INFECTION
AND
Vol. 26, No. 2
IMMUNITY, Nov. 1979, p. 492-497
0019-9567/79/1140492/06$02.00/0
Alterations in Lung Macrophage Antimicrobial Activity Associated with Viral Pneumonia GLENN A. WARR* AND GEORGE J. JAKAB Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 Received for publication 6 August 1979
Secondary bacterial infections are a common sequelae of viral pneumonias. To study 2 functions of the phagocytic defenses of the lung, macrophages were obtained by lung savage from parainfluenza 1 virus-infected and noninfected mice. The phagocytic capacities (binding, ingestion, and killing) of these cells were assessed in vitro against viable Candida krusei. Viral pneumonia resulted in a progressive suppression (through day 7 of the infection) of the ability of macrophages to bind candida to their surfaces by nonimmunological or complement receptors; ingestion and intracellular killing of candida were also decreased. After day 7, all these functions returned and, in fact, cells with enhanced activities were present on day 17. After introduction of virus into the lungs, the lung macrophage population increased significantly between days 3 and 7 of infection. This resulted in an increase in the phagocytic potential of the lung, despite the virus-associated suppression of the phagocytic activity in a portion of the macrophages. However, the ability of the macrophages to kill ingested microorganisms was also reduced, resulting in an overall deficiency in the lung macrophage defenses. It was concluded that viral pneumonia was associated with at least two suppressive effects on the lung macrophage-decreased receptor activity and microbicidal activityresulting in a deficiency in the lung phagocytic defenses represented by these cells. These effects were maximal 1 week after infection and could account for the increased susceptibility of these lungs to secondary bacterial pneumonias. In contrast, during the period of convalescence, the lung macrophage antimicrobial activities were increased and reflected in enhanced resistance of the lungs to infections.
In murine models of viral pneumonia, establishment of parainfluenza 1 (Sendai) and influenza virus infections in the lungs leads to suppression of pulmonary antibacterial defenses, and concurrently secondary bacterial pneumonias often develop (5, 7). The lung macrophage (LM) phagocytic system constitutes a primary portion of the lung defenses against bacterial infections (3, 4); therefore, the development of bacterial pneumonias indicates either suppression of normal functions of the phagocytes or circumvention of their defenses. During viral penumonia, in vivo studies have demonstrated a virus-induced defect in the lung phagocytic defense system (9, 20). The macrophages possess both immunological (19) and noniimunological (1, 21) membrane receptors that are involved in the recognition and ingestion of foreign particles. In the nonimmune host, recognition ofbacterial cell wall components enables the phagocytes to bind and ingest the microorganisms (1). Once the particle
is ingested, multiple intracellular microbicidal mechanisms (10, 18) interact to kill the microorganisms. The importance of nonimmune phagocytosis is exemplified by the early (preimmune) defense stages against candida infection (12). In the series of experiments reported here, further support for the concept of a virus-induced phagocytic defect was sought by examining the ability of LM to bind untreated and complementtreated yeast cells and to ingest and kill these yeast cells in the presence of complement. The numbers of LM and their functional capabilities were determined at specified times during virfl replication in the lungs, at the time of maximum suppression of lung antibacterial defenses, and during convalescence from the ensuing pneu-
492
monia.
MATERIALS AND METHODS Experimental animals. Female outbred Swiss mice, 18 to 21 g (Microbiological Associates, Bethesda, Md.), were used in these experiments. The animals
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VIRAL SUPPRESSION OF LM DEFENSES
(free from Sendai virus) were housed in filter-topped cages and given food and water ad libitum. Their sera were negative for agglutinating antibody against the assay microorganism Candida krusei. Viral infection. Mice were infected by aerosol inhalation of a sublethal dose of parainfluenza 1 (Sendai) virus by previously described methods (8). Infected and noninfected animal were housed in separate rooms. Pulmonary lavage. Virus-infected and noninfected mice were killed by cervical dislocation and exsanguinated by cardiac puncture. The lungs were excised in toto and ravaged with three 1.5-ml portions of fluid, consisting of isotonic saline supplemented with 0.1% glucose0.1% ethylenediaminetetraacetate disodium and buffered to pH 7.4 with 25 mM N-2hydroxyethylpiperazine-AN'-2-ethanesulfonic acid (HEPES; Sigma Chemical Co., St. Louis, Mo.). Each individual volume was introduced into and recovered from the lungs three times, so that each lung was washed nine times. Thereafter, the savage fluids from each individual animal were centrifuged for 10 min at 200 x g, and the cells were suspended in tissue culture medium 199 (TCM-199; GIBCO Laboratories, Grand Island, N. Y.) buffered with HEPES. Direct cell counts were performed in a hemocytometer, and differential cell counts were performed on Wright-Giemsa-stained cell monolayers. Preparation of yeast suspensions. Two yeast preparations were used: untreated viable cells and viable cells treated with complement. C. krusei (a laboratory strain) was grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) with constant shaking for 18 h at 371C. The yeast were washed three times and resuspended in TCM-199 at a concentration of 107 cells/ml (based on direct cell counts using a hemocytometer). This preparation was used for the nonimmunological receptor binding studies. For the LM binding, ingestion, and microbicidal studies with complement-coated candida, the washed yeast suspensions were incubated with 5% guinea pig serum (obtained from Cordis Laboratories; this concentration of serum contained complement at 12.5 50% hemolytic complement units/ml) for 30 min at 370C. Yeast cell walls activate the alternate complement pathway (14). The complement-coated candida (candida-C') were centrifuged and suspended at 107 cells/ ml in either TCM-199 for LM (candida-C') binding studies or TCM-199 supplemented with 10% heat-inactivated fetal calf serum and 5% guinea pig serum for phagocytosis and microbicidal studies. Heat-inactivated fetal calf serum and guinea pig complement did not contain agglutination antibody against C. krusei. Treatment with complement did not reduce the viability of the candida as determined by their ability to exclude methylene blue dye (11, 16). Viability of all yeast preparations was greater than 99%. Macrophage-candida binding assays. Monolayers of the LM were prepared by incubating 0.2-ml portions of the cell suspensions (5 x 106 LM/ml of TCM-199) on 22-mm2 cover glasses for 30 min at 371C. This allowed the cells to adhere and spread on the
493
glass surfaces. The LM preparations were then brought to a temperature of 220C. Trplicate LM monolayers were prepared for each assay on each LM suspension. The fluid covering the LM monolayers was removed, and 0.2 ml of either the unsensitized candida or candida-C' suspension was layered over the LM (2.0 x 106 Candida/106 LM) and allowed to incubate for 1 h at 221C. To remove free candida, each cell culture was then washed five times with an isotonic saline solution supplemented with 0.1% gelatin (NSG), rapidly air dried, fixed with absolute methanol, and processed and Wright-Giemsa stain. A minimum of 100 LM on each cover glass was randomly selected and read microscopically to quantitate the percentage of LM with associated candida and the mean number of candida bound to active LM. The values for macrophage-associated candida include all yeast particles associated, (surface bound and ingested) with the phagocytes. To determine the mean binding activity of the LM populations, an LM-candida binding index was calculated for each LM suspension, using the following formula: mean number of candida bound 100 total LM
number of LM binding candida 100 LM mean number of candida bound X (1) LM
Macrophage-candida phagocytosis and microbicidal assays. Monolayers of the LM were challenged with candida-C' (20 yeast cells/LM) suspended in TCM-199 supplemented with 10% heat-inactivated fetal calf serum and 5% guinea pig serum (complement source) for 30 min at 370C. The cell cultures were then washed three times with warm NSG fluid to remove the nonadherent candida. At this time, some monolayers were assessed for ingestion and microbicidal activity. The remaining monolayers were replenished with fresh medium and incubated for an additional 2.5 h at 370C. The viability of the candida was determined by the methylene blue dye exclusion method (11, 16). After the 2.5-h incubation, the cells were incubated in medium containing 0.064 mg of methylene blue per ml for 5 min at 370C. The cover glasses supporting the LM monolayers were then inverted, placed on microscope slides, and examined by phase microscopy. The percentage of ingested yeast that were killed (stained blue), the percentage of the LM actively phagocytic, and the mean number of candida ingested per LM were determined. These parameters are indicators of LM functional activity; however, to examine LM activity in perspective with alterations in the phagocytic defense capabilities for each lung, it was necessary to take into account fluctuations in both total numbers and capabilities of LM. The phagocytic defense capabilities for each lung are represented by lung indices, which were calculated by the following formulas:
494
INFECT. IMMUN.
WARR AND JAKAB
number of phagocytic LM/lung number of phagocytic LM 100 LM x total number of LM recovered from lung (2) index of number of candida ingested/lung number of phagocytic LM lung number of candida ingested phagocytic LM lung microbicidal capacity index of number of x
mean
candida killed/lung
=
number of candida ingested lung
x number of ingested candida killed 100 ingested candida
In all of the studies, control (noninfected) mice were always assayed simultaneously with the virus-infected groups. Also, all mononuclear phagocyte populations recovered from the lungs of both normal and virus infected animals are designated as lung macrophages (LM).
RESULTS Viral infection. The lungs of mice were lavaged at 3, 7, and 17 days after aerogenic admin-
istration of virus. These times correlate with viral replication, maximum suppression of lung antimicrobial defenses, and convalescence, respectively (6, 7). At 3 days, no gross consolidation of the lung surface was observed. By day 7, however, the lungs of all animals had lesions indicative of severe viral pneumonitis with consolidation involving between 25 and 75% of the surface areas of the lungs. Lung consolidation had resolved by day 17 of the infection. LM receptor activity. In these studies, the
LM binding of untreated and complementtreated candida was followed by examining three parameters: (i) number of active LM expressed as percentage of LM binding candida; (ii) mean number of candida bound per active LM; and (iii) the LM-candida binding index, which was dependent upon the first two values (see formula 1). The LM recovered from noninfected control mice were able to recognize and bind untreated and complement-treated candida in the absence of serum factors (Table 1). Pretreatment of the candida with complement resulted in a significant increase (P < 0.05, Student's t test) in the LM-candida binding index. This was due to an increase in the number of candida-C' bound to the LM and not to an increase in the percentage of LM capable of binding. By day 3, the viral infection had resulted in significant suppression in the LM-candida binding indices for both the untreated and complement-treated candida (Table 1), binding of the untreated candida being the more sensitive to the effects of virus infection. The decreased indices were a result of suppression in both the percentage of LM binding candida and the mean number of candida bound to active LM. Maximum suppression of the LM-candida binding occurred on day 7. During the convalescent period (day 17), the percentage of LM capable of binding the untreated and complement-treated candida had returned to normal levels. However, at this time, the mean numbers of candida bound per LM were significantly increased over control values for untreated and complement-treated candida (57 and 34% increases, respectively). LM microbicidal activity. In the presence of nonimmune serum and complement factors (at 3700), LM from noninfected mice actively ingested and killed the yeast cells (Table 2). This microbicidal activity was suppressed by the
TABLE 1. Alterations of LM receptors during viral pneumonia % LM binding candida Lung source of LM
. N Nonmnmune
receptors
Noninfected controls (n = 12) Virus infected
83.3 ± 2.2
Complerecepment
Mean no. of candida/LM . Nommmune
tors
receptors
85.2 i 1.2
3.5 ± 0.2
Complerecepment
LM-candida binding index . Nonmmune
tors
receptors
4.1 i 0.2
292 ± 21
Complerecepment tors
352 ± 21
207±8c 59±5C 34.0±1.5c 64.3±4.7c 1.7±O.lc 3.2±O.2c Day3(n=6) 55 ± 5c 35 ± 5c 2.5 ±0.c 1.8 ±0.c 18.8 ± 2.1c 21.3 ± 1.5c Day 7 (n =24) 418 ± 26 429 ± 43c 5.5 ± 0.3c 77.2 ± 5.3 76.0 ± 4.1 5.5 ± 0.3c Day 17 (n = 12) aBinding of viable C. krusei to LM (21 candida associated/LM) upon incubation for 1 h at 22"C in absence factors. Values expressed as mean ± standard error. of serum b Binding index represents the total number of candida cells bound/100 total LM (including active and inactive LM). Values significantly (P < 0.025) decreased or elevated with respect to control values as determined by c
Student's t test.
TABLz 2. Alteration of LM microbicidal activity Lung source of LM.,
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VIRAL SUPPRESSION OF LM DEFENSES
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LM
~~Phago-
cytic (
Mean no. Igse of.cd candida killed (%) %)
6.9 i 0.3 54.5 ± 3.0 Noninfected con- 91.3 ± 1.5 trols (n - 12) Virus infected 30.0 4.4b 7.0 ± 0.4 89.3 ± 1.0 Day 3 (n -12) 12.7 2.Ob 5.1 ± 0.2 55.0 ± L. Day 7 (n -30) 9.4 ± 0.3' 35.1 ± 2.5b 83.2 ± 2.2 Day 17 (n - 20) a Macrophage microbicidal activity in vitro after 3-h incuat 371C. Values expressed as mean ± standard error. bation b Values significantly (P < 0.001, Student's t test) reduced below control macrophage activity. 'Value significantly (P < 0.001) elevated above control macrophage activity.
virus infection. On day 3 of the infection, the LM had normal ingestion activity, but their ability to kill the engulfed yeast cells was reduced to 55 ± 8% (P < 0.001) of the control microbicidal activity. By day 7, the percentage of LM actively phagocytic, the mean number of candida ingested per phagocytic LM, and the percentage of the internalized candida killed were all significantly (P < 0.001) reduced below control values. Ten days later, during the convalescent period, the percentage ofLM ingesting yeast cells had increased (1.5-fold over day 7 values) to 83.2 ± 2.2%. The phagocytic activity (number of yeast engulfed) of these cells had also increased, demonstrating a value 36% higher than for the control LM. However, although these LM killed a greater percentage of the internalized yeast than the LM recovered on either day 3 or day 7, they killed a lower percentage of the ingested candida than did the control LM. This may, in part, reflect the fact that by day 17 the LM were capable of ingesting more yeast cells than the control LM and therefore had more yeast cells to kill. The above data describe alterations in the mean LM functional capacities associated with viral pneumonia. To relate these observations to the ability of the lungs to handle microbial invasions, fluctuations in the numbers of LM were considered and lung indices were calculated (formulas 2, 3, and 4). These data are presented in Fig. 1. The recovered savage volumes from the lungs of the virus-infected mice did not significantly vary from those recovered from control animals. The lungs from noninfected mice yielded 7.58 ± 0.40 x 105 LM (day 0, Fig. 1). Of these, 6.92 ± 0.11 x 10' cells were phagocytic, ingesting 4.75 ± 0.21 x 106 candida, of which 2.59 + 0.14 x 10" were killed within a 3-h period. On the 3rd day of virus infection, the total numbers of LM, actively phagocytic LM, and ingested candida
did not significantly differ from the controls. However, the numbers of ingested yeast cells that were subsequently killed were significantly (P < 0.005) reduced (59.5% control values). By day 7, the total numbers of LM recovered had signiicantly (P < 0.001) increased (274% control values), resulting in a concomitant increase in the total numbers of actively phagocytic LM and candida ingested (166.2 and 125.9% control, respectively) (Fig. 1). This occurred despite the decrease in the percentage of LM that were phagocytic and decreased numbers of candida ingested per LM (Table 2). However, although the phagocytic potential of the lungs was increased during this period, the total numbers of ingested candida killed were further reduced (35.5% control). Thus, the virus infection was 107
CANWDA GOESTED t
1--l
5-
INGESTED CANDDA KLLED~
/
0
/0
z
/0
k
0
/
z 9-
I0 10
_
5.
I
O3
DAYS
7 AFTER
4 17 VIRUS
FIG. 1. LM cellular response and antimicrobial activity during parainfluenza 1 (Sendai) virus pneumonia. Symbols: A), LM), total number of macrophages recovered from each lung by lavage; P, phagocytic LM), total number of the LM that actively phagocytosed C. krusei in vitro, in the presence of nonimmune serum and complement; (A, candida ingested), index of the potential total number of candida ingested by the complement of lung LM; (*, ingested candida killed), index of the potential total lung microbicidal capacity as represented by the number of ingested candida killed by the complement of lung LM with in a 3-h period. Results are expressed as the mean ± standard error, n = 12 to 30. Activity was determined with LM recovered by lung lavage from noninfected animals (day 0) and from mice previously infected with virus (days 3, 7, and 17 after infection).
496
INFECT. IMMUN.
WARR AND JAKAB
associated with a significant suppression of the lung antimicrobial activity. The suppression of the lung (LM) antimicrobial activity was reversed on the 17th day of the infection. At this time, the numbers of LM were declining from that obtained on day 7, although they remained elevated above normal values (212.4% control). The numbers of actively phagocytic LM and ingested candida were elevated above the levels in all ofthe experimental groups examined (193.6 and 264.2% control, respectively). In addition, the LM microbicidal index was significantly increased (169.9% control, P = 0.005), demonstrating that pulmonary antimicrobial activity was enhanced during the convalescent period of viral pneumonia. DISCUSSION The LM recovered from normal healthy mice expressed an innate capacity to bind the micro.organism, C. krusei, to their surface membranes in the absence of serum factors. In addition to the nonimmunological receptors, the presence of complement components on a particle enhances its binding by phagocytes (19), and the interaction of the candida with complement resulted in increased LM binding activity (Table 1). Viral pneumonia suppressed LM binding of both untreated and complement-treated candida, indicating alterations in both the nonixnmunological and complement receptors. However, equivalent suppression did not occur, the complement receptor being more active than the nonimmunological receptor on day 3 (Table 1). Maximum suppression of LM-candida binding occurred on the 7th day of the infection; however, active LM were still present. Since there was an increase in the LM populations during the pneumonia (Fig. 1), these active cells may have represented newly recruited phagocytes and not resident cells that were resistant to the virus effect. Conversely, the mechanism responsible for this suppression is not known; thus, natural or acquired LM resistance may also occur, or the cells with lower activity may represent a new population of immature inflammatory monocytes. By day 17, the percentage of LM active had returned to normal, whereas the mean number of candida bound per LM was significantly elevated above normal, demonstrating that convalescence was associated with LM having increased binding activity. The microbicidal assays included a fresh source of complement not only to enhance possible ingestion of the yeast by the LM, but also because complement factors may play a role in the intracellular killing of at least some candida species (2, 23). Since the viral infection was
associated with suppression of the ability of LM to bind yeast cells, it was not surprising that phagocytic ingestion was also decreased (Table 2). Because of the nature ofthese studies, further experiments are required to determine whether there is an additional defect which suppresses ingestion of a particle once it becomes attached to the surfaces of the phagocytes. Nevertheless, from these data it was clear that after virus infection, the ability of the LM to kill an organism once it had been engulfed did not correlate with the ingestion ability of the LM (Table 2). During the viral pneumonia, many of the LM which exhibited the capacity to engulf particles had a diminished ability to kill the internalized organisms.
These in vitro assays on LM recovered from virus-infected animals have documented functional defects in the ability of portions of the LM population to bind, ingest, and kill the microorganism, C. krusei. The magnitude of these defects was related to the time interval after virus infection. Clinically, even though all of the animals with viral pneumonia demonstrate signs of illness and a portion develop secondary bacterial pneumonias, many survive (6, 7). By calculating lung antimicrobial indices from the data in this study (Fig. 1), we were better able to analyze the ability of the lungs of virus infected animals to defend against secondary opportunistic bacterial infections. Because the LM populations increased during the course of the viral penumonia, the actual lung phagocytic capacity was increased. However, because of the additional suppression of intracellular killing mechanisms, especially on the 7th day of infection, the lungs were compromised. Thus, these data agreed with the observed in vivo models of virus suppression of lung antimicrobial activity (6, 7). During the period that the lungs were compromised, the ultimate recovery from illness was finely balanced between the numerous virulence factors of any invading organisms and the ability of the lung defense mechanisms to respond. Immunological mechanisms participate in the lung defense mechanisms as indicated by the increase in T lymphocytes (22), antiviral antibody, and interferon (15) during viral pneumonia. Lymphokines (13) and interferon (17) are known to activate macrophages, resulting in an enhancement of their phagocytic capabilities. In this study, by the 17th day of the infection, the LM population had binding and phagocytic activity increased above normal (Tables 1 and 2). These factors, coupled with the increased numbers of LM on day 17, resulted in an enhanced lung antimicrobial capacity (Fig. 1). The dynamics of the response of the host
VIRAL SUPPRESSION OF LM DEFENSES
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defenses during viral pneumonia are complex, and numerous interactions among the defense systems, the infecting virus, and opportunistic bacteria occur. This study established a virusassociated suppression of the activity of lung mononuclear phagocytes' antimicrobial activity and correlated this defect with the observed in vivo suppression of lung antibacterial capacity. During the convalescent period after recovery from the virus infection, the antimicrobial activity of a portion of the lung phagocyte population was enhanced. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant HL22029 from the National Heart, Lung, and Blood Institute. This work was conducted during the tenure of G. A. W. as a postdoctoral research fellow (Public Health Service award 1 F32 HL05404-01 from the National Heart, Lung, and Blood Institute). G. A. W. is the recipient of Public Health Service young investigator award HL22823-01 from the same institute. G. J. J. is the recipient of Public Health Service research career development award HL00415 from the National Heart, Lung, and Blood Institute.
LITERATURE CITED 1. Freimer, N. B., HI M. Ogmundsdotter, C. C. Blackwell, I. W. Sutherland, L Graham, and D. M. Weir. 1978. The role of cell wall carbohydrates in binding of microorganisms to mouse peritoneal exudate macrophages. Acta Pathol. Microbiol. Sect. B 86:53-57. 2. Gelfand, J. A., D. L. Hurley, A. S. Fauci, and M. M. Frank. 1978. Role of complement in host defense against experimental disseminated candidiasis. J. Infect. Dis. 138:9-16. 3. Goldstein, E., W. Lippert, and D. Warshauer. 1974. Pulmonary alveolar macrophage. Defender against bacterial infection of the lung. J. Clin. Invest. 54:519-528. 4. Green, G. M., and E. H. Kass. 1964. The role of the alveolar macrophage in the clearance of bacteria from the lung. J. Exp. Med. 119:167-176. 5. Hers, J. F. P., J. Mulder, N. Masurel, L Kuip, and D. A. J. Tyrrell. 1962. Studies on the pathogenesis of influenza virus pneumonia in mice. J. Pathol. Bacteriol. 83:207-217. 6. Jakab, G. 1974. Effect of sequential inoculations of Sendai virus and Pasteurella pneumotropia in mice. Am. Vet. Med. Assoc. 164:723-728. 7. Jakab, G. J. 1977. Pulmonary defense mechanisms and the interaction between viruses and bacteria in acute
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respiratory infections. Bull. Eur. Physiopathol. 13:119135. 8. Jakab, G. J., and G. AL Green. 1972. The effect of Sendai virus infection on bacterial and transport mechanisms of the murine lung. J. Clin. Invest. 51:19891998. 9. Jakab, G. J., and G. Green. 1976. Defect in intracellular killing of Staphylococcus aureus within alveolar macrophages in Sendai virus-infected murine lungs. J. Clin. Invest. 57:1533-1539. 10. Lehrer, R. L 1975. The flngicidal mechanisms of human monocytes. I. Evidence for myeloperoxidase-linked and myeloperoxidase-independent candidacidal mechanisms. J. Clin. Invest. 55:33-346. 11. Lehrer, R. L, and M. J. Cline. 1969. Interaction of Candida albicana with human leukocytes and serum. J. Bacteriol. 98:996-1004. 12. Miyake, T., K. Takeya, K. Nomoto, and S. Muraoka. 1977. Cellular elements in resistance to candida infection in mice. I. Contribution of T lymphocytes and phagocytes at various stages of infection. Microbiol. Immunol. 21:703-725. 13. North, R. J. 1978. The concept of the activated macrophage. J. Immunol. 121:806-809. 14. Pillemer, L, L. Blum, L H. Lepow, 0. A. Ross, E. W. Todd, and A. C. Wardlaw. 1954. The properdin system and immunity I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 120:279-285. 15. Robinson, T. W. E., R. J. R. Cureton, and R. B. Health. 1968. The pathogenesis of Sendai virus infection in the mouse lung. J. Med. Microbiol. 1:89-95. 16. Schmid, L., and K. Brune. 1974. Assessment of phagocytic and antimicrobial activity of human granulocytes. Infect. Immun. 10:1120-1126. 17. Schultz, R. M., K A. Chirigos, and U. L. Heine. 1978. Functional and morphological characteristics of interferon-treated macrophages. Cell. Immunol. 35:84-91. 18. Stossel, T. P. 1974. Phagocytosis. N. Engi. J. Med. 290: 774-780. 19. Stossel, T. P. 1975. Phagocytosis: recognition and ingestion. Semin. Hematol. 12:83-116. 20. Warshauer, D. E. Goldstein, T. Akers, W. Lippert, and K. Kim. 1977. Effect of influenza viral infection on the ingestion and killing of bacteria by alveolar macrophages. Am. Rev. Respir. Dis. 115:269-277. 21. Weir, D. M., and H. Ogmundedotter. 1977. Non-specific recognition mechanisms by mononuclear phagocytes. Clin. Exp. Immunol. 30:323-329. 22. Wyde, P. R., D. L Peavy, and T. R. Cate. 1978. Morphological and cytochemical characterization of cells infiltrating mouse lungs after influenza infection. Infect. Iimmun. 21:140-146. 23. Yamamura, AL, and HE Valdimarson. 1977. Participation of C3 in intracellular killing of Candida albicans. Scand. J. Immunol. 6:591-594.
AND
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IMMUNITY, Nov. 1979, p. 492-497
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Alterations in Lung Macrophage Antimicrobial Activity Associated with Viral Pneumonia GLENN A. WARR* AND GEORGE J. JAKAB Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205 Received for publication 6 August 1979
Secondary bacterial infections are a common sequelae of viral pneumonias. To study 2 functions of the phagocytic defenses of the lung, macrophages were obtained by lung savage from parainfluenza 1 virus-infected and noninfected mice. The phagocytic capacities (binding, ingestion, and killing) of these cells were assessed in vitro against viable Candida krusei. Viral pneumonia resulted in a progressive suppression (through day 7 of the infection) of the ability of macrophages to bind candida to their surfaces by nonimmunological or complement receptors; ingestion and intracellular killing of candida were also decreased. After day 7, all these functions returned and, in fact, cells with enhanced activities were present on day 17. After introduction of virus into the lungs, the lung macrophage population increased significantly between days 3 and 7 of infection. This resulted in an increase in the phagocytic potential of the lung, despite the virus-associated suppression of the phagocytic activity in a portion of the macrophages. However, the ability of the macrophages to kill ingested microorganisms was also reduced, resulting in an overall deficiency in the lung macrophage defenses. It was concluded that viral pneumonia was associated with at least two suppressive effects on the lung macrophage-decreased receptor activity and microbicidal activityresulting in a deficiency in the lung phagocytic defenses represented by these cells. These effects were maximal 1 week after infection and could account for the increased susceptibility of these lungs to secondary bacterial pneumonias. In contrast, during the period of convalescence, the lung macrophage antimicrobial activities were increased and reflected in enhanced resistance of the lungs to infections.
In murine models of viral pneumonia, establishment of parainfluenza 1 (Sendai) and influenza virus infections in the lungs leads to suppression of pulmonary antibacterial defenses, and concurrently secondary bacterial pneumonias often develop (5, 7). The lung macrophage (LM) phagocytic system constitutes a primary portion of the lung defenses against bacterial infections (3, 4); therefore, the development of bacterial pneumonias indicates either suppression of normal functions of the phagocytes or circumvention of their defenses. During viral penumonia, in vivo studies have demonstrated a virus-induced defect in the lung phagocytic defense system (9, 20). The macrophages possess both immunological (19) and noniimunological (1, 21) membrane receptors that are involved in the recognition and ingestion of foreign particles. In the nonimmune host, recognition ofbacterial cell wall components enables the phagocytes to bind and ingest the microorganisms (1). Once the particle
is ingested, multiple intracellular microbicidal mechanisms (10, 18) interact to kill the microorganisms. The importance of nonimmune phagocytosis is exemplified by the early (preimmune) defense stages against candida infection (12). In the series of experiments reported here, further support for the concept of a virus-induced phagocytic defect was sought by examining the ability of LM to bind untreated and complementtreated yeast cells and to ingest and kill these yeast cells in the presence of complement. The numbers of LM and their functional capabilities were determined at specified times during virfl replication in the lungs, at the time of maximum suppression of lung antibacterial defenses, and during convalescence from the ensuing pneu-
492
monia.
MATERIALS AND METHODS Experimental animals. Female outbred Swiss mice, 18 to 21 g (Microbiological Associates, Bethesda, Md.), were used in these experiments. The animals
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VIRAL SUPPRESSION OF LM DEFENSES
(free from Sendai virus) were housed in filter-topped cages and given food and water ad libitum. Their sera were negative for agglutinating antibody against the assay microorganism Candida krusei. Viral infection. Mice were infected by aerosol inhalation of a sublethal dose of parainfluenza 1 (Sendai) virus by previously described methods (8). Infected and noninfected animal were housed in separate rooms. Pulmonary lavage. Virus-infected and noninfected mice were killed by cervical dislocation and exsanguinated by cardiac puncture. The lungs were excised in toto and ravaged with three 1.5-ml portions of fluid, consisting of isotonic saline supplemented with 0.1% glucose0.1% ethylenediaminetetraacetate disodium and buffered to pH 7.4 with 25 mM N-2hydroxyethylpiperazine-AN'-2-ethanesulfonic acid (HEPES; Sigma Chemical Co., St. Louis, Mo.). Each individual volume was introduced into and recovered from the lungs three times, so that each lung was washed nine times. Thereafter, the savage fluids from each individual animal were centrifuged for 10 min at 200 x g, and the cells were suspended in tissue culture medium 199 (TCM-199; GIBCO Laboratories, Grand Island, N. Y.) buffered with HEPES. Direct cell counts were performed in a hemocytometer, and differential cell counts were performed on Wright-Giemsa-stained cell monolayers. Preparation of yeast suspensions. Two yeast preparations were used: untreated viable cells and viable cells treated with complement. C. krusei (a laboratory strain) was grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) with constant shaking for 18 h at 371C. The yeast were washed three times and resuspended in TCM-199 at a concentration of 107 cells/ml (based on direct cell counts using a hemocytometer). This preparation was used for the nonimmunological receptor binding studies. For the LM binding, ingestion, and microbicidal studies with complement-coated candida, the washed yeast suspensions were incubated with 5% guinea pig serum (obtained from Cordis Laboratories; this concentration of serum contained complement at 12.5 50% hemolytic complement units/ml) for 30 min at 370C. Yeast cell walls activate the alternate complement pathway (14). The complement-coated candida (candida-C') were centrifuged and suspended at 107 cells/ ml in either TCM-199 for LM (candida-C') binding studies or TCM-199 supplemented with 10% heat-inactivated fetal calf serum and 5% guinea pig serum for phagocytosis and microbicidal studies. Heat-inactivated fetal calf serum and guinea pig complement did not contain agglutination antibody against C. krusei. Treatment with complement did not reduce the viability of the candida as determined by their ability to exclude methylene blue dye (11, 16). Viability of all yeast preparations was greater than 99%. Macrophage-candida binding assays. Monolayers of the LM were prepared by incubating 0.2-ml portions of the cell suspensions (5 x 106 LM/ml of TCM-199) on 22-mm2 cover glasses for 30 min at 371C. This allowed the cells to adhere and spread on the
493
glass surfaces. The LM preparations were then brought to a temperature of 220C. Trplicate LM monolayers were prepared for each assay on each LM suspension. The fluid covering the LM monolayers was removed, and 0.2 ml of either the unsensitized candida or candida-C' suspension was layered over the LM (2.0 x 106 Candida/106 LM) and allowed to incubate for 1 h at 221C. To remove free candida, each cell culture was then washed five times with an isotonic saline solution supplemented with 0.1% gelatin (NSG), rapidly air dried, fixed with absolute methanol, and processed and Wright-Giemsa stain. A minimum of 100 LM on each cover glass was randomly selected and read microscopically to quantitate the percentage of LM with associated candida and the mean number of candida bound to active LM. The values for macrophage-associated candida include all yeast particles associated, (surface bound and ingested) with the phagocytes. To determine the mean binding activity of the LM populations, an LM-candida binding index was calculated for each LM suspension, using the following formula: mean number of candida bound 100 total LM
number of LM binding candida 100 LM mean number of candida bound X (1) LM
Macrophage-candida phagocytosis and microbicidal assays. Monolayers of the LM were challenged with candida-C' (20 yeast cells/LM) suspended in TCM-199 supplemented with 10% heat-inactivated fetal calf serum and 5% guinea pig serum (complement source) for 30 min at 370C. The cell cultures were then washed three times with warm NSG fluid to remove the nonadherent candida. At this time, some monolayers were assessed for ingestion and microbicidal activity. The remaining monolayers were replenished with fresh medium and incubated for an additional 2.5 h at 370C. The viability of the candida was determined by the methylene blue dye exclusion method (11, 16). After the 2.5-h incubation, the cells were incubated in medium containing 0.064 mg of methylene blue per ml for 5 min at 370C. The cover glasses supporting the LM monolayers were then inverted, placed on microscope slides, and examined by phase microscopy. The percentage of ingested yeast that were killed (stained blue), the percentage of the LM actively phagocytic, and the mean number of candida ingested per LM were determined. These parameters are indicators of LM functional activity; however, to examine LM activity in perspective with alterations in the phagocytic defense capabilities for each lung, it was necessary to take into account fluctuations in both total numbers and capabilities of LM. The phagocytic defense capabilities for each lung are represented by lung indices, which were calculated by the following formulas:
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INFECT. IMMUN.
WARR AND JAKAB
number of phagocytic LM/lung number of phagocytic LM 100 LM x total number of LM recovered from lung (2) index of number of candida ingested/lung number of phagocytic LM lung number of candida ingested phagocytic LM lung microbicidal capacity index of number of x
mean
candida killed/lung
=
number of candida ingested lung
x number of ingested candida killed 100 ingested candida
In all of the studies, control (noninfected) mice were always assayed simultaneously with the virus-infected groups. Also, all mononuclear phagocyte populations recovered from the lungs of both normal and virus infected animals are designated as lung macrophages (LM).
RESULTS Viral infection. The lungs of mice were lavaged at 3, 7, and 17 days after aerogenic admin-
istration of virus. These times correlate with viral replication, maximum suppression of lung antimicrobial defenses, and convalescence, respectively (6, 7). At 3 days, no gross consolidation of the lung surface was observed. By day 7, however, the lungs of all animals had lesions indicative of severe viral pneumonitis with consolidation involving between 25 and 75% of the surface areas of the lungs. Lung consolidation had resolved by day 17 of the infection. LM receptor activity. In these studies, the
LM binding of untreated and complementtreated candida was followed by examining three parameters: (i) number of active LM expressed as percentage of LM binding candida; (ii) mean number of candida bound per active LM; and (iii) the LM-candida binding index, which was dependent upon the first two values (see formula 1). The LM recovered from noninfected control mice were able to recognize and bind untreated and complement-treated candida in the absence of serum factors (Table 1). Pretreatment of the candida with complement resulted in a significant increase (P < 0.05, Student's t test) in the LM-candida binding index. This was due to an increase in the number of candida-C' bound to the LM and not to an increase in the percentage of LM capable of binding. By day 3, the viral infection had resulted in significant suppression in the LM-candida binding indices for both the untreated and complement-treated candida (Table 1), binding of the untreated candida being the more sensitive to the effects of virus infection. The decreased indices were a result of suppression in both the percentage of LM binding candida and the mean number of candida bound to active LM. Maximum suppression of the LM-candida binding occurred on day 7. During the convalescent period (day 17), the percentage of LM capable of binding the untreated and complement-treated candida had returned to normal levels. However, at this time, the mean numbers of candida bound per LM were significantly increased over control values for untreated and complement-treated candida (57 and 34% increases, respectively). LM microbicidal activity. In the presence of nonimmune serum and complement factors (at 3700), LM from noninfected mice actively ingested and killed the yeast cells (Table 2). This microbicidal activity was suppressed by the
TABLE 1. Alterations of LM receptors during viral pneumonia % LM binding candida Lung source of LM
. N Nonmnmune
receptors
Noninfected controls (n = 12) Virus infected
83.3 ± 2.2
Complerecepment
Mean no. of candida/LM . Nommmune
tors
receptors
85.2 i 1.2
3.5 ± 0.2
Complerecepment
LM-candida binding index . Nonmmune
tors
receptors
4.1 i 0.2
292 ± 21
Complerecepment tors
352 ± 21
207±8c 59±5C 34.0±1.5c 64.3±4.7c 1.7±O.lc 3.2±O.2c Day3(n=6) 55 ± 5c 35 ± 5c 2.5 ±0.c 1.8 ±0.c 18.8 ± 2.1c 21.3 ± 1.5c Day 7 (n =24) 418 ± 26 429 ± 43c 5.5 ± 0.3c 77.2 ± 5.3 76.0 ± 4.1 5.5 ± 0.3c Day 17 (n = 12) aBinding of viable C. krusei to LM (21 candida associated/LM) upon incubation for 1 h at 22"C in absence factors. Values expressed as mean ± standard error. of serum b Binding index represents the total number of candida cells bound/100 total LM (including active and inactive LM). Values significantly (P < 0.025) decreased or elevated with respect to control values as determined by c
Student's t test.
TABLz 2. Alteration of LM microbicidal activity Lung source of LM.,
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VIRAL SUPPRESSION OF LM DEFENSES
VOL. 26, 1979
LM
~~Phago-
cytic (
Mean no. Igse of.cd candida killed (%) %)
6.9 i 0.3 54.5 ± 3.0 Noninfected con- 91.3 ± 1.5 trols (n - 12) Virus infected 30.0 4.4b 7.0 ± 0.4 89.3 ± 1.0 Day 3 (n -12) 12.7 2.Ob 5.1 ± 0.2 55.0 ± L. Day 7 (n -30) 9.4 ± 0.3' 35.1 ± 2.5b 83.2 ± 2.2 Day 17 (n - 20) a Macrophage microbicidal activity in vitro after 3-h incuat 371C. Values expressed as mean ± standard error. bation b Values significantly (P < 0.001, Student's t test) reduced below control macrophage activity. 'Value significantly (P < 0.001) elevated above control macrophage activity.
virus infection. On day 3 of the infection, the LM had normal ingestion activity, but their ability to kill the engulfed yeast cells was reduced to 55 ± 8% (P < 0.001) of the control microbicidal activity. By day 7, the percentage of LM actively phagocytic, the mean number of candida ingested per phagocytic LM, and the percentage of the internalized candida killed were all significantly (P < 0.001) reduced below control values. Ten days later, during the convalescent period, the percentage ofLM ingesting yeast cells had increased (1.5-fold over day 7 values) to 83.2 ± 2.2%. The phagocytic activity (number of yeast engulfed) of these cells had also increased, demonstrating a value 36% higher than for the control LM. However, although these LM killed a greater percentage of the internalized yeast than the LM recovered on either day 3 or day 7, they killed a lower percentage of the ingested candida than did the control LM. This may, in part, reflect the fact that by day 17 the LM were capable of ingesting more yeast cells than the control LM and therefore had more yeast cells to kill. The above data describe alterations in the mean LM functional capacities associated with viral pneumonia. To relate these observations to the ability of the lungs to handle microbial invasions, fluctuations in the numbers of LM were considered and lung indices were calculated (formulas 2, 3, and 4). These data are presented in Fig. 1. The recovered savage volumes from the lungs of the virus-infected mice did not significantly vary from those recovered from control animals. The lungs from noninfected mice yielded 7.58 ± 0.40 x 105 LM (day 0, Fig. 1). Of these, 6.92 ± 0.11 x 10' cells were phagocytic, ingesting 4.75 ± 0.21 x 106 candida, of which 2.59 + 0.14 x 10" were killed within a 3-h period. On the 3rd day of virus infection, the total numbers of LM, actively phagocytic LM, and ingested candida
did not significantly differ from the controls. However, the numbers of ingested yeast cells that were subsequently killed were significantly (P < 0.005) reduced (59.5% control values). By day 7, the total numbers of LM recovered had signiicantly (P < 0.001) increased (274% control values), resulting in a concomitant increase in the total numbers of actively phagocytic LM and candida ingested (166.2 and 125.9% control, respectively) (Fig. 1). This occurred despite the decrease in the percentage of LM that were phagocytic and decreased numbers of candida ingested per LM (Table 2). However, although the phagocytic potential of the lungs was increased during this period, the total numbers of ingested candida killed were further reduced (35.5% control). Thus, the virus infection was 107
CANWDA GOESTED t
1--l
5-
INGESTED CANDDA KLLED~
/
0
/0
z
/0
k
0
/
z 9-
I0 10
_
5.
I
O3
DAYS
7 AFTER
4 17 VIRUS
FIG. 1. LM cellular response and antimicrobial activity during parainfluenza 1 (Sendai) virus pneumonia. Symbols: A), LM), total number of macrophages recovered from each lung by lavage; P, phagocytic LM), total number of the LM that actively phagocytosed C. krusei in vitro, in the presence of nonimmune serum and complement; (A, candida ingested), index of the potential total number of candida ingested by the complement of lung LM; (*, ingested candida killed), index of the potential total lung microbicidal capacity as represented by the number of ingested candida killed by the complement of lung LM with in a 3-h period. Results are expressed as the mean ± standard error, n = 12 to 30. Activity was determined with LM recovered by lung lavage from noninfected animals (day 0) and from mice previously infected with virus (days 3, 7, and 17 after infection).
496
INFECT. IMMUN.
WARR AND JAKAB
associated with a significant suppression of the lung antimicrobial activity. The suppression of the lung (LM) antimicrobial activity was reversed on the 17th day of the infection. At this time, the numbers of LM were declining from that obtained on day 7, although they remained elevated above normal values (212.4% control). The numbers of actively phagocytic LM and ingested candida were elevated above the levels in all ofthe experimental groups examined (193.6 and 264.2% control, respectively). In addition, the LM microbicidal index was significantly increased (169.9% control, P = 0.005), demonstrating that pulmonary antimicrobial activity was enhanced during the convalescent period of viral pneumonia. DISCUSSION The LM recovered from normal healthy mice expressed an innate capacity to bind the micro.organism, C. krusei, to their surface membranes in the absence of serum factors. In addition to the nonimmunological receptors, the presence of complement components on a particle enhances its binding by phagocytes (19), and the interaction of the candida with complement resulted in increased LM binding activity (Table 1). Viral pneumonia suppressed LM binding of both untreated and complement-treated candida, indicating alterations in both the nonixnmunological and complement receptors. However, equivalent suppression did not occur, the complement receptor being more active than the nonimmunological receptor on day 3 (Table 1). Maximum suppression of LM-candida binding occurred on the 7th day of the infection; however, active LM were still present. Since there was an increase in the LM populations during the pneumonia (Fig. 1), these active cells may have represented newly recruited phagocytes and not resident cells that were resistant to the virus effect. Conversely, the mechanism responsible for this suppression is not known; thus, natural or acquired LM resistance may also occur, or the cells with lower activity may represent a new population of immature inflammatory monocytes. By day 17, the percentage of LM active had returned to normal, whereas the mean number of candida bound per LM was significantly elevated above normal, demonstrating that convalescence was associated with LM having increased binding activity. The microbicidal assays included a fresh source of complement not only to enhance possible ingestion of the yeast by the LM, but also because complement factors may play a role in the intracellular killing of at least some candida species (2, 23). Since the viral infection was
associated with suppression of the ability of LM to bind yeast cells, it was not surprising that phagocytic ingestion was also decreased (Table 2). Because of the nature ofthese studies, further experiments are required to determine whether there is an additional defect which suppresses ingestion of a particle once it becomes attached to the surfaces of the phagocytes. Nevertheless, from these data it was clear that after virus infection, the ability of the LM to kill an organism once it had been engulfed did not correlate with the ingestion ability of the LM (Table 2). During the viral pneumonia, many of the LM which exhibited the capacity to engulf particles had a diminished ability to kill the internalized organisms.
These in vitro assays on LM recovered from virus-infected animals have documented functional defects in the ability of portions of the LM population to bind, ingest, and kill the microorganism, C. krusei. The magnitude of these defects was related to the time interval after virus infection. Clinically, even though all of the animals with viral pneumonia demonstrate signs of illness and a portion develop secondary bacterial pneumonias, many survive (6, 7). By calculating lung antimicrobial indices from the data in this study (Fig. 1), we were better able to analyze the ability of the lungs of virus infected animals to defend against secondary opportunistic bacterial infections. Because the LM populations increased during the course of the viral penumonia, the actual lung phagocytic capacity was increased. However, because of the additional suppression of intracellular killing mechanisms, especially on the 7th day of infection, the lungs were compromised. Thus, these data agreed with the observed in vivo models of virus suppression of lung antimicrobial activity (6, 7). During the period that the lungs were compromised, the ultimate recovery from illness was finely balanced between the numerous virulence factors of any invading organisms and the ability of the lung defense mechanisms to respond. Immunological mechanisms participate in the lung defense mechanisms as indicated by the increase in T lymphocytes (22), antiviral antibody, and interferon (15) during viral pneumonia. Lymphokines (13) and interferon (17) are known to activate macrophages, resulting in an enhancement of their phagocytic capabilities. In this study, by the 17th day of the infection, the LM population had binding and phagocytic activity increased above normal (Tables 1 and 2). These factors, coupled with the increased numbers of LM on day 17, resulted in an enhanced lung antimicrobial capacity (Fig. 1). The dynamics of the response of the host
VIRAL SUPPRESSION OF LM DEFENSES
VOL. 26, 1979
defenses during viral pneumonia are complex, and numerous interactions among the defense systems, the infecting virus, and opportunistic bacteria occur. This study established a virusassociated suppression of the activity of lung mononuclear phagocytes' antimicrobial activity and correlated this defect with the observed in vivo suppression of lung antibacterial capacity. During the convalescent period after recovery from the virus infection, the antimicrobial activity of a portion of the lung phagocyte population was enhanced. ACKNOWLEDGMENTS This work was supported by Public Health Service research grant HL22029 from the National Heart, Lung, and Blood Institute. This work was conducted during the tenure of G. A. W. as a postdoctoral research fellow (Public Health Service award 1 F32 HL05404-01 from the National Heart, Lung, and Blood Institute). G. A. W. is the recipient of Public Health Service young investigator award HL22823-01 from the same institute. G. J. J. is the recipient of Public Health Service research career development award HL00415 from the National Heart, Lung, and Blood Institute.
LITERATURE CITED 1. Freimer, N. B., HI M. Ogmundsdotter, C. C. Blackwell, I. W. Sutherland, L Graham, and D. M. Weir. 1978. The role of cell wall carbohydrates in binding of microorganisms to mouse peritoneal exudate macrophages. Acta Pathol. Microbiol. Sect. B 86:53-57. 2. Gelfand, J. A., D. L. Hurley, A. S. Fauci, and M. M. Frank. 1978. Role of complement in host defense against experimental disseminated candidiasis. J. Infect. Dis. 138:9-16. 3. Goldstein, E., W. Lippert, and D. Warshauer. 1974. Pulmonary alveolar macrophage. Defender against bacterial infection of the lung. J. Clin. Invest. 54:519-528. 4. Green, G. M., and E. H. Kass. 1964. The role of the alveolar macrophage in the clearance of bacteria from the lung. J. Exp. Med. 119:167-176. 5. Hers, J. F. P., J. Mulder, N. Masurel, L Kuip, and D. A. J. Tyrrell. 1962. Studies on the pathogenesis of influenza virus pneumonia in mice. J. Pathol. Bacteriol. 83:207-217. 6. Jakab, G. 1974. Effect of sequential inoculations of Sendai virus and Pasteurella pneumotropia in mice. Am. Vet. Med. Assoc. 164:723-728. 7. Jakab, G. J. 1977. Pulmonary defense mechanisms and the interaction between viruses and bacteria in acute
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respiratory infections. Bull. Eur. Physiopathol. 13:119135. 8. Jakab, G. J., and G. AL Green. 1972. The effect of Sendai virus infection on bacterial and transport mechanisms of the murine lung. J. Clin. Invest. 51:19891998. 9. Jakab, G. J., and G. Green. 1976. Defect in intracellular killing of Staphylococcus aureus within alveolar macrophages in Sendai virus-infected murine lungs. J. Clin. Invest. 57:1533-1539. 10. Lehrer, R. L 1975. The flngicidal mechanisms of human monocytes. I. Evidence for myeloperoxidase-linked and myeloperoxidase-independent candidacidal mechanisms. J. Clin. Invest. 55:33-346. 11. Lehrer, R. L, and M. J. Cline. 1969. Interaction of Candida albicana with human leukocytes and serum. J. Bacteriol. 98:996-1004. 12. Miyake, T., K. Takeya, K. Nomoto, and S. Muraoka. 1977. Cellular elements in resistance to candida infection in mice. I. Contribution of T lymphocytes and phagocytes at various stages of infection. Microbiol. Immunol. 21:703-725. 13. North, R. J. 1978. The concept of the activated macrophage. J. Immunol. 121:806-809. 14. Pillemer, L, L. Blum, L H. Lepow, 0. A. Ross, E. W. Todd, and A. C. Wardlaw. 1954. The properdin system and immunity I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science 120:279-285. 15. Robinson, T. W. E., R. J. R. Cureton, and R. B. Health. 1968. The pathogenesis of Sendai virus infection in the mouse lung. J. Med. Microbiol. 1:89-95. 16. Schmid, L., and K. Brune. 1974. Assessment of phagocytic and antimicrobial activity of human granulocytes. Infect. Immun. 10:1120-1126. 17. Schultz, R. M., K A. Chirigos, and U. L. Heine. 1978. Functional and morphological characteristics of interferon-treated macrophages. Cell. Immunol. 35:84-91. 18. Stossel, T. P. 1974. Phagocytosis. N. Engi. J. Med. 290: 774-780. 19. Stossel, T. P. 1975. Phagocytosis: recognition and ingestion. Semin. Hematol. 12:83-116. 20. Warshauer, D. E. Goldstein, T. Akers, W. Lippert, and K. Kim. 1977. Effect of influenza viral infection on the ingestion and killing of bacteria by alveolar macrophages. Am. Rev. Respir. Dis. 115:269-277. 21. Weir, D. M., and H. Ogmundedotter. 1977. Non-specific recognition mechanisms by mononuclear phagocytes. Clin. Exp. Immunol. 30:323-329. 22. Wyde, P. R., D. L Peavy, and T. R. Cate. 1978. Morphological and cytochemical characterization of cells infiltrating mouse lungs after influenza infection. Infect. Iimmun. 21:140-146. 23. Yamamura, AL, and HE Valdimarson. 1977. Participation of C3 in intracellular killing of Candida albicans. Scand. J. Immunol. 6:591-594.
