed and replaced with 2.5#104 50%tissue culture infectious doses of SFV. After a further two days incubation, cellswere examined microscopically and the titre was read as the reciprocal of thedilution giving 50% protection to the cell monolayer. IFN-a/b titres werestandardized relative to an international reference preparation of mouse IFN-a(Ga02-901-535, National Institute of Allergy and Infectious Diseases, Bethesda,MD, USA).

Differential analysis of lavage cellsPeritoneal exudate cells (PECs) were harvested by washing of the peritonealcavity with 5ml of ice-cold RPMI 1640 medium supplemented with 30mgml–1

gentamicin and 10Uml–1 heparin (RPG-H). PECs were treated with Tris-NH4Cl (0.14M NH4Cl in 17mM Tris, adjusted to pH 7.2) to lyse erythrocytes,washed and resuspended in cold DMEM/F-12 medium and approximately5#104 cells were centrifuged onto glass microscope slides, air dried and stainedwith Diff-Quick (Lab Aids, Narrabeen, Victoria, Australia). Slides were ana-lyzed using a light microscope and a minimum of 100 cells in 4–8 randomfields were counted. MF, lymphocytes and neutrophils were identified by theirdistinct nuclear morphologies.

Cytotoxicity assaysPECs were tested for cytotoxic activity against P815 (a MF-sensitive masto-cytoma cell line) or YAC-1 (an NK cell-sensitive cell line) targets in a standard51Cr release assay. Target cells (2#106) were labeled with 200mCi of Na251CrO4

(Amersham Corp., Sydney, New South Wales, Australia), washed to remove freelabel and adjusted to 105 cells per ml. Serial dilutions of PECs were incubated intriplicate cultures with 104 target cells in a total volume of 200ml in round-bottomed 96-well plates (Nunc, Glostrup, Denmark) at 37 1C in 5% CO2. After4 h (YAC-1 targets) or 18h (P815 targets) plates were spun to pellet cells and100ml of supernatant was assayed for 51Cr release. Results are expressed asmean percent-specific lysis, defined as (experimental c.p.m."spontaneousc.p.m.)/(maximum detergent c.p.m."spontaneous c.p.m.). Maximum deter-gent c.p.m. was calculated by adding 100ml 4% Triton-X to target cells,and spontaneous lysis was determined by incubation of target cells inmedium alone.

Nitric oxide (NO) productionPEC suspensions were counted, adjusted to 2#106 cells per ml and culturedovernight in sterile flat-bottomed 96-well plastic trays (Nunc, Roskilde, Den-mark) in a volume of 50ml. NO synthesis in cultures was determined bymeasurement of nitrite (NO2

") in culture supernatants, a stable product ofNO, as described elsewhere.36

Flow cytometric analysis of cell surface and intracellular antigensFor live cell stains, PEC suspensions were incubated with supernatant fromhybridoma 2.4G2 to block Fc receptors and then stained with appropriatecombinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE), allo-phycocyanin (APC) or biotinylated monoclonal antibodies to NK1.1 (clonePK136), CD3e (clone 145-2C11), mouse I-Ad (Clone AMS-32.1), CD80 (clone16-10A1) or CD86 (clone GL1) and F4/80 (clone BM8). All antibodies werepurchased from BD Pharmingen (North Ryde, NSW, Australia), except F4/80,which was purchased from Caltag Laboratories (Burlingame, CA, USA). Afterwashing, propidium iodide (PI; 10mgml–1) was added and cells were analyzedon a FACS Calibur flow cytometer. A minimum of 50 000 living cells (PI")were collected.

To detect intracellular IFN-g from NK cells, PEC cells were incubatedwithout stimulation in 10mgml–1 Brefeldin A (Sigma-Aldrich) for 4 h at37 1C and stained for cell surface expression of CD3e and NK1.1, and, followingpermeabilization with 0.2% saponin, for intracellular expression of mouse IFN-g (XMG1.2, BD Pharmingen) as described.37 To detect infection of PEC MF insitu, mice were inoculated i.p. with 107 PFU of BJx109 or PR8, or with anequivalent volume of PBS, and 6 h later PECs were obtained by peritonealwashout. PECs were incubated with supernatants from hybridoma 2.4G2 toblock Fc receptors, stained with F4/80-APC and, following permeabilization in0.2% saponin, with anti-NP mAb A-3 followed by FITC-conjugated sheep anti-mouse immunoglobulin. Cells were analyzed on a FACS Calibur flow cyt-ometer and a minimum of 20 000 F4/80+ PECs were collected.

Statistical analysisFor the comparison of two sets of values, a Student’s t-test (two-tailed, two-sample equal variance) was used. When comparing three or more sets of values,data were analyzed by one-way ANOVA (nonparametric) followed by post hocanalysis using Tukey’s multiple comparison test. A P-value of p0.05 wasconsidered statistically significant.

ACKNOWLEDGEMENTSThis study was supported by Project Grant no.400226 from The NationalHealth and Medical Research Council (NH&MRC) of Australia. PCR is aNH&MRC RD Wright Research Fellow. The Melbourne WHO CollaboratingCentre for Reference and Research on Influenza is supported by the AustralianGovernment Department of Health and Ageing.

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