Influenza Virus-Induced Immune Complexes Suppress Alveolar Macrophage Phagocytosis

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1 JOURNAL OF VIROLOGY, May 1984, p X/84/5287-6$2./ Copyright 1984, American Society for Microbiology Vol. 5, No. 2 Influena Virus-Induced Immune Complexes Suppress Alveolar Macrophage Phagocytosis CALVIN L. ASTRYt* AND GEORGE J. JAKAB Department of Environmental Health Sciences, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 2125 Received 6 September 1983/Accepted 22 January 1984 Immune complexes in the lungs are capable of inducing adverse responses. Herein e have detailed the formation of immune complexes in the lungs of influena virus-infected mice and examined their effect on alveolar macrophage defenses. On days 3, 7, 1, 15, and 3 after aerosol infection ith influena A/PR8/34 virus, the acellular pulmonary lavage fluid as tested for viral antigen, specific viral antibody, and immune complexes by immunoassays. Whereas peak viral antigen (day 3) diminished to undetectable levels by day 1, specific viral antibody remained at a lo concentration until day 1, after hich it rapidly increased. Immune complex concentrations increased through day 7, peaked at day 1, and gradually returned to the control level by day 3. These data demonstrate that immune complexes of detectable sie are induced by influena virus infection during the interface beteen antigen excess and antibody excess conditions. Since alveolar macrophages are the pivotal phagocytic defense cells in the lung, the ability of normal alveolar macrophages to ingest opsonied erythrocytes as quantitated in the presence of immune complexes from lavage fluid. Immune complexes from day 1 virus-infected lungs caused a dose-dependent suppression of antibody-mediated phagocytosis to 3% of control values. In contrast, although these immune complexes also markedly decreased the phagocytosis of antibody-coated yeast cells, they did not significantly impair the antibody-independent ingestion of unopsonied yeast cells by macrophages. The suppressive effects of immune complexes on alveolar macrophages may, in part, explain the phagocytic dysfunction that occurs 7 to 1 days after influena virus pneumonia. Influena virus infections transiently suppress pulmonary antibacterial defenses by causing dysfunctions in the alveolar macrophage phagocytic system (6, 29). The groth of virus in the lungs is not immediately accompanied by the impairment of phagocyte function. Instead, the macrophage defect is associated ith the period of time of rapidly declining pulmonary virus titers and the concomitant development of the antiviral immune response in the lungs (9, 13). This temporal relationship suggests that alveolar macrophage dysfunction does not result from a direct effect of virus on phagocytes (19) and that the host immune response may play a role in the defect (11). Indeed, recently e have demonstrated that the transient defect in alveolar macrophage phagocytosis is, in part, mediated by the humoral (1) and cell-mediated (13) antiviral immune response of the host. Formation of immune complexes is a normal immunological response to viral antigens (2), and immune complexes have been shon to induce phagocytic defects in macrophages (7, 17) and polymorphonuclear leukocytes (26). Hoever; to produce the defect, the immune complexes have to be at or near their equivalence, since small soluble complexes found in antigen or antibody excess are generally innocuous to normal phagocytic function (26). Herein, e tested the hypotheses that immune complexes are formed in the lungs during the course of influena virus infection and that the immune complexes are at the greatest detectable concentration during the time of maximal virus-induced suppression of alveolar macrophage phagocytosis. We also demonstrated * Corresponding author. t Present address: Department of Medicine, Louisiana State University School of Medicine, Ne Orleans, LA that incubation of these immune complexes ith normal alveolar macrophages suppresses phagocytosis. MATERIALS AND METHODS Animals. Female Siss mice (Harlan Sprague-Daley, Walkersville, Md.) eighing beteen 2 and 25 g ere used in this study. The animals ere fed rat cho and ater ad libitum. Infected and uninfected animals ere housed in separate rooms to prevent cross contamination. Viral infections. Influena A/PR8/34 virus as gron and titrated in the allantoic fluid of 1-day-old embryonated chicken eggs by standard methods (15). The virus had a titer of 17-5 median egg infectious doses per ml and as stored in small portions at -8 C. Mice ere infected by a 3-min aerosol inhalation of a 1:5 dilution of the virus as previously described (12). The resultant infection induced a moderate to severe pneumonia that as sublethal to the animals. Pulmonary lavage. On days 3, 7, 1, 15, and 3 after the onset of viral infection, infected and noninfected mice ere sacrificed by cervical dislocation and exsanguinated by cardiac puncture. Serum as separated and stored at -2 C. The lungs ere surgically removed and lavaged ith three 1.5-ml portions of fluid as described previously (1). After centrifugation (2 x g for 1 min) lavaged cells from normal mice ere suspended in Hanks balanced salt solution and counted in a hemacytometer. A portion of the collected alveolar lavage fluid as stored at -8 C for later analysis for viral antigen and antiviral antibody by immunoassay. The remaining portion of the lavage fluid as mixed ith equal volumes of 12% polyethylene glycol (PEG; Sigma Chemical Co., St. Louis, Mo.) in borate buffer (ph 5.8), and the mixture as left overnight at 4 C to precipitate immune complexes. This 6% PEG solution caused the precipitation of large-molecular-eight immune complexes, but it exclud- Donloaded from on November 8, 218 by guest

2 288 ASTRY AND JAKAB ed monomeric immunoglobulin G (IgG) (4). Although PEG precipitation of immune complexes from bronchoalveolar lavage fluid has not been documented, this method has been applied to precipitate immune complexes from synovial fluid (16). The glycoprotein mucin, hich is found in synovial fluid, did not appear to interfere ith the recovery of immune complexes. Precipitated immune complexes ere collected by centrifugation (1, x g for 2 min), ashed in 6% PEG, and suspended in Hanks balanced salt solution. These isolated virus-induced immune complexes ere used to incubate normal alveolar macrophages before the in vitro phagocytic assays, as described belo. Immunoglobulin and sera. Lyophilied chicken antibody to influena A/PR8/34 virus (National Institutes of Health research reagent, catalog no. V ) and alkaline phosphatase-labeled goat antibodies to rabbit IgG or mouse IgG (Kirkegaard and Perry, Gaithersburg, Md.) ere reconstituted in sterile distilled ater and stored in small portions at -2 C. Rabbit influena A/PR8/34 virus antiserum as prepared by intravenous injection of Madin Darby canine kidney tissue culture-gron virus eekly (3) into Ne Zealand White rabbits for 3 consecutive eeks. After the final injection, the animals ere exsanguinated by cardiac puncture, and the serum as separated and stored at -2 C. The serum had a hemagglutination inhibition titer of 1:256 against PR8 virus ith chicken erythrocytes. Immunoassays. Influena A/PR8/34 viral antigen and specific antiviral antibody ere detected in samples of lavage fluid and serum by a modification of the double-sandich enyme-linked immunosorbent assay techniques previously described (25). (i) Antigen detection. Chicken antibody against influenla A/PR8/34 virus as diluted to 1:4, ith carbonate buffer (ph 9.6) and alloed to adhere to polyvinyl microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.) overnight at 4 C. The microtiter ells ere ashed three times ith phosphate-buffered saline (ph 7.4) plus.5% Teen 2 (PBS-Teen) to remove excess antibody. A 1-,u sample of lavage supernatant fluid from normal and virus-infected mice as incubated in duplicate in the antibody-coated microtiter ells for 2 h at 37 C. After each ell as ashed three times ith PBS-Teen to remove excess lavage fluid, a 1-pA sample of rabbit antiserum against influena A/PR8/34 virus (diluted 1:1, ith PBS-Teen plus 1% normal mouse serum) as added to the microtiter ells for ca. 1 h at 37 C. Each ell as rinsed three times ith PBS-Teen, and 1 RI of alkaline phosphatase-labeled goat antibody to rabbit IgG (diluted 1:4 ith PBS-Teen) as added for 1 h at 37 C. Each microtiter ell as rinsed three times and incubated ith 1 pa of p-nitrophenyl phosphate (1 mg/ml) in diethanolamine buffer (ph 9.8) for 3 min at 37 C. A sample as considered positive for viral antigen if the resultant absorbance at 45 nm as greater than 2 standard deviations above control samples (32). (ii) Antibody detection. Microtiter ells ere precoated ith chicken antibody against influena A/PR8/34 and then incubated ith the egg-gron stock virus (diluted 1:5 in PBS-Teen) for 2 h at 37 C. Each ell as rinsed three times ith PBS-Teen to remove excess virus. Three dilutions from each lavage supernatant fluid ere incubated in separate ells for 1 h at 37 C. After the microtiter ells ere ashed three times ith PBS-Teen, a 1 RI sample of alkaline phosphatase-labeled goat antibody against mouse IgG as added for 1 h at 37 C. The ells ere rinsed and incubated ith 1 RI of p-nitrophenyl phosphate for 3 min at 37 C. The resultant relative absorbances at 45 nm ere J. VIROL. used to calculate the specific antibody titer against influena virus by extrapolating to the dilution at hich the relative absorbance as no longer greater than 2 standard deviations above that of lavage fluid from noninfected control animals. (iii) Immune complex detection. Immune complexes in the lavage fluid and serum of virus-infected mice ere quantified by methods previously described (31). Briefly, ra bovine semen (generously donated by Sire Poer, Frederick, Md.) as stored in small portions at -2 C. Before each assay, the semen as ashed three times ith PBS; the spermatooa ere counted ith a hemacytometer and suspended to 17 spermatooa per ml in PBS. A 1-pl sample of the spermatooan suspension as added to each ell of a polyvinyl microtiter plate, hich as then centrifuged (1, x g for 1 min) and submerged in a.25% glutaraldehyde- PBS solution for 5 min. The plate as inverted and snapped to remove the fixative and then rinsed three times ith PBS- Teen. A 5-pA sample of lavage fluid or serum, diluted ith an equal volume of PBS-Teen, as added to each spermcoated ell, and the ells ere incubated at 37 C for 2 h. After each ell as rinsed three times ith PBS-Teen, 1 pul of alkaline phosphatase-labeled goat antibody to mouse IgG (diluted 1:4 ith PBS-Teen) as added, and the ells ere incubated at 37 C for 1 h. The microtiter ells ere rinsed three times ith PBS-Teen, and a 1-pA sample of p-nitrophenyl phosphate substrate solution as incubated in each ell for 3 min at 37 C. The relative absorbance at 45 nm as used to measure the concentration of immune complexes. For a more complete quantitation of immune complexes, mouse IgG as heat aggregated at 63 C for 2 min and then separated from monomeric immunoglobulin by precipitation in 6% PEG (molecular eight, 8). The precipitated aggregates of IgG have properties similar to those described for antigen-antibody complexes (21, 22). Therefore, the standard curve that defined the relationship beteen optical density (by immunoassay) and protein concentration of aggregated IgG (immune complex) as used to quantitate immune complexes from virus-infected mice as micrograms of aggregated IgG protein. Phagocytic assays. The effect that virus-induced immune complexes had on antibody-mediated (Fc receptor) and antibody-independent phagocytosis by alveolar macrophages as tested in vitro ith both erythrocytes and yeast cells as phagocytic particles. Sheep erythrocytes (SRBCs; Cordis Laboratories, Miami, Fla.) ere radiolabeled ith chromium-51 and opsonied ith specific goat antibody (Cordis Laboratories) as previously described (5). Candida krusei, gron from stock portions in tryptic soy broth overnight at 37 C, as ashed three times ith PBS and suspended in Hanks balanced salt solution. One portion of the yeast cell preparation as opsonied for 3 min at 37 C ith specific anti-c. krusei rabbit IgG (agglutination titer, 1:32) hich had been isolated from other immunoglobulins ith immobilied concanavalin A as previously described (2). The phagocytic assays ere performed ith 15 alveolar macrophages from noninfected control mice. The macrophages ere adhered to glass cover slips (22 by 22 mm) at 37 C for 3 min. After the nonadherent cells ere removed, the resultant cell monolayer consisted of more than 98% macrophages, as determined by morphological criteria. The cell monolayer as first incubated ith 1 pa of immun complexes (isolated from virus-infected mice) for 1 h at 37 C and then challenged ith 2 x 17 SRBCs (2 SRBCs per macrophage) for 2 h at 37 C. After the lysis of extracellular SRBCs by hypotonic shock, the released radiolabel as removed in five ashes ith PBS plus 1% gelatin. Total Donloaded from on November 8, 218 by guest

3 VOL. 5, 1984 VIRAL IMMUNE COMPLEXES SUPPRESS PHAGOCYTOSIS 289 monolayer cell lysis as accomplished by treatment ith PBS plus.5% Triton X-1. The amount of SRBCs ingested by the macrophages as quantitated by measuring total radioactivity in the macrophage lysate ith liquid scintillation counting techniques. Similarly, after preincubation ith immune complex lysates, macrophage monolayers ere challenged ith 2 x 16 unopsonied or antibody-coated C. krusei cells (2 yeast cells per macrophage) for 3 min at 37 C. After vigorous agitation ith five ashes of PBS-1% gelatin to remove extracellular yeast cells, the cell monolayers ere fixed in methanol and stained ith a Wright-Giemsa solution. Phagocytic activity as quantitated microscopically by determining both the percentage of phagocytic macrophages (containing three or more yeast cells) and the number of yeast cells phagocytied per macrophage. The phagocytic index as calculated by multiplying the number of phagocytic macrophages per 1 cells by the average number of yeast cells per macrophage. Statistical method. Data ere tested by analysis of variance, regression analysis, or Student's t test (24). All statements of significance are at P <.5. RESULTS The use of bovine spermatooa as a solid phase for binding free immune complexes is demonstrated in Fig. 1. Various amounts of mouse IgG, aggregated by heat (63 C for 3 min) and separated from monomeric IgG by precipitation in 6% PEG solution, ere bound by fixed spermatooa. As little as 1,ug of aggregated IgG (A-IgG) could be accurately measured by the resultant alkaline phosphatase activity, closely reflecting previously published findings (31). Since monomeric IgG has been shon to offer little interference in this immune complex assay (31), the data in Fig. 1 ere used in making a standard curve to quantitate complexes in the lungs of virus-infected mice. The temporal relationship among the concentrations of viral antigen, antiviral IgG, and immune complexes in the acellular lung lavage fluid during the course of influena virus infection is presented in Fig. 2. The high concentrations of influena virus antigen that existed during the early stages of infection (day 3) rapidly declined to undetectable levels by day 1. In contrast, antiviral IgG as only detected at lo concentrations during eek 1 of infection. Hoever, after day 7, the antibody levels continued to rapidly increase I- cm a -J A ~~~~~~~A// A A - a -.1 1o JG of A-IgG FIG. 1. Standard dose-dependent curve of A-IgG as detected by bovine spermatooa. Each value represents the mean optical density from the resultant alkaline phosphatase activity in duplicate experiments. -J -J >.3 P Z.2 a.1 < Z C! cc C SL4 -r I sor-i VNIMs A I VIRAL ANd I DAYS AFTER VIRUS INFECTION 3 FIG. 2. Temporal relationship among viral antigen (), antiviral IgG (U), and immune complexes (A) in the acellular lavage fluid from influena virus-infected mice. Each value represents the mean ± standard error of at least five determinations. through the 3-day assay period. Immune complex detection as limited to a period of time beteen eeks 1 and 2 of infection, hen free viral antigen as aning and antiviral IgG as beginning to accumulate. Concentrations of immune complexes peaked beteen days 7 and 1 at ca. 18 p.g of A-IgG per 1 RI of lavage fluid. Subsequently, the immune complex concentration gradually diminished, so that by day 3, a time of maximal antiviral IgG levels, less than 1,ug of A-IgG remained. To determine hat effect the presence of virus-induced immune complexes have on pulmonary phagocytic function, normal alveolar macrophages ere preincubated ith immune complexes isolated from lung lavage fluids of virusinfected mice and challenged ith opsonied SRBCs as the phagocytic particle (Fig. 3). Preliminary assays ere performed ith lung lavage fluid directly incubated ith normal macrophages, and a slight inhibition of phagocytosis as observed. By using PEG precipitation techniques on lung lavage fluid, the large dilution factor inherent to lavage techniques as circumvented, hich alloed the concentration of immune complexes. After concentration, a doseresponse relationship of phagocytosis inhibition could still be demonstrated (Fig. 3, insert), consistent ith the previously described properties of immune complexes (7). Although e did not directly establish that the parameters of the PEG precipitation techniques are the same for both serum and lung lavage fluid, our experimental results are consistent ith the fact that a surfactant did not alter the precipitation of immune complexes from lavage fluid. Antibody-mediated phagocytosis of SRBCs as depressed by 68 ± 7% in the presence of immune complexes from day 1 virus-infected animals-tice the percent depression caused by immune complexes from virus-infected mice on other days. The inhibitory properties of day 1 immune complexes ere dose dependent beteen.1 and 15,ug of protein (Fig. IuIrN,,I\ _ \ \ a / I ANTIVIRAL IgG IMMUNE COMPLEXES /\I IC\- A klitlt'%ki ai Donloaded from on November 8, 218 by guest

4 29 ASTRY AND JAKAB 1- CO UJ 8 -J.- 6 O 4 I. I-. UJ2 'W (1 15l3 DAYS AFTER VIRUS INFECTION i ~- < r = Wo M 2 * O UG of PROTEIN/ ASSAY FIG. 3. Inhibition of antibody-mediated phagocytosis of SRBCs by macrophages in the presence of immune complexes from virusinfected mice. Each value represents the percentage of control ingestion as determined by the ratio of radiotracer activity. Immune complexes from five mice per time point ere pooled and run in triplicate. The inset shos the relationship beteen inhibition of macrophage phagocytosis and various concentrations of day 1 immune complexes. 3, insert), causing phagocytosis impairment of up to 75%. Microscopic observation shoed that over 9% of all macrophages (including controls) remained phagocytic during these assays. Therefore, a major suppressive effect of immune complexes from virus-infected mice appears to be a decrease in the number of engulfed SRBCs per macrophage. To test hether virus-induced immune complexes interfered ith antibody-independent phagocytosis, alveolar macrophage monolayers ere challenged ith unopsonied C. krusei. In the presence of immune complexes lavaged from infected mice, antibody-independent phagocytosis as not significantly altered (Fig. 4). In contrast, immune complexes did interfere ith the ingestion of specifically opsonied C. krusei (Fig. 4). In fact, immune complexes from day 1 virus-infected mice depressed macrophage ingestion of antibody-coated yeast cells by 41%. As in the erythrocyte assays, immune complexes did not significantly change the number of phagocytic macrophages (Fig. 4); therefore, the defect in the phagocytic index reflects only a decrease in the number of yeast cells ingested per phagocyte. DISCUSSION As a consequence of influena virus infection, there exists a transient impairment of alveolar macrophage phagocytic activity (28). Previous studies in our laboratory have demonstrated a failure of Fc receptor-mediated ingestion by alveolar macrophages that occurred ca. 9 days after the onset of virus infection in mice (28). Concomitantly, Fc receptor binding activity remained normal, and cytophilic Fc and complement receptor activities ere enhanced, thus suggesting that the virus-induced defect as not a generalied metabolic dysfunction but a receptor-specific phenomenon. The observed suppression of phagocytosis occurs predominantly during eek 2 of the viral infection (11) in animals ith intact humoral (1) and cellular immune (13) responses. Since the infectious state of the virus infection is virtually resolved after eek 1 (3), it seems likely that the virus acts indirectly to produce its adverse effects on phagocytosis. The present study tested the hypothesis that immune complex formation, knon to cause dysfunction in macrophage and neutrophil phagocytic function in vitro (7), may be one mechanism by hich influena virus interferes ith phagocyte function. There are numerous assays by hich immune complexes can be measured (14). Most established methods bind complexes via Fc receptors (18) or complement receptors (8). Similarly, bovine spermatooa have Fc receptors that bind predominantly complexed antibody and not monomeric antibody (31). We have demonstrated (Fig. 1 and 2) not only that this immune complex assay as quantitatively sensitive to various concentrations of immune complex-like A-IgG, but also that it as not affected by the accumulated levels of monomeric IgG that existed at day 3 after virus infection. Hoever, like other assays, bovine spermatooa bound any immune complex present and did not sho specificity for antigen. Using the bovine spermatooa assay, e found that influena virus infection induced immune complex formation (recoverable by lavage) predominantly during eek 2 (Fig. 2). This corresponded to a time hen the lung fluid as changing from viral antigen excess to antiviral antibody excess as immunological defenses ere becoming ell established. This suggests that a previous report failed to detect immune complexes by immunofluorescence in the lungs of virus-infected mice, possibly because the experiments did not go beyond day 5 of infection (23). Others have shon that the sie, solubility, and characteristics of immune complexes are determined by the ratio of antigen to antibody (21). Therefore, during the course of influena virus infection, there existed a continuum of changing conditions that resulted in the formation of immune complexes of x I- - I C) İ J. VIROL DAYS AFTER VIRUS INFECTION FIG. 4. Ingestion of unopsonied (solid bars) or IgG-coated (hatched bars) C. krusei by macrophages in the presence of immune complexes from virus-infected mice. Each bar represents the mean phagocytic index + standard error of five individual determinations. The number in parentheses indicates the percentage of macrophages that remained phagocytic in the presence of immune complexes. Donloaded from on November 8, 218 by guest

5 VOL. 5, 1984 VIRAL IMMUNE COMPLEXES SUPPRESS PHAGOCYTOSIS 291 various sies and composition. The importance of immune complex sie on interaction ith phagocytes has been idely reported (7, 21). Large immune complexes, formed in vitro near the equivalence point, ere much more able to impair phagocytosis (7) and to stimulate release of oxygen metabolites (26) from macrophages and neutrophils than ere small immune complexes formed in antigen or antibody excess. Similarly, e have demonstrated that hen the amount of detectable immune complexes in virus-infected mice as at a maximum, the ability of these complexes to impair antibody-mediated phagocytosis by macrophages as greater than at any other time (Fig. 3 and 4). Whether the difference in macrophage phagocytic impairment as due to immune complex sie or simply to the absolute amount of immune complexes recovered is important. As e have demonstrated, inhibition of phagocytosis is dose dependent (Fig. 3, insert). Hoever, immune complex concentrations from day 7 virus-infected mice ere almost equal to those from day 1 animals, yet day 7 immune complexes had only a small inhibitory action against phagocytosis. This, most likely, is due to the fact that since the bovine spermatooa assay cannot differentiate beteen a fe large immune complexes and many smaller immune complexes, to samples that are approximately equal by this detection assay may indeed contain different sies of immune complexes and consequently affect macrophage function differently. Not all phagocytic functions of macrophages ere affected by immune complexes. Ingestion of C. krusei yeast cells is mediated through mannose-type receptors (27) and is not altered significantly by immune complexes (Fig. 4). From this and previous reports (7), it appears that only antibodymediated phagocytosis is paralyed in the presence of immune complexes and that the consequences of this transient phenomenon in virus-infected animals may be limited in scope. Hoever, hen a phagocytic particle that can be phagocytied via alternate receptors is coated ith antibody, immune complexes inhibit its ingestion (Fig. 4). Therefore, antibody-mediated phagocytosis becomes dominant, possibly because the antibody molecule protrudes aay from the particle. This predominance in specific receptors may mean that in the lungs of virus-infected mice, particles or opportunistic microorganisms are protected from ingestion by opsoniing antibody. This study documents the formation of immune complexes in influena virus-infected mice and demonstrates macrophage dysfunction caused by immune complexes isolated from these mice. LITERATURE CITED 1. Astry, C. L., G. A. Warr, and G. J. Jakab Impairment of polymorphonuclear leukocyte immigration as a mechanism of alcohol-induced suppression of pulmonary antibacterial defenses. Am. Rev. Respir. Dis. 128: Boyle, M. D. P., and J. J. Langone A simple procedure to use hole serum as a source of either IgG- or IgM-specific antibody. J. Immunol. Methods 32: Choppin, P. W Replication of influena virus in a continuous cell line: high yield of infectious virus from cells inoculated at high multiplicity. Virology 39: Creighton, W. D., P. H. Lambert, and P. A. Miescher Detection of antibodies and soluble antigen-antibody complexes by precipitation ith polyethylene glycol. J. Immunol. 111: Fischer, D. G., and H. S. Koren Quantitative immune phagocytosis by macrophages, p In H. B. Herscourt, H. T. Holden, J. A. Bellante, and A. Ghaffer (ed.), Manual of macrophage methodology. Marcel Dekker, Inc., Ne York. 6. Gardner, I. D Suppression of antibacterial immunity by infection ith influena virus. J. Infect. Dis. 144: Griffin, F. M Effects of soluble immune complexes on Fc receptor and C3b receptor-mediated phagocytosis by macrophages. J. Exp. Med. 152: Inada, Y., M. Kamiyama, T. Kanemitsu, and W. S. Clark Detection of circulating immune complexes: a ne application of immune adherence haemagglutination. Ann. Immunol. 132: Jakab, G. J Pulmonary defense mechanisms and the interaction beteen viruses and bacteria in acute respiratory infections. Bull. Eur. Physiopathol. Respir. 13: Jakab, G. J Immune impairment of alveolar macrophage phagocytosis during influena virus pneumonia. Am. Rev. Respir. Dis. 126: Jakab, G. J Viral-bacterial interactions in pulmonary infection. Adv. Vet. Sci. Comp. Med. 26: Jakab, G. J., and G. M. Green The effect of Sendai virus infection on bacterial and transport mechanisms of the murine lung. J. Clin. Invest. 51: Jakab, G. J., and G. A. Warr The participation of antiviral immune mechanisms in alveolar macrophage dysfunction during viral pneumonia. Bull. Eur. Physiopathol. Respir. 19: Lambert, P. H., F. J. Dixon, R. H. Zubler, V. Agnello, C. Cambiaso, P. Casali, J. Clark, J. S. Codery, F. C. McDuffie, F. C. Hay, I. C. M. MacClennan, P. Masson, H. G. Muller- Eberhard, K. Pentinen, M. Smith, C. Tappeiner, A. N. Theofilopoulos, and P. Verroust A WHO collaborative study for the evaluation of eighteen methods for detecting immune complexes in serum. J. Clin. Lab. Immunol. 1: McKee, A. P Virological methods, p In Society of American Bacteriologists Committee on Bacteriological Technic (ed.), Manual of microbiological methods. McGra- Hill Book Co., Ne York. 16. Meretey, K., U. Bohm, A. Falus, and S. Bosoky Radioimmune double PEG precipitation technique for detecting complexed IgE. J. Immunol. Methods 26: Michl, J., J. C. Unkeless, M. M. Preconka, and S. C. Silverstein Modulation of Fc receptors of mononuclear phagocytes by immobilied antigen-antibody complexes. J. Exp. Med. 157: Milgrom, F., and K. Kano Comparison of various procedures for the detection of antigen-antibody complexes. Int. Arch. Allergy Appl. Immunol. 56: Nugent, K. M., and E. L. Pesanti Effect of influena infection on the phagocytic and bactericidal activities of pulmonary macrophages. Infect. Immun. 26: Pernice, W., H. Schmit, F. Schindera, F. Behrens, and H. H. Sedlacek Antigen-specific detection of immune complexes in patients ith hepatitis B, influena A and rubella. Behring Inst. Mitt. 64: Pestel, J., M. Joseph, J. P. Dessaint, and A. Capron Macrophage triggering by aggregated immunoglobulins 1. Delayed effects of IgG aggregates or immune complexes. J. Immunol. 126: Poskitt, P. K. F., and T. R. Poskitt A quantitative enyme-linked immunoassay for serum immune complexes. J. Clin. Lab. Immunol. 5: Semko, R., and J. Wilcynski Detection and tissue localiation of components of the immune complex in animals infected and immunied ith influena virus. Acta Virol. (Prague) (Engl. Ed.) 23: Steel, R. D. G., and J. H. Torrie Principles and procedures of statistics. McGra-Hill Book Co., Ne York. 25. Voller, A., D. Bidell, and A. Bartlett Microplate enyme immunoassays for the immunodiagnosis of virus infections, p In N. R. Rose and N. Frieman (ed.), Manual of Clinical Immunology. American Society for Microbiology, Washington, D.C. 26. Ward, P. A., R. E. Duque, M. D. Sulavik, and K. J. Johnson. Donloaded from on November 8, 218 by guest

6 292 ASTRY AND JAKAB In vitro and in vivo stimulation of rat neutrophils and alveolar macrophages by immune complexes. Am. J. Pathol. 11: Warr, G. A A macrophage receptor for (mannose/glucosamine)-glycoproteins of potential importance in phagocytic activity. Biochem. Biophys. Res. Comm. 93: Warr, G. A., G. J. Jakab, and J. E. Hearst Alterations in lung macrophage immune receptor(s) activity associated ith viral pneumonia. J. Reticuloendothel. Soc. 26: Warshauer, D., E. Goldstein, T. Akers, W. Lippert, and M. Kim Effect of influena viral infection on the ingestion and killing of bacteria by alveolar macrophages. Am. Rev. Respir. J. VIROL. Dis. 115: Wells, M. A., P. Albrecht, and F. A. Ennis Recovery from a viral respiratory infection: I. Influena pneumonia in normal and T-deficient mice. J. Immunol. 126: Witkin, S. S., S. K. Shahani, S. Gupta, R. A. Good, and N. K. Day Demonstration of IgG Fc receptors on spermatooa and their utiliation for the detection of circulating immune complexes in human serum. Clin. Exp. Immunol. 41: Yolken, R. H., and V. Torsch Enyme-linked immunosorbent assay for the detection and identification of Coxsackie B antigen in tissue cultures and clinical specimens. J. Med. Virol. 6: Donloaded from on November 8, 218 by guest