Haruo Fujisawa* Department of Microbiology, Hyogo College of Medicine, , 1-1 Mukogawa-cho, Nishinomiya City, Hyogo Prefecture, Japan

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1 JOURNAL OF VIROLOGY, Mar. 2008, p Vol. 82, No X/08/$ doi: /jvi Copyright 2008, American Society for Microbiology. All Rights Reserved. Neutrophils Play an Essential Role in Cooperation with Antibody in both Protection against and Recovery from Pulmonary Infection with Influenza Virus in Mice Haruo Fujisawa* Department of Microbiology, Hyogo College of Medicine, , 1-1 Mukogawa-cho, Nishinomiya City, Hyogo Prefecture, Japan Received 2 June 2007/Accepted 19 November 2007 The role of polymorphonuclear leukocytes (PMN) in protection in the early phase and recovery in the late phase of influenza A virus infection was investigated by the depletion of PMN in, and passive transfer of anti-influenza virus antiserum to, mice with pulmonary infections. The depletion of PMN in normal mice by treatment with monoclonal antibody RB6-8C5 both increased the mortality rate and pulmonary virus titers from the early to the late phase after infection and delayed virus elimination in the late phase. The passive transfer of the antiserum to normal mice before or after infection abolished pulmonary virus propagation in the early phase, during 3 days, or rapidly decreased high virus titers in the plateau phase, on days 3 to 5, as well as accelerated virus elimination in the late phase, on day 7, after infection, respectively. The passive transfer of the antiserum to PMN-depleted mice could neither prevent the more rapid virus propagation in the early phase, diminish the higher virus titers in the plateau phase, nor accelerate the markedly delayed virus elimination in the late phase after infection in comparison to those for controls. The antibody responses to the virus began to increase on day 7 after infection in normal and PMN-depleted mice. The prevention of virus replication, cytotoxic activity in virus-infected cell cultures, and phagocytosis of the virus in vitro by PMN were all augmented in the presence of the antiserum. These results indicate that PMN play an essential role in virus elimination in both protection against and recovery from infection, in cooperation with the antibody response. Protection against influenza virus infection involves primarily the production of antibody to a surface glycoprotein, hemagglutinin (HA) (3, 56), which is responsible for the adsorption of virions in the initial stage of infection. Recovery from the primary influenza virus infection is dependent on the specific acquired immunity based on T and B cells (11, 18, 53). The significance of the responding effector cells or molecules in acquired immunity to influenza virus has been progressively investigated in murine models in which each effector is depleted from or deficient in the host by means such as treatment with specific antibodies (1) or specific chemicals (38) and/or the use of immunologically deficient or transgenic mice (27, 52). However, the relative importance and cooperation of the various defense mechanisms in the control of the infection in the intact host are not entirely resolved. The role of phagocytes, including neutrophils (polymorphonuclear leukocytes [PMN]) and macrophages, in the innate host defense against generalized virus infection, including influenza virus infection, is also unclear despite the existence of a thorough analysis demonstrating their significance in protection against various types of bacterial infection. Since Toll-like receptors (TLRs), which play an important role in innate immune recognition and protect against several types of pathogens, have been discovered to be receptor molecules on phagocytes (50), several studies have examined the role of TLRs in virus infection and * Present address: Pharmaceutical Division, Kumano Hospital, , Shimooichi-nishimachi, Nishinomiya City, Hyogo Prefecture, Japan. Phone: Fax: haruofujisawa@bird.ocn.ne.jp. Published ahead of print on 9 January have been increasing in significance in the protective roles of phagocytes in the early phase of infection (6, 21). The two series of phagocytes contribute to differing degrees of protection against individual species of pathogens during bacterial infection. In terms of the relative contributions to early protection against bacterial infection, the roles of phagocytes were investigated using the susceptibilities of PMN to gamma irradiation and carrageenan. Gamma irradiation-sensitive and carrageenan-resistant PMN contributed primarily to early protection against extracellular bacteria such as Pseudomonas aeruginosa (44), Escherichia coli (47), and Streptococcus pneumoniae (22), while protection against intracellular bacteria such as Listeria monocytogenes was highly dependent on tissue-fixed gamma-irradiation-resistant and carrageenan-sensitive macrophages (25, 44). This is consistent with the observation that early protection against intracellular bacteria is also dependent on PMN, based on an analysis using recombinant granulocyte colony-stimulating factor (5, 19, 40). Recently, the protective role of PMN against bacterial infection has been further analyzed using a specific monoclonal antibody (MAb) to PMN (10, 12, 26, 45). In contrast, since the protective role of PMN in virus infection was first reported in bovine herpesvirus infection (36), most subsequent work consisted of in vitro studies that investigated mainly human herpesvirus (24, 36, 37). A very few reports have analyzed the role of PMN in the innate host defense against generalized virus infections based on in vivo studies with selective depletion of PMN, such as those using the specific anti-pmn MAb (48, 49). The aim of this study is to elucidate the role of PMN in host defense against virus infection by using mice infected with influenza A virus as a model. The investigations were initiated on the basis of the observations that PMN are nonpermissive 2772

2 VOL. 82, 2008 ROLE OF PMN IN PROTECTION AGAINST INFLUENZA VIRUS 2773 to virus infection and capable of preventing the multiplication of several types of virus (16, 35, 46). In contrast, both macrophages and lymphocytes, which are responsible for innate and acquired immunity, respectively, are permissive to virus infections and act as target cells (15, 16, 33, 34). Previous studies using gamma-irradiated and carrageenan-treated mice strongly suggest that PMN are the primary cells involved in protection in the early phase of pulmonary infection with either a low ( PFU) or a high ( PFU) inoculum of influenza virus and that alveolar macrophages could also contribute to this early protection against virus infection with a high inoculum (16). An earlier study indicated that PMN can prevent influenza virus from propagating in the lungs of tumor-bearing mice with neutrophilic leukocytosis and that they play a significant role in host defense in the early to the stationary (plateau) phase against primary pulmonary infection with the virus (14). More recently, in a study using a recombinant human H1N1 virus with genes from the 1918 influenza A virus bearing pathogenicity for mice, Tumpey et al. (48) reported that both types of phagocytes contributed to early protection against primary infection with a sublethal dose. However, the roles of the two types of phagocytes in the host defense against virulent strains of influenza A virus have not been entirely clarified. Indeed, the influenza A virus used in our earlier studies was a low-virulence strain of the virus. Furthermore, little is known about the functional role of PMN in the mechanisms of virus elimination during the recovery stage of primary infection and in early protection against reinfection with the virus after generation of the specific acquired immunity. This study was undertaken to clarify the cooperative contributions of PMN and the antibody response to the elimination of a virulent strain of influenza A virus from mice infected with either a low or a high inoculum by means of passive transfer of an antiserum against influenza virus to control and PMN-depleted mice. The results revealed that PMN preferentially contributed, together with the specific antibody response, to virus elimination in the early- to the late-phase protection against reinfection as well as to late-phase recovery from the primary infection. MATERIALS AND METHODS Mice, viruses, and cell lines. Female BALB/c mice and nude mice of BALB/c background, 6 to 12 weeks old, were purchased from Charles River Japan, Inc. (Kanagawa, Japan). A virulent strain of influenza A virus, A/PR/8/34 (H1N1) (PR8), was obtained from the Osaka Prefectural Institute of Public Health (Osaka, Japan), and a virus pool was prepared by two passages of allantoic fluid from 10-day-old fertile hen s eggs inoculated 48 h before and stored at 70 C. Madin-Darby canine kidney (MDCK) cells were obtained from the National Institute of Infectious Diseases (Tokyo, Japan) and the RIKEN (Institute of Physical and Chemical Research) Cell Bank (Tsukuba, Ibaraki, Japan) and were maintained in culture with Eagle s minimal essential medium (MEM; Nissui Pharmaceutical Co., Tokyo, Japan, and Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco), 100 U/ml of penicillin G, and 100 g/ml of streptomycin (supplemented MEM). The hybridoma RB6-8C5, which produces a rat immunoglobulin G2b (IgG2b) anti-mouse granulocyte MAb reacting with mouse Ly-6G and Ly-6C (also known as Gr-1), referred to below as MAb RB6, was given by the Office of Technology Licensing, Stanford University (Palo Alto, CA), was obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University (Sendai, Japan), and was maintained in culture with RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (Gibco), 100 U/ml of penicillin G, and 100 g/ml of streptomycin (supplemented RPMI 1640), 2 mm L-glutamine (Nacarai Tesque Co. Ltd., Kyoto, Japan), 100 g/ml sodium pyruvate (Gibco), 100 nonessential amino acids (Gibco), 2 g/ml amphotericin B (Gibco), and 25 M 2-mercaptoethanol (Sigma, St. Louis, MO). Antisera and antibodies. Mice were intraperitoneally (i.p.) inoculated with PFU of the virus and were boosted intravenously twice at 1-week intervals. One week later, mice were lightly anesthetized with diethyl ether, blood was collected from the axillary artery and vein, and sera were separated from the pooled blood by centrifugation. The titer of neutralizing antibody (50% neutralizing dose; ND 50 ) in the serum (referred to below as anti-influenza virus antiserum, or anti-pr8 antiserum) was 1:2,048 to 1:4,096 in the MDCK cell plaque assay (14, 16). Normal mouse serum (NMS) was collected and prepared in the same way as the sera from PR8-immunized mice and was used as a control serum. The rat IgG2b MAb RB6 was prepared from the culture supernatant and ascites of pristane-primed BALB/c nude mice transplanted with the hybridoma by affinity chromatography with protein G-agarose (Fast Flow column; Millipore Co., Billerica, MA) and was quantified using an enzyme-linked immunosorbent assay quantitation kit for rat IgG2b antibody (Bethyl Laboratories, Inc., Montgomery, TX). As a control, the same amount of rat IgG (Sigma) was used in the experiment for PMN depletion in vivo. Virus infection and titration. The mice were anesthetized with diethyl ether and inoculated intranasally with 50 l of a virus suspension diluted in cold MEM. After the bleeding of the axillary artery and vein, a lung homogenate (10%) prepared in phosphate-buffered saline (PBS) was dispersed by sonication and centrifuged, and then the supernatant was stored at 70 C until titration. Tenfold dilutions of the supernatant were prepared in PBS supplemented with 0.25% bovine serum albumin (Sigma, St. Louis, MO). The infectivity of the pulmonary virus in the supernatant was titrated by an MDCK cell plaque assay (14, 16). The results were expressed as the mean PFU standard deviation (SD) per lung for 6 to 8 mice/group. Preparation of PMN and alveolar macrophages. Heparinized peripheral blood was collected from the axillary artery and vein or from the retro-orbital venous plexus of lightly anesthetized mice. Peritoneal exudate cells were prepared from the cavities of mice that had received 1.5 ml of glycogen solution (0.1% in saline) 3 to 4 h previously. The peripheral blood and peritoneal exudate PMN were partially purified by centrifugation after being layered onto Mono-Poly resolving medium ((Flow Laboratories Inc., McLean, VA), and the neutrophil-enriched fraction was collected. Alveolar macrophages were prepared by washing the lungs of mice. After the chest cavity was opened, the trachea was cannulated with a needle and anchored by suturing. The lung was washed with PBS containing 0.02% EDTA (10 ml), and the cells were collected by centrifugation. The PMN and alveolar macrophages were resuspended in supplemented RPMI 1640 and used in subsequent experiments (14, 16). Preparation of cells infiltrating the lungs of mice infected with influenza virus. Single-cell suspensions of the lungs were prepared as follows. Freshly resected lungs were minced with scissors to a fine slurry and enzymatically digested by incubation at 37 C for 1 h in supplemented RPMI 1640 medium (15 ml/lung) containing type A collagenase (1 mg/ml; Wako Pure Chemicals Co., Osaka, Japan) and DNase I (30 U/ml; Wako Pure Chemicals). The cell suspensions were drawn up and down 20 times in 10-ml syringes to disperse the cells mechanically, and the suspensions were pelleted, resuspended, and passed through nylon mesh filters. The isolated cells were counted using a hemocytometer, and cell suspensions were first applied to glass slides and then dried and fixed with methanol and stained with Giemsa. The differential cell counts were determined under a microscope, and the absolute number of a leukocyte subtype was determined by multiplication of the percentage of that cell type by the total number of lung leukocytes in the sample. Depletion of PMN, passive transfer of anti-pr8, and treatment with fmlp. MAb RB6 treatment was carried out by daily i.p. injections of 400 g/0.4 ml of the antibody from day 2 to day 8 after virus infection (6 to 8 mice/group). This treatment resulted in a decrease in the PMN counts in the peripheral blood of mice before virus infection to less than 0.5% of the total cell counts. As a control, the same amount of rat IgG (Sigma) was used in the experiment for PMN depletion in vivo. For passive transfer, 0.4 to 0.6 ml of the anti-pr8 antiserum was injected i.p. before or after infection. The same amount of NMS was used as a control. Formylmethionyl-leucyl-phenylalanine (fmlp) ( M; 50 l/mouse) (Wako Pure Chemicals), a tripeptide produced by several kinds of bacteria and a potent chemoattractant and functional activator for PMN and macrophages (9, 29, 39), was intranasally administered to the mice on days 1 and 3 after infection in order to enhance PMN infiltration into the lungs of mice in early infection. Measurement of the inhibitory effect of neutrophils on virus propagation in vitro. One day after the seeding of MDCK cells ( /well) into multiwell culture plates (Nunclon 3033; Nunc), cells were infected with PR8 at a multiplicity of infection (MOI) of 0.1, 1.0, or 10 for 1 h at room temperature, washed with MEM, and incubated in RPMI 1640 supplemented with 10 mm HEPES and 10% fetal calf serum (supplemented RPMI 1640) at 37 C under a 5% CO 2 atmosphere for 6 h. PMN, alveolar macrophages, or spleen cells of normal mice

3 2774 FUJISAWA J. VIROL. FIG. 1. Survival rates of PMN-depleted and control mice infected with a small ( PFU) (A) or a large ( PFU) (B) inoculum of influenza A virus strain PR8 and receiving passive transfer of anti-pr8 antiserum or NMS (or neither) 3 days (3d) after infection. BALB/c mice (8 to 10/group) were daily injected i.p. with MAb RB6 or control rat IgG from day 2 to day 8 after virus infection. Mice were intranasally (i.n.) infected with either inoculum of PR8 on day zero. suspended in supplemented RPMI 1640 ( to cells/well) were added to the infected MDCK cell cultures. To analyze the synergistic inhibition of virus propagation by PMN and antibody, anti-pr8 antiserum inactivated at 56 C for 30 min was diluted in RPMI 1640 and added to the culture with phagocytes or spleen cells, and fmlp ( M) was added from the initiation of the culture (3 to 5 cultures/group). The cells were cultured at 37 C under a 5% CO 2 atmosphere for 24 or 48 h, harvested with the culture supernatant after freezing and thawing, and disrupted by sonication (200 W, 2 A, for 5 min; Insonator model 200M; Kubota K.K., Japan). After centrifugation, the virus titer in a 10-fold dilution of the supernatant was titrated by an MDCK cell plaque assay (16, 46). Results were expressed as the mean PFU per culture SD for triplicate to quintuplicate cultures from at least three independent experiments. Assessments of the cytotoxicity and ADCC of PMN. MDCK cells ( / well) seeded into flat-bottom 96-well microplates (Nunclon; Nunc) 18 h previously were incubated with 30 l ofna 2 51 CrO 4 (610 mci/mm) at 37 C for 1 h, washed four times with MEM, and subsequently cultured for 3 h. Cells were infected with PR8 at an MOI of 10 at 25 C for 1 h, washed twice with MEM, and cultured in supplemented RPMI 1640 for 6 h. Peritoneal or peripheral blood PMN were added to the cultures in the presence or absence of heat-inactivated anti-pr8 antiserum (1:200) or NMS and fmlp ( M), respectively, and were subsequently incubated for 6 to 18 h (triplicate cultures/group). The radioactivity in the culture supernatant was determined using a gamma counter, and the cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) of PMN were expressed as percent 51 Cr release. Cytotoxic activity was calculated as (release by PMN spontaneous release) 100/(total count spontaneous release). ADCC activity was calculated as (release by PMN plus sera spontaneous release) 100/(total count spontaneous release). The spontaneous release from the target cells infected with the virus alone usually ranged from 3 to 5% of the total counts. The total counts were determined by target cell lysis after freezing and thawing of the cultured cells with the supernatant. Results were expressed as means SDs for triplicate cultures from at least three independent experiments. Measurement of chemiluminescent response. The chemiluminescent response of neutrophils to PR8 was measured by a slight modification of a method described previously (14). A PMN suspension ( cells/0.1 ml) was mixed with an equal volume of luminol solution ( M; Laboscience, Tokyo, Japan) in a counting tube, and the tube was placed in a lumiphotometer (Packard, Downers Grove, IL). Intact PR8 ( to PFU) or PR8 pretreated with heat-inactivated NMS or anti-pr8 antiserum (1:100) at 4 C for 15 min was added to the tube, and then the chemiluminescent emission was measured immediately at 37 C (triplicate cultures/group). fmlp ( M) and zymosan (ZAP; Packard) were also used as standard stimulants. Results were expressed as means in triplicate from at least three independent experiments. Statistical analysis. Data from quantitative analyses were expressed as means SDs (6 to 8 mice/group for in vivo experiments or 3 to 5 cultures/group for in vitro cultures). Statistical analyses were performed using Student s t test, and P values of 0.05 were considered to be significant. RESULTS Survival rates of mice infected with a virulent strain of influenza A virus, PR8. After infection with a low ( PFU) (Fig. 1A) or a high ( PFU) (Fig. 1B) inoculum of PR8, the survival rate of control mice consecutively treated with rat IgG was 80 or 20%, respectively, on day 14 after the infection. Consecutive treatments of PR8-infected mice with MAb RB6 resulted in decreases in their survival rates (Fig. 1A

4 VOL. 82, 2008 ROLE OF PMN IN PROTECTION AGAINST INFLUENZA VIRUS 2775 FIG. 2. Pulmonary virus titers of mice infected with a small inoculum of influenza A virus strain PR8 ( PFU) and effects of passive transfer of anti-pr8 or NMS into mice on the virus titers. MAb RB6-treated mice (triangles) and rat IgG-treated control mice (circles) were intranasally (i.n.) infected with PR8 virus and received passive transfer of anti-pr8 (open symbols) or NMS (solid symbols) at 1 h before (A), and on day 1 (B) and 3 (C) after, PR8 infection. Results are expressed as means SDs for 6 to 8 mice per group. Asterisks indicate that the titers in the lungs of PMN-depleted and/or anti-pr8-transferred mice are significantly different (P 0.05) from the titers in the lungs of control mice. and B). The passive transfer of anti-pr8 (0.6 ml of antiserum) to rat IgG-treated mice on day 3 after infection with either inoculum increased the survival rate to 100%, while passive transfer (0.6 ml) to MAb RB6-treated mice led to a slight increase in the survival rate but not to complete survival (Fig. 1A and B). The transfer of NMS to these rat IgG-treated or MAb RB6-treated mice failed to demonstrate any effect on either survival rate (Fig. 1A and B). Cooperative contributions of PMN and antibody to elimination of influenza A virus strain PR8. When a nearly sublethal dose of PR8 (below LD 20 ), PFU, and an almost-lethal dose of PR8 (above LD 80 ), PFU, were inoculated into rat IgG-treated mice, the virus titers in the lungs of mice increased markedly from day 1 after the infection, reached a plateau on days 3 to 5, and began to decrease gradually from day 7 (Fig. 2 and 3). Following infection of MAb RB6-treated mice with PFU of the virus, the PMN depletion resulted in significant elevations of pulmonary virus titers on day 1, day 5, and thereafter, and titers decreased gradually after day 7 (Fig. 2A to C). For the infection with PFU, the MAb RB6 treatment led to elevations of pulmonary virus titers on days 1 to 5, but the increases over titers in control IgG-treated mice were not significant (approximately fivefold), whereas virus titers on day 7 were significantly increased by the treatment (Fig. 3A to C). When anti- PR8 (0.6 ml of antiserum) was passively transferred to rat IgG-treated mice at 1 h before or on day 1 after PR8 infection with either inoculum, the increase in pulmonary virus titers was clearly abolished and the virus titers decreased significantly on day 1 or 3 and continued to decrease thereafter (Fig. 2A and B and 3A and B). Although passive transfer of anti-pr8 (0.6 ml) into MAb RB6-treated mice at 1 h before virus infection with either inoculum resulted in pulmonary virus titers significantly lower, from day 1, than those for control rat IgG-treated or MAb RB6-treated mice without antiserum transfer, the transfer could neither prevent the progressive increase in the virus titers nor suppress their moderate level for 5 days after the infection; the titers declined gradually from day 7 (Fig. 2A and 3A). NMS transfer to rat IgG-treated or MAb RB6-treated mice at 1 h before infection with either inoculum had no effect on pulmonary virus titers in control mice (Fig. 2A and 3A). Passive transfer of anti-pr8 (0.6 ml of antiserum) to MAb RB6-treated mice on day 1 after virus infection with either inoculum could not prevent the progressive increase in the titers in the early phase by day 3 after virus infection and could not decrease the high level of the titers through day 7 (Fig. 2B and 3B). Passive transfer (0.6 ml) into MAb RB6-treated mice on day 3 after infection with either inoculum could not decrease the high virus titers in the lung through day 7 after virus infection (Fig. 2C and 3C). The virus titers of MAb RB6- treated mice transferred with anti-pr8 (0.6 ml of antiserum) on days 1 and 3 after virus infection decreased significantly on day 9 after infection compared to those of MAb RB6-treated control mice (Fig. 2B and C and 3B and C), and the decrease in the virus titers of mice infected with the high inoculum from day 7 was markedly delayed in comparison to that for mice infected with the low inoculum (Fig. 2B and C and 3B and C). These results clearly indicated that PMN in cooperation with the antibody prevented the early-phase infection as well as eliminating the virus in the late phase.

5 2776 FUJISAWA J. VIROL. FIG. 3. Pulmonary virus titers of mice infected with a large inoculum of influenza A virus strain PR8 ( PFU) and effects of passive transfer of anti-pr8 or NMS into mice on the virus titers. MAb RB6-treated mice (triangles) and rat IgG-treated control mice (circles) were intranasally (i.n.) infected with PR8 virus and received passive transfer of anti-pr8 (open symbols) or NMS (solid symbols) at 1 h before (A), and on day 1 (B) and 3 (C) after, PR8 infection. Results are expressed as means SDs for 4 to 8 mice per group. Asterisks indicate that the titers in the lungs of PMN-depleted and/or anti-pr8-transferred mice are significantly different (P 0.05) from the titers in the lungs of control mice. Downloaded from Titers of neutralizing antibody in sera of mice receiving or not receiving anti-pr8 antiserum. When rat IgG-treated and MAb RB6-treated mice were infected with a sublethal inoculum ( PFU) of the virus and injected with NMS before or after the infection, the ND 50 began to rise on day 7 and reached 1:32 or 1:64, respectively, on day 9 (Fig. 4) (1:16 in either group of mice infected with PFU [data not shown]). Passive transfer of anti-pr8 (0.6 ml of antiserum) into rat IgG-treated or MAb RB6-treated mice infected with PFU of the virus led to the steady elevation of neutralizing antibody titers in both mouse groups to 1:512 on consecutive days (Fig. 4A to C) (1:1,024 in either group of mice infected with PFU [data not shown]). These results indicated that the passive transfer of anti-pr8 could provide a steady and high level of anti-pr8 antibody, and they implied that the delay of virus elimination in the late phase of the infection in MAb RB6-treated mice with or without anti-pr8 transfer was not due to a failure of anti-pr8 antibody response in those mice. Enhancement of PMN infiltration into the lungs of mice infected with influenza A virus by passive transfer of antiserum and failure of PMN infiltration due to treatment with MAb RB6. After infection of rat IgG-treated mice with PFU of virus, the number of viable leukocytes infiltrating the lungs of mice increased rapidly on day 1 to more than 10 7 cells, gradually increased until day 5, and declined on day 7 (Fig. 5A). The PMN infiltrating the lungs of mice on day 1 after infection constituted 15 to 20% of the total infiltrated cells and increased to approximately 30% on day 4 to 5 (Fig. 5A). When anti-pr8 (0.5 ml of antiserum) was passively transferred on day 1 after infection to rat IgG-treated mice infected with PFU of the virus, the number of cells infiltrating the lungs of mice reached a plateau more swiftly, on days 2 to 4, than those for control mice receiving NMS and decreased on day 7 to the level observed on day 1. The PMN levels also increased more rapidly to levels higher (approximately 40% of total infiltrated cells) than those in control mice, and the higher levels were maintained by day 7 after infection (Fig. 5A). When MAb RB6-treated mice were infected with the virus, the total number of cells infiltrating the lungs of mice was lower than that in rat IgG-treated control mice by day 5 and gradually increased by day 7 after infection (Fig. 5A and B). However, the number of infiltrating cells for those mice on day 7 was higher than that in untreated control mice (Fig. 5A and B). In contrast, passive transfer of anti-pr8 (0.5 ml of antiserum) to MAb RB6- treated mice after virus infection resulted in markedly increased infiltrations of mononuclear cells, approximately twofold greater than those in MAb RB6-treated mice without the transfer, with a peak on days 3 to 5 after infection (Fig. 5B). The level of PMN infiltrating the lungs of MAb RB6-treated mice with or without anti-pr8 transfer was below 5 to 10% of the level of total infiltrating cells through the whole period of this experiment (Fig. 5B). The ND 50 in the serum after the transfer of anti-pr8 (0.5 ml of antiserum) to mice with or on July 3, 2018 by guest

6 VOL. 82, 2008 ROLE OF PMN IN PROTECTION AGAINST INFLUENZA VIRUS 2777 FIG. 4. ND 50 s in the sera of mice infected with PFU of influenza A virus strain PR8 before or after passive transfer of anti-pr8. Mice treated with MAb RB6 (triangles) and control mice treated with rat IgG (circles) received NMS (solid symbols) or anti-pr8 antiserum (open symbols) at 1 h before (A), and on day 1 (B) and 3 (C) after, virus infection. Results are expressed as ND 50 s of pooled sera from 6 to 8 mice per group. without MAb RB6 treatment revealed 1:512 of the same level (data not shown). These results indicated that the antiserum transfer preferentially augmented PMN infiltration of, and accumulation in, the lungs of mice after virus infection. Acceleration of pulmonary influenza A virus elimination based on the cooperation of PMN and antibody by fmlp administration in vivo. To further confirm the primary contribution of PMN in cooperation with the antibody to virus elimination, fmlp was intranasally administered to mice with or without anti-pr8 transfer after virus infection was performed. fmlp, a potent chemoattractive peptide for PMN and macrophages that is produced by several kinds of bacteria (9, 29, 39), has been shown to be capable of preferentially causing PMN to accumulate in the lungs of mice infected intranasally with influenza A virus in my previous study (14) and this study (Fig. 6C). When fmlp was intranasally administered to normal mice on day 1 after infection with PFU of the virus, the increase in the pulmonary virus titers from day 2 after infection was significantly suppressed for 2 days after fmlp treatment but relapsed on day 5 after infection (Fig. 6A). fmlp treatment in addition to anti-pr8 transfer (0.5 ml of antiserum) on day 1 after virus infection further decreased the virus titer from day 2 after virus infection and accelerated virus elimination compared with that in mice with anti-pr8 transfer alone (Fig. 6A). Consecutive treatment with fmlp on days 1 and 3 after infection resulted in a decrease in the virus titer on day 5, while the survival rate of mice treated consecutively with fmlp decreased from that for the normal-mouse group (Fig. 6B). However, the passive transfer of anti-pr8 to mice treated consecutively with fmlp after infection abolished this decrease in the survival rate (Fig. 6B). The single and consecutive administrations of fmlp preferentially increased PMN accumulation in the lungs to high levels, the same as those in mice receiving anti-pr8 transfer after virus infection (Fig. 6C), and fmlp treatment plus anti-pr8 transfer also further increased PMN infiltration and accumulation (Fig. 6C). These results clarified that PMN mainly contributed to virus elimination in cooperation with antibody, but excessive accumulation of PMN decreased the survival of virus-infected mice, and that the antibody transfer could reduce this decrease in the survival rate due to excessive infiltration. Synergistic prevention of virus propagation with PMN and anti-influenza virus antiserum in vitro. When MDCK cells infected with the virus at an MOI of 0.1 or 1.0 were cultured with PMN at a PMN-to-MDCK cell ratio of 10 for 2 days after infection, the in vitro propagation of virus in cultures was markedly inhibited compared with that in MDCK cell cultures without PMN (Table 1). When anti-pr8 antiserum (1:200) was added to MDCK cell cultures infected at an MOI of 0.1 or 1.0 at 6 h after infection, the virus titer decreased to approximately 1/10 of that in the control culture (Table 1). The addition of PMN to the infected MDCK cell cultures in the presence of the antiserum markedly abolished viral propagation and diminished titers to less than 1/1,000 of the level in MDCK cells alone (Table 1). The addition of fmlp to cultures with PMN alone or PMN plus antiserum led to a more marked inhibition of

7 2778 FUJISAWA J. VIROL. FIG. 5. Counts of total cells and PMN infiltrating and accumulating in the lungs of rat IgG-treated control mice (A) and MAb RB6-treated mice (B) with or without passive transfer of anti-pr8 antiserum or NMS on day 1 after virus infection ( PFU). Open symbols, total-cell counts; solid symbols, PMN counts; triangles, anti-pr8; circles, NMS. Results are means from three experiments for 3 mice per group. propagation compared with that for the corresponding controls (Table 1). The addition of normal alveolar macrophages or spleen cells to the infected MDCK cell cultures in the presence or absence of antiserum did not influence the propagation of virus compared with that in each control culture (Table 1). These findings further confirmed that PMN but not alveolar macrophages could prevent the virus from propagating in MDCK cell cultures and that the prevention of virus propagation by PMN was markedly facilitated by the presence of antibody. Cytotoxic and ADCC activities of PMN against MDCK cells infected with influenza virus. The addition of PMN to MDCK cell cultures infected with the virus led to weak cytotoxic activity, approximately 4 to 8%, against the target cells (Fig. 7). When PMN were cocultured with infected cells in the presence of anti-pr8, the cytotoxic activity of the PMN toward the target cells increased approximately twofold over that in the absence of the antiserum, although the addition of PMN to the MDCK cell cultures in the presence of heat-inactivated NMS did not influence cytotoxic activity (Fig. 7). The addition of fmlp to the infected MDCK cell cultures in the presence of PMN either alone or with antiserum further augmented both cytotoxic and ADCC activities (Fig. 7). The cytotoxic activity of PMN in the presence of NMS was also augmented by the addition of fmlp to the infected target cells (Fig. 7). This indicated that PMN could be an effector cell population for cytotoxicity and ADCC toward influenza virus-infected cells. Facilitation of virus phagocytosis by PMN in the presence of anti-influenza virus antiserum. The addition of the virus to PMN suspensions at an MOI of 1.0 led to a peak chemiluminescent response in the presence of luminol within 5 min and was maintained even after 10 min. The pattern of chemiluminescence was similar to that induced by zymosan rather than to that induced by fmlp (Fig. 8A). No chemiluminescent response was induced when the virus was added at an MOI of 10 or 25 (Fig. 8A). In contrast, the addition of virus pretreated with anti-pr8 to the PMN suspension at an MOI of 1.0 or 10 resulted in a more potent and prolonged chemiluminescent response than that with untreated infective virus (Fig. 8B). Pretreatment with NMS did not give rise to such an augmentation of the response (Fig. 8). These results indicated that PMN could phagocytose not only a small amount of virus particles in the absence of antibody but also a large amount of virus particles in the presence of antibody. DISCUSSION Although previous studies in this series (16, 45) revealed the protective roles of PMN and alveolar macrophages in the early phase of primary infection with the low-virulence strain PR8, the role of both types of phagocytes in early protection against primary infection with a virulent strain of influenza A virus remained unclear until Tumpey et al. recently reported their study (48). That study indicated that PMN and macrophages play a critical role in early-phase protection against a sublethal infection with a recombinant human influenza virus (H1N1) bearing 1918 influenza virus HA and NA (pathogenic to mice) in mice depleted of PMN and/or macrophages by treatment with MAb RB6 and/or an anti-macrophage agent, respectively. In the present study, the depletion of PMN by MAb RB6 treatment resulted in a significant increase in pulmonary virus titers in the early to intermediate phases of infection with a low inoculum of the virulent strain PR8 but not in infection with a high inoculum, despite showing a tendency toward an increase in virus titers (Fig. 2 and 3). This indicated that PMN contrib-

8 VOL. 82, 2008 ROLE OF PMN IN PROTECTION AGAINST INFLUENZA VIRUS 2779 FIG. 6. Effects of intranasal (i.n.) treatment with fmlp on pulmonary virus titers (A), survival rates of mice (B) (8 to 10 mice per group), and counts of viable cells infiltrating the lungs of mice (C) after infection with influenza A virus strain PR8 ( PFU) with or without passive transfer of anti-pr8 antiserum. (A and B) Solid and shaded symbols, mice without anti-pr8 passive transfer; open symbols, mice receiving anti-pr8 transfer on day 1 after virus infection. Circles, virus-infected normal mice; triangles, normal mice treated with fmlp on day 1; squares, normal mice treated with fmlp on days 1 and 3. Results in panel A are means SDs for 5 to 6 mice per group. (C) Counts of mononuclear cells (shaded bars) and PMN (solid bars) infiltrating the lungs of PR8-infected mice with or without fmlp treatment (on day 1 only or on days 1 and 3) and/or passive transfer of anti-pr8 on day 1 after infection. Results are means from three experiments with 3 mice per group. uted primarily to early protection against sublethal primary infection with the low inoculum of the virulent strain PR8 but not with the high inoculum. This result also seemed to reflect a limitation of PMN function in that PMN alone might not be able to protect against high-dose infection with more than nearly lethal infection of the virulent virus. In the late recovery phase after virus infection with either inoculum, MAb RB6 treatment led to a marked delay in elimination of the virus (Fig. 2 and 3), strongly suggesting that PMN might contribute to late-phase clearance of pulmonary virus. However, it is necessary to further analyze the role of PMN in virus elimination in the late phase of infection, since it has been reported very recently that MAb RB6 treatment resulted in partial depletions of Gr1 CD8 T cells and Gr1 monocytes (12). Therefore, in addition to the sole protective role of PMN in innate immunity to the virulent virus, the cooperative role of PMN with the antibody response in virus infection was further investigated. The passive transfer of the antiserum into PMN-depleted mice before and after virus infection with either inoculum could neither suppress rapid virus propagation and high virus titers nor accelerate virus elimination, although the transfer into control mice markedly enhanced early protection and late recovery over the whole period of the infection, even with a high inoculum of the virus (Fig. 2 and 3). The neutralizing antibody titer in the serum (ND 50 ) after antiserum transfer was sufficiently elevated to neutralize even a high level of pulmonary virus titers (Fig. 2, 3, and 4), while for MAB RB6-treated or control mice receiving NMS, low and similar ND 50 s (1:32 or 1:64) in the sera, which could not decrease high virus titers in MAb RB6-treated mice and control mice transferred with NMS were detectable on day 7 after infection with either inoculum (Fig. 4) (data for the high inoculum not shown). Therefore, the delay in virus elimination in the late phase for MAb RB6-treated mice was not attributable to a failure of the antibody response in PMN-depleted mice due to MAb RB6 treatment (Fig. 2, 3, and 4). Furthermore, antiserum transfer into control mice but not into PMN-depleted mice swiftly and preferentially enhanced PMN infiltration of the lung from the early to the late phase after virus infection (Fig. 5A and B). Quite recently, Buchweitz et al. (7) reported that the kinetics of PMN infiltration into the lungs of C57BL/6 mice infected with the A/PR/8 strain resembled that in the present study. These results clearly indicate that the antibody transfer and pulmonary PMN accumulation are well correlated with the decreases in pulmonary virus titers (Fig. 2B and C and 3B and C) and in the mortality rate of mice (Fig. 1). Therefore, the present study has clarified the most notable evidence that PMN primarily cooperate with the antibody response in the protection against and recovery from both primary infection and reinfection with the virulent virus.

9 2780 FUJISAWA J. VIROL. TABLE 1. Synergistic prevention of influenza virus propagation by PMN and anti-influenza virus antiserum in vitro MOI Effector cell type (E:T ratio) a fmlp Antiserum Virus titer (10 4 PFU)/culture b 0.1 None Spleen (20:1) Al M (10:1) PMN (10:1) NMS None NMS NMS Spleen (20:1) Al M (10:1) PMN (10:1) NMS NMS a PMN, alveolar macrophages (Al M ), or spleen cells (effector-to-target cell E:T ratio, 10:1 or 20:1) obtained from normal mice were added to MDCK cell cultures that had been infected 6 h earlier with influenza virus at an MOI of 0.1 or 1.0. Where indicated, anti-influenza virus antiserum or a control (NMS), both at a 1:200 dilution, and fmlp were added to the cultures concomitantly with the effector cells. Cultures were subsequently incubated for 2 days. b Virus titers in the cultures were determined by an MDCK cell plaque assay 48 h after infection. Results are means SDs for triplicate cultures. The propagation of virulent influenza virus in vitro was inhibited by the addition of PMN to virus-infected MDCK cell cultures, and the inhibition was synergistically facilitated in the presence of heat-inactivated antiserum but not of NMS in the cultures (Table 1), while the addition of alveolar macrophages or spleen cells from normal mice could neither inhibit virus propagation nor exhibit such potent inhibition with the antiserum (Table 1). This indicated that the potent preventive effect on virus propagation was attributable neither to nonspecific disturbances by other leukocytes without PMN nor to nonspecific inhibitors in the serum (2, 35, 42). The weak cytotoxic and more potent ADCC activities of PMN in the presence of antiserum were also observed in MDCK cell cultures infected with influenza virus (Fig. 7), suggesting that PMN could be a cytotoxic effector cell population responsible for the virus-infected target cells and could efficiently inhibit the spread of virus in the infective site in the presence of antibody. This result seemed to be supported by evidence that PMN could adhere to influenza virus-infected cells (32). Furthermore, a chemiluminescent response of PMN was elicited with a small amount of influenza virus (MOI, 1.0), but was abolished by large numbers of infective virus (MOI, 10). However, a potent and prolonged chemiluminescent response to the large amount of virus was induced by the pretreatment of the virus with the antiserum (Fig. 8A and B). My previous study also indicated that PMN were capable of phagocytosing influenza virus based on the chemiluminescent response of PMN to the virus and that PMN were nonpermissive to virus replication, although alveolar and peritoneal macrophages are permissive to the virus but not as susceptible as MDCK cells (46). Cassidy et al. (8) also indicated that PMN infected with influenza virus were abortive and nonpermissive to the virus infection. These results indicated that PMN could phagocytose and inactivate a large amount of the virus in the presence of antibody, supporting the notion that PMN cooperated with antibody in eliminating swiftly the high virus titers in the lungs of mice infected with either inoculum of the virus (Fig. 2 and 3). In the present study, indeed, treatments with fmlp in vitro markedly potentiated the preventive effect of PMN on influenza virus propagation and both the cytotoxic and ADCC activities of PMN in the presence and absence of antibody (Fig. 7; Table 1). Furthermore, intranasal administration of fmlp to normal mice following infection resulted not only in an increase in PMN infiltration of the lung (Fig. 6C) but also in a significant decrease in virus titers in the early phase of infection (Fig. 6A), though in the intermediate phase they relapsed to a high level similar to that for control mice (Fig. 6A). This result was probably due to the short half-life of fmlp in the body (9). Further consecutive treatments (on days 1 and 3) of mice with fmlp increased the mortality rate of mice in spite of the prolongation of the decrease in virus titers (Fig. 6A and B). Since previous studies have suggested that tissue damage and destruction in the lung could be elicited by excessive infiltration and accumulation of PMN and macrophages (4, 28), the increase in the mortality of mice by consecutive fmlp treatment seems to reflect that interpretation (Fig. 6). In contrast, antiserum transfer in addition to fmlp treatment not only eliminated the increased mortality rate of mice elicited by the fmlp treatment alone (Fig. 6B) but also more markedly decreased virus titers and accelerated virus elimination (Fig. 6A) and further augmented the inflammation and accumulation of PMN compared to that observed with antiserum transfer or fmlp treatment alone (Fig. 6C). Thus, PMN play an essential role in the host defense in cooperation with the potent antibody response even against infection with a high inoculum of virulent influenza A virus. Assuming that highly pathogenic influenza viruses such as H5N1 avian virus and the 1918 pandemic virus lead to marked virus propagation in the lung and multiple organs and to excessive accumulation of PMN and macrophages in the lung and that they cause functional deficiency of organs and ultimately death, these results strongly suggest that monoclonal or polyclonal antibodies to highly pathogenic influenza viruses will be utilized for prophylaxis and therapy in pandemics and epidemics of the virus. Tumpey et al. (48) also reported that alveolar macrophage depletion or total-phagocyte depletion in mice resulted in more rapid and higher mortality of mice and in higher virus titers than those in naïve mice with depleted PMN alone after primary infection with the human H1N1 virus. These results did not elucidate the role of macrophages in virus elimination in cooperation with antibody but demonstrated the significant role of macro-

10 VOL. 82, 2008 ROLE OF PMN IN PROTECTION AGAINST INFLUENZA VIRUS 2781 FIG. 7. Cytotoxic and ADCC activities of PMN in vitro. A PMN suspension and either anti-pr8 antiserum (1:200) or NMS (also at a 1:200 dilution) were concomitantly added to influenza virus-infected MDCK cell cultures at 6 h after infection. The release of 51 Cr from target MDCK cells was measured at 12 h after the addition of PMN and expressed as a percentage of total cell counts. Dark shaded bars, ADCC activity in the presence of antiserum; light shaded bars, cytotoxic activity in the presence of NMS; open bars, cytotoxic activity in the absence of serum. Results are means SDs for triplicate or quadruplicate cultures. phages in early protection against primary infection with the virulent H1N1 virus. My previous study had already shown the significance of alveolar macrophages in early-phase protection against primary infection with a high inoculum of the low-virulence virus (16). For mice infected with a virulent virus, however, the cooperative protective role of alveolar macrophages and/or mononuclear phagocytes with antibody was not clarified by the present study. Furthermore, in this study, although the passive transfer of anti-pr8 to MAb RB6-treated mice after virus infection elicited remarkable infiltration of the lung by alveolar macrophages and mononuclear phagocytes (monocytes from morphological observations) in the early to intermediate phases of infection (Fig. 5B), pulmonary virus titers in PMN-depleted mice were demonstrated to be higher than those in control mice (Fig. 2A to C). These data and the studies cited above suggested that alveolar macrophages and/or mononuclear phagocytes might effectively cooperate with PMN in virus elimination in the presence and absence of antibody. In a study using Fc receptor knockout (FcR / ) mice, Huber et al. (23) reported that FcR / mice were significantly more susceptible to lethal respiratory reinfection with a virulent influenza A/PR8 virus than FcR / mice after intranasal immunization with a influenza vaccine plus interleukin-12. In addition, using passive transfer of immune serum to naïve FcR / and FcR / mice, they further showed that macrophages are capable of ingesting opsonized virus by Fc receptor-mediated phagocytosis and that FcR / mice were highly susceptible to influenza virus infection even in the presence of anti-influenza virus antibody (23). The FcR knockout mice used in their studies lacked two Fc receptors, Fc RI and Fc RIII (43), and FcεRI (31), and all of these FcRs were also expressed on the surfaces of PMN (17, 51). From their study, therefore, it appears likely that not only macrophages but also PMN might be included among the FcR-positive cells responsible for antibody-mediated viral clearance, and their results did not contradict the results of the present study. It was recently reported that murine peritoneal macrophages could phagocytose apoptotic HeLa cells induced by influenza A virus infection and then diminish the virus multiplicity in the culture (13). Most recently, Hashimoto et al. (20) reported that both PMN and macrophages accumulated in the lungs of mice infected with influenza virus A/WSN (H1N1) could phagocytose the infected apoptotic cells and that alveolar macrophages from virus-infected mice had greater phagocytic activity than those from uninfected mice. The preventive effect of PMN on virus propagation in vivo and in vitro in this study (Fig. 2 and 3; Table 1) might include phagocytosis of apoptotic cells induced by virus infection, while inhibition of virus propagation by alveolar macrophages was not observed in