Phagocytosis by Guinea Pig Alveolar Macrophages

Transcription

1 INFECTION AND IMMUNITY, Apr. 1982, p Vol. 36, No /82/ $02.00/0 Release of Mycoplasma pneumoniae Substances After Phagocytosis by Guinea Pig Alveolar Macrophages M. KIST,* E. JACOBS, AND W. BREDT Institut fur Allgemeine Hygiene und Bakteriologie, Zentrum fur Hygiene, Universitat Freiburg, D-7800 Freiburg, West Germany Received 27 July 1981/Accepted 4 December 1981 Antibody-opsonized Mycoplasma pneumoniae cells with various radioactive markers were sedimented onto monolayers of guinea pig alveolar macrophages (AM). After 2 h of incubation, about 50% of the activity of [3H]palmitate-labeled mycoplasmas was associated with AM. Nonspecific attachment of the opsonized mycoplasmas to AM-free plastic surface areas was negligible. The occurrence of phagocytosis was proven by electron microscopy and monitoring of AM surfacebound antigen by 125I-labeled F(ab)2 fragments. The activity of [3H]palmitic acidlabeled mycoplasmas was only slowly released into the supernatant. About 55% of the activity remained AM-associated up to 70 h after phagocytosis. After phagocytosis of [3H]thymidine-labeled cells, about 70% of the radioactivity found in the supernatant was released within 8 h. More than 80% of this activity was non-precipitable by trichloracetic acid. 3H-amino acid-labeled protein was released to 50% within 8 h. Supernatants and AM were tested for M. pneumoniae antigen with enzyme-linked immunosorbent assay. Considerable amounts of antigenically active material could be found in the supernatant within 8 h. This antigen was totally inactivated by heat (80 C). Trypsin treatment (1 mg/ml, 10 min) reduced the antigenicity by 80%. The results suggest a selective release of microbial material after phagocytosis. Alveolar macrophages (AM) are the first line of cellular defense against infection from Mycoplasma pneumoniae. The interaction of these phagocytes (6, 8, 12) or of macrophages of other origins (1, 5, 7, 9, 10, 14) with mycoplasmas has been studied by several authors, but effective phagocytosis has only been observed after opsonization (5, 6, 8, 9, 10, 12, 14). However, little is known about the fate of the ingested microbial substances. The release of [3H]thymidine was used as marker of cellular destruction (9), but no sufficient data are available on other mycoplasmal components, such as membrane glycolipids or proteins. Even less is known about the biological activity of such native or modified molecules released by the macrophage. These molecules may still be able to activate complement (2, 3), react with antibody, or attach to host cell membranes. This study describes the characteristics of a phagocytosis system using guinea pig AM and M. pneumoniae. MATERIALS AND METHODS Mycoplasmas. M. pneumoniae strain FH was grown in Roux bottles in 100-ml Hayflicks medium containing 1,000 U/ml of penicillin, 0.05% thallium acetate, and 1% glucose. Radioactive substances were added according to the experimental schedule. A frozen stock inoculum (-70 C) containing about 108 colony-forming units per ml was used for each culture. After 48 h at 37 C, the glass adherent mycoplasmas were washed four times with 5 ml of cold, sterile phosphate-buffered saline (PBS), ph 7.2, and harvested into 3 ml of tissue culture medium (TCM) as described below. The suspension was diluted to an optical density of 0.15 at 660 nm before being used. Radioactive labeling of mycoplasma cells. For experiments with labeled M. pneumoniae cells, 50,uCi of [3H]palmitic acid or 150,uCi of [6_3H]thymidine was added to 100 ml of the growth medium before incubation. The mycoplasmas were harvested as previously described and tested for specific activity. For protein labeling, the glass adherent mycoplasmas of one Roux bottle were harvested into 1 ml of amino acid-free TCM containing 2% horse serum and substituted with 0.1,uCi of a 3H-amino acid mixture or 1.Ci with 357 [14C]methionine, respectively. After 4 h of incubation at 37 C, the mycoplasma cells were centrifuged and washed three times with PBS. Radioactive amino acids not bound to the proteins of mycoplasma cells were separated by passage of the suspension through a Sephadex G-25 column (9.1-ml bed volume). Preparation and labeling of anti-m. pneumoniae F(ab)2 fragments with "2I. Rabbit anti-m. pneumoniae immunoglobulin G (IgG; 50 mg) purified by gel filtration was digested with 3 mg of pepsin in 0.1 M sodium acetate buffer, ph The digestion products were gel filtrated (Sephacryl S 300), and the antibody activity of the F(ab)2 fraction was determined in an indirect immunofluorescent assay on glass-grown M. pneumoniae cells. The absence of Fc fragments was proven

2 358 KIST, JACOBS, AND BREDT by the negative complement fixation test with M. pneumoniae as antigen. F(ab)2 fragments (0.5 mg) were iodinated with 0.5 mci of carrier-free 125I (Eidgenossisches Institut fur Reaktorforschung, Wurenlingen, Switzerland) by using the chloramin T method (11). Unbound 1251 was removed by gel filtration on Bio-Gel P10 (1 by 30 cm). The specific antibody activity of iodinated F(ab)2 fragments was again tested by immunofluorescence on M. pneumoniae and the absence of binding onto AM monolayers. AM. The lungs of anaesthesized guinea pigs were washed four times with 20 ml of cold TCM containing antibiotics. The cells were centrifuged in siliconized vials for 10 min at 300 x g, resuspended in 10 ml of TCM, and adjusted to 106 cells per ml. Samples of this suspension (2 ml) were put into plastic petri dishes (Nuncolon, Nunc, Denmark; 35 by 8 mm) and allowed to settle at 37 C in a CO2 atmosphere. After 2 h the cultures were vigorously washed four times with PBS to remove nonadherent cells (about 40%). These monolayers were incubated for 15 to 20 h at 37 C in a CO2 atmosphere before use and considered to be AM. The viability of the cultures was monitored by measuring the lactate dehydrogenase concentration of the supernatant and by using the trypan blue exclusion test. Chemicals and media. Medium 199 (Flow Laboratories, Inc., Rockville, Md.) with glutamine and sodium bicarbonate, and 2 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) was used as TCM. The 3H-labeled substances were obtained from Amersham-Buchler (Braunschweig, Germany). As coating buffer for enzyme-linked immunosorbent assay, a sodium carbonate-bicarbonate buffer (ph 9.6) was used. As a substrate buffer, a diethanolamin buffer (ph 9.8) was used (97 ml of diethanolamin, 100 mg of MgCl2 6H2O, and 800 ml of distilled water). Cytochalasin B, trypsin, and trypsin inhibitor were purchased from Serva (Heidelberg, West Germany). Phagocytosis. The M. pneumoniae suspension was opsonized by rabbit anti-m. pneumoniae antiserum in a final concentration of 1:200 (15 min at 37 C) with intermittent mixing. Amounts of 900 RI each of these suspensions were centrifuged onto AM monolayers (300 x g for 10 min at room temperature). After 2 h of incubation at 37 C, the cultures were washed four times with 2 ml of sterile PBS and refilled with 2 ml of TCM. This was termed zero hour. Electron microscopy. After 2 h of phagocytosis, AM were washed three times with PBS (2 ml), fixed in situ with 3% glutaraldehyde in PBS (ph 7.2) for 30 min on ice, and then treated with 1% OS04 in 0.1 M cacodylate buffer (ph 7.2) for 2 h. During dehydration with increasing concentrations of ethanol, the cultures were prestained with 0.5% uranyl acetate and 1% phosphotungstic acid at the 70% ethanol step. After centrifugation (800 x g for 10 min), the pellet was embedded in Epon. Sections were cut on the LKB microtome, stained with uranyl acetate and lead citrate, and examined in a Siemens Elmiscop 102. M. pneumoniae antigen bound onto AM surfaces. Phagocytosis of M. pneumoniae was performed as previously described. At various times after the 2-h phagocytosis period, the cultures were thoroughly washed with PBS. Diluted anti-m. pneumoniae F(ab)2 fragments (100,l) labeled with 1251 were added and the cultures were incubated for 20 min at room temperature. Unbound F(ab)2 fragments were removed by washing them with PBS, and the remaining radioactivity was determined. The total amount of AM-associated M. pneumoniae could be estimated by simultaneous [3H]palmitic acid labeling of the mycoplasma cells. AM monolayers without M. pneumoniae were used as controls. Experiments were done in triplicate. Detection of antigen. An enzyme-linked immunosorbent assay was used for the detection of M. pneumoniae antigen. Enzyme-linked immunosorbent assay microplates (Dynatech Laboratories, Nuirtingen, West Germany) were coated by putting 200,u of coating buffer containing 100,ug/ml anti-m. pneumoniae IgG purified by gel filtration (Sephacryl S 300, Pharmacia, Inc., Freiburg, West Germany) in each well. The plates were incubated at 4 C for 24 h and washed with PBS containing 0.05% Tween 20. The samples diluted in PBS-Tween buffer containing 0.5% bovine serum albumen put in the wells and incubated overnight at 4 C. The plates were then washed as described above and treated with the IgG-phosphatase conjugate (200,ug protein per ml). After reaction (2 h at 37 C and then 4 h at 4 C), the unbound conjugate was removed by washing, and the substrate p-nitrophenylphosphate (Boehringer Mannheim Corp.; 1 mg/ml) was added. After 30 min at room temperature, the plate was read at 405 nm in a Dynatech Multiscan. Buffer, conjugate, and standard mycoplasma suspensions were used as appropriate controls. IgG conjugate. The isolated IgG fraction was coupled with alkaline phosphatase by the modified method of Avrameas as described by Clark and Adams (4). Antigen treatment by heat and trypsin. Four hours after phagocytosis, supernatants and AM (sonicated three times for 1 min, power 4, Branson Sonifier Cell Disrupter B15; Branson Instruments Co.) were heated at different temperatures for 10 min or treated with trypsin (1 mg of trypsin per ml for 10 min at 37 C). RESULTS INFECT. IMMUN. Characteristics of the phagocytosis system. About 50 to 75% of the harvested alveolar cells became adherent after 2 h of cultivation, and 90% of them excluded trypan blue. During the phagocytosis experiments, the viability decreased, usually within 48 h, to about 70% of the adherent cells. Additionally, the number of glass-attached AM was reduced to about 80% after 20 h and about 70% after 48 h. The effectiveness of the phagocytosis system was tested with [3H]palmitic acid-labeled mycoplasmas. After centrifugation, incubation (2 h), and washing, about 50% of the activity was found to be associated with the AM monolayer (Fig. 1A). An additional complement treatment (10% guinea pig serum) in an experiment with [3H]thymidinelabeled mycoplasmas removed only a minor part of this AM-associated activity, suggesting that most of the mycoplasmas were internalized or tightly bound to the macrophage surface. Electron microscopy. Numerous mycoplasmas were found in phagocytic vacuoles (Fig. 2). In

3 VOL. 36, 1982 M. PNEUMONIAE RELEASE AFTER AM PHAGOCYTOSIS 359 C- _20 EL 10 A B 3H- PALMITATE Ic ac ie z n 4 e i c c 1U ou 11 Time after phagecytesi (hairs) FIG. 1. Phagocytosis of [3H]palmitic acid-labeled M. pneumoniae by guinea pig AM. The results of two experiments (I and II) are shown. (A) Characteristics of the phagocytosis system. Symbols: a, total amount of radioactivity applied per petri dish; b, AM-associated activity after 2 h of phagocytosis; c, non-specifically bound activity in control dishes without macrophages. (B) Release of [3H]palmitic acid-labeled substances. Sampling of supernatant started after 2 h of phagocytosis (not included in time) and removal of mycoplasma suspension. The supernatant activity was plotted cumulatively. some of the phagocytized mycoplasmas, typical structural elements, e.g., the tip structure, remained visible, and some other organisms showed peripheral chromatin clumping or the beginning of degradation. The phagosomes increased in size during time, probably by fusion with surrounding lysosomes. Disappearance of M. pneumoniae antigen from AM surface during phagocytosis. 125I-labeled F(ab)2 fragments of anti- M. pneumoniae IgG were used to detect antigenic material on the macrophage surface. The F(ab)2 labeling of surface-bound antigenicity is possible only after the initial 2-h phagocytosis period has ended and the supernatant of opsonized M. pneumoniae cells has been removed. At this time much of the mycoplasma material has already been internalized, and consequently, the antigen exposed on the macrophage surface at this particular moment is only a fraction of the total macrophageassociated antigen. The results are demonstrated in Table 1. The total amount of mycoplasma material (inside and outside the macrophage) is represented by the 3H counts of the palmitic acid-labeled mycoplasmas. The activity of 1251 represents only the mycoplasma antigen bound to the macrophage surface. The data show a considerable reduction of this material to about 15% (1251) after 22 h, whereas the total amount of AM-associated mycoplasma material apparently drops only to 70% (3H). In the presence of cytochalasin B, a different pattern is seen. The AM-associated activity is reduced to a comparable level (about 83%), but about 40% of the initial surface-bound antigenicity is still detectable after 22 h. Furthermore, the proportion of antigen remaining at the surface is apparently much higher under the influence of the inhibitor, as indicated by a lower 3H/125I ratio (nine versus four) at time 0. The results suggest an effective inhibition of phagocytosis by cytochalasin B and continuous internalization under normal conditions. Release of [3H]thymidine-labeled substances. After phagocytosis of [3H]thymidine-labeled mycoplasmas, most of the label was found in the supernatant within a few hours (Fig. 3). Of the activity released within 50 h, about 65 to 75% was detected within the first 8 h. Of this activity, 84 to 93% was not precipitable by 10% trichloracetic acid. When cytochalasin B (10,ug per ml) was added to prevent phagocytosis, the release of 3H-labeled material was reduced by about 20 to 40%. Release of [3H]palmitic acid-labeled substances. In contrast to the thymidine experiments, the activity of the [3H]palmitic acid-labeled mycoplasmas was only slowly released into the supernatant after phagocytosis (Fig. 1B). About 55% of the radioactive material remained associated with the adherent AM for as long as 70 h. After centrifugation of the supernatants at 12,000 x g (5 min), 14 to 28% of the activity was found in the pellet, probably representing detached AM. Release of 3H-amino acid-labeled substances. The release pattern of amino acid-labeled mycoplasmas was comparable to that of thymidine. Within the first 8 h, about 50% of the activity was found in the supernatant (Fig. 4). After 20 h, only 20 to 28% of the total activity was still bound to the macrophages. Antigen detection. Supernatants and residual AM monolayers were tested for M. pneumoniae antigen. The occurrence of antigenically active material in the supernatant is shown in Fig. 5. Apparently most (about 80%) of the detectable material was released within 8 h. Characteristics of released and AM-associated antigen. The antigenic activity found in the supernatant (4 h) was destroyed by being heated at 90 C for 10 min. Additional experiments showed that the inactivation began at 54 C and was completed at 80 C. When the activity of the AMassociated antigen (4 h) was tested under comparable conditions, antigenicity was reduced to 10% at 80 C, and no further reduction was seen at 100 C. This corresponds with the results obtained on sonicated suspensions of whole M. pneumoniae cells. The antigenicity of the supernatant was reduced to 20% by treatment with 1 mg of trypsin per ml (10 min at 37 C). Furthermore, ammonium sulfate in a concentration of

4 360 KIST, JACOBS, AND BREDT Mf~ ~ ~ ~ ~ ~ *' 0s. * R '..!.,.: :....:.c M *C.r.. k ; Mp'''."* : F INFECT. IMMUN. z : '':i :: '. f.. e N..:... s*e\ 'I:.!tR:!... :S..S... S$; a: :; mk: *e.e FIG. 2. Electron micrograph of AM after 2 h of phagocytosis of opsonized M. pneumoniae (Mp). Abbreviations: CM, cytoplasma membrane; N, nucleus. Bar = 1,.m. 80% (wt/vol) precipitated 86% of the antigenic activity. DISCUSSION Quantitative studies of phagocytosis systems are often hampered because some of the microorganisms are not ingested, thereby influencing the results of viable counts and other parameters. This problem is even more complicated with mycoplasmas, especially M. pneumoniae. Their ingestion cannot be monitored sufficiently by light microscopy, and furthermore, they stick effectively to those areas of the plastic surface which are not covered by the macrophages. Four systems are conceivable for maximal phagocytosis of M. pneumoniae: (i) phagocytosis with both reaction partners in suspension (8); (ii) macrophages sedimented onto sheets of glass-grown mycoplasmas (1); (iii) monolayers of macrophages infected with mycoplasmas which are subsequently opsonized (5, 6, 8, 9, 10, 12); and (iv) monolayers of macrophages onto which opsonized mycoplasmas are sedimented. Preliminary experiments suggested the use of the last method, mainly because the portion of mycoplasmas attaching "unspecifically" could be kept to a minimum. At the same time the macrophages could be precultivated for longer times, reducing the number of nonphagocytic cells to a minimum. On the other hand, opsonized mycoplasmas often form small aggregates and are therefore more difficult to quantitate TABLE 1. Effect of cytochalasin B on internalization of M. pneumoniae by AM No inhibitor Cytochalasin B (10 pkg/ml) Controla Time (h) Mycoplasma AM- Mycoplasma after end of AM-associated Myconlasma AM- Mycop AM without phagocytosis mycoplasmas mycoplasmas period (3H cpm) AM surface mycoplasmas AM surface (1251 cpm) pefiod ('H cpm) ~~~(1251 cpm) (3H cpm) (125i cpm) 0 14,335 ± 325b 1,578 ± 96 8,784 ± 278 2,100 ± ± ,179 ± 537 1,210 ± 95 6,762 ± 141 1,747 ± ± ,048 ± ± 24 7,313 ± ± ± 53 a AM without mycoplasmas treated with F (ab)2 fragments. b Mean value cpm ± SD.

5 VOL. 36, Time after phagocytosis (hours) FIG. 3. Release of [3H]thymidine-labeled substances from M. pneumoniae after phagocytosis by guinea pig AM. The phagocytosis took 2 h, and the time of release began after removal of the mycoplasma suspension. The results of two experiments (I and II) are shown. (this property was apparently not of major importance in this study). The data obtained in our experiments suggest a sufficient uptake of opsonized mycoplasmas by the macrophages. Electron microscopy and staining with fluorescent dyes (unpublished observations) show an internalization even of aggregates of considerable size. The number of M. pneumoniae cells found outside the macrophages after the 2-h phagocytosis period was small enough not to influence the results significantly. Even these cells, mostly aggregates, were probably taken up by the phagocytes during the following hours of incubation, as suggested by the disappearance of most of the mycoplasma antigen from the macrophage surface. During the first hours of phagocytosis, mycoplasma material may be released in two ways: (i) by lysis of microorganisms which are attached to the phagocyte surface or (ii) by release of such substances from within the phagocyte. The first way is probably more efficient in mycoplasmas than in other bacteria because the lack of a cell wall permits a direct attack of excreted lysosomal enzymes on the mycoplasma membrane. The considerable amount of thymidine label found in the supernatant, even in the presence of cytochalasin B, suggests a certain degree of such extracellular lysis. However, the inhibitor itself is apparently triggering an increased release of lysosomal enzymes (13, 15, 16) so that the data are probably not completely representative of the unmodified phagocytosis. The results with labeled thymidine and amino M. PNEUMONIAE RELEASE AFTER AM PHAGOCYTOSIS 361 acids during the first hours of phagocytosis suggest a relatively fast release, mainly of cytoplasmic material. A different pattern was observed with the palmitate label. Apparently, the mycoplasma lipids are retained within the macrophage for a much longer time. They may be stored there in the form of intact mycoplasma substance, as suggested by the antigen content of the macrophages, or the labeled fatty acid could possibly be used by the phagocyte for its own lipid synthesis. The substances released into the supernatant in the hours after phagocytosis are at least partially mycoplasma antigens with properties which indicate their protein nature. They are apparently not degraded by the action of lysosomal enzymes and are therefore immediately available for further biological interactions with the humoral and cellular factors of the host. The fate and further role of the substances retained in the macrophage for the time of observation is unknown. The few data available suggest that a considerable part of these materials are of nonprotein nature, consisting possibly of membrane lipids, but they may also contain some membrane proteins, which at least partly may be retained within the phagocytic cell. The fast or delayed release of products of the phagocytic process could have some biological importance for the interaction between parasite and host in the early stages and in the sequelae of infectious diseases. Besides being spread X.20 c E CT 1X0 A a*b Zeit mach Phagozytose (Stunden) FIG. 4. Release of 3H-amino acid mixture and [14C]methionine-labeled substances after 2 h of phagocytosis. Time of release began after removal of mycoplasma suspension. (A) Characteristics of macrophages. Symbols: a, AM-associated activity after 2 h of phagocytosis; b, AM-associated activity after 20 h. (B) Characteristics of the supernatant showing release of 3H-amino acids (I) and [14C]methionine- (II) labeled substances. Cumulative plotting of the supernatant activity was done.

6 362 KIST, JACOBS, AND BREDT 3.0 ;, i M._ go g::::~~d~~.:. A Time after phagocytosis (hours) FIG. 5. Release of M. pneumoniae antigen from AM after 2 h of phagocytosis. Cumulative plotting was done, and the amount of antigen was measured by enzyme-linked immunosorbent assay. Symbols: 0, supernatant; 0, supernatant heated at 90 C for 10 min; A, control (release from dishes treated with M. pneumoniae suspension, but without AM). Mean values were calculated from triplicate tests. The standard deviation was 7 to 10%. throughout the tissues of the host, these substances may act as immunogenic stimuli, reacting with effector systems such as complement (2, 3), and they could be toxic for the host cell membraneb, alter the surface of the host cell, or interfere with immunological reactions. They therefore certainly deserve further investigation. LITERATURE CITED 1. Bredt, W Phagocytosis by macrophages of Mycoplasma pneumoniae after opsonization by complement. Infect. Immun. 12: Bredt, W., and D. Bitter-Suermann Interactions INFECT. IMMUN. between Mycoplasma pneumoniae and guinea pig complement. Infect. Immun. 11: Bredt, W., B. Wellek, H. Brunner, and M. Loos Interactions between Mycoplasma pneumoniae and the first component of complement. Infect. Immun. 15: Clark, M. F., and A. N. Adams Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34: Cole, B. C., and J. R. Ward Interaction of Mycoplasma arthritidis and other mycoplasmas with murine peritoneal macrophages. Infect. Immun. 7: Erb, P., and W. Bredt Interaction of Mycoplasma pneumoniae with alveolar macrophages: viability of adherent and ingested mycoplasmas. Infect. Immun. 25: Howard, C. J., and G. Taylor Variation in the virulence of strains of Mycoplasma pulmonis related to susceptibility to killing by macrophages in vivo. J. Gen. Microbiol. 114: Howard, C. J., G. Taylor, J. Collins, and R. N. Gourlay Interaction of Mycoplasma dispar and Mycoplasma agalactiae subsp. bovis with bovine alveolar macrophages and bovine lacteal polymorphonuclear leucocytes. Infect. Immun. 14: Jones, T. C., and J. G. Hirsch The interaction in vitro of Mycoplasma pulmonis with mouse peritoneal macrophages and L-cells. J. Exp. Med. 133: Jones, T. C., S. Yeh, and J. G. Hirsch Studies on attachment and ingestion phases of phagocytosis of Mycoplasma pulmonis by mouse peritoneal macrophages. Proc. Soc. Exp. Biol. Med. 139: McConahey, P. J., and F. J. Dixon A method of trace iodination of proteins for immunologic studies. Int. Arch. Allergy 29: Powell, D. A., and W. A. Clyde, Jr Opsoninreversible resistance of Mycoplasma pneumoniae to in vitro phagocytosis by alveolar macrophages. Infect. Immun. 11: Sorber, W. A Effect of complement fixation on the release of lysosomal enzymes from rabbit alveolar macrophages. Infect. Immun. 19: Taylor, G., and C. J. Howard Interaction of Mycoplasma pulmonis with mouse peritoneal macrophages and polymorphonuclear leucocytes. J. Med. Microbiol. 13: Temple, A., G. Loewi, P. Davies, and A. Howard Cytotoxicity of immune guinea-pig cells. Immunology 24: Zurier, R. B., S. Hoffstein, and G. Weissmann Cytochalasin B: effect on lysosomal enzyme release from human leukocytes. Proc. Nat. Acad. Sci. U.S.A. 70: