INFECTION AND IMMUNrrY, Feb. 1975, p. 405-414 Copyright i 1975 American Society for Microbiology

Vol. 11, No. 2 Printed in U.S.A.

Phagocytosis of Mycoplasma salivarium by Human Polymorphonuclear Leukocytes and Monocytes CLAYTON F. PARKINSON'* AND PAUL B. CARTER Department of Biology, Utah State University, Logan, Utah 84322 Received for publication 30 August 1974

Mycoplasmacidal activity was exhibited by human peripheral blood leukocytes in the absence of detectable specific antiserum. After incubation of varying concentrations of Mycoplasma salivarium with leukocytes, changes in colony-forming units (CFU) of this species per milliliter occurred. The most noticeable decrease in CFU per milliliter was then the incubation mixtures contained five mycoplasmas per leukocyte. At this ratio, the mycoplasmacidal activity resulted in a fivefold decrease in numbers of viable mycoplasmas after 90 min of incubation. Continued incubation demonstrated a tenfold decrease in CFU per milliliter by 4 h. Electron micrographs of incubated mixtures of human leukocytes and M. salivarium showed this mycoplasma to be phagocytized by monocytes and neutrophils whenever mutual contact or pseudopodial formation occurred. The process was continuous. Numerous phagocytic vacuoles developed which contained multiple ingested microorganisms. After the cytoplasmic granules of the leukocytes fused with the phagocytic vacuole, the phagocytized mycoplasmas became disrupted and unrecognizable. Results involving interactions between mycoplasmas and mammalian phagocytes are contradictory. Electron microscopic studies by Zucker-Franklin et al. (19, 20) indicated that Mycoplasma pneumoniae, M. neurolyticum, and M. gallisepticum rapidly adsorbed to human blood leukocytic plasma membranes, which then developed long cytoplasmic processes not seen in the controls. Phagocytosis occurred in the absence of specific antiserum with accompanying degranulation of leukocytes. While studying tissue responses to M. pulmonis, Jones and Hirsch (10) observed the adsorption of this species to mouse peritoneal macrophages and reported that homologous rabbit antiserum was required for ingestion and killing. Simberkoff and Elsbach (17) reported that M. hominis and M. arthritidis did not adsorb to human or rabbit polymorphonuclear leukocytes within 2 h. No killing of mycoplasmas was observed either in the presence or absence of rabbit antimycoplasmal sera, but preincubation of mycoplasmas and leukocytes impaired subsequent leukocytic activity against Escherichia coli. Evidence was presented by Cole and Ward (5) that neither mouse nor rat peritoneal macrophages were able to kill M. arthritidis in the absence of specific hyperim-

mune rabbit antiserum. Specific hyperimmune rabbit antiserum initiated phagocytic action of this species and resulted in a 50-fold decrease in viable mycoplasmas in 6 h. M. salivarium appears to be a gingival sulci and plaque inhabitant that may produce gingivitis and periodontal disease (7, 13, 16). Mergenhagen and Snyderman (15) suggested that microbial products residing in the dental plaque activate the host's effector systems and can lead to inflammation in the periodontal tissue. The inflammatory process may be perpetuated by the phagocytic process as migratory polymorphonuclear leukocytes (18) release intracellular lysosomal enzymes capable of tissue destruction

(4).

These observations indicate a need to assess the capability of human leukocytes to phagocytize M. salivarium. The present investigation was designed to determine mycoplasmacidal activity and phagocytosis of this species by human leukocytes. MATERIALS AND METHODS Organism. All experiments were performed with M. salivarium strain PG 20, obtained from the National Institutes of Health, Bethesda, Md. Confirmation of species was accomplished by the growth-inhibition method described by Clyde (3) using paper disks saturated with homologous and heterologous antimycoplasmal sera. These sera were obtained from Baltimore Biological Laboratories

' Present address: Department of General Dentistry, University of Connecticut Health Center, Farmington, Conn. 06032.

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INFECT. IMMUN.

PARKINSON AND CARTER

(BBL) and the National Institutes of Health. The growth precipitation test described by KrogsgaardJensen (12), which has a high degree of specificity, was periodically employed to test culture purity. Medium, enumeration, and mycoplasma collection. The medium used in culturing the mycoplasmas contained 80% pleuropneumonia-like organisms (PPLO) broth or agar (BBL), 10% agamma horse serum (BBL), and 10% of a 1% Albimi Laboratories yeast autolysate solution. Additions to the medium included 500 U of penicillin per ml and 250 mg of thallium acetate per liter. After transfer of the original culture to broth media, 10-ml portions were stored at -90 C for re-inoculation and use. Enumeration and viability of mycoplasmas were determined from broth, phosphate-buffered saline suspensions, or incubation mixtures taken at various times, serially diluted in PPLO broth free of additives and supplements, and plated on agar. The inoculated agar was incubated anaerobically at 37 C for 48 h and then inspected for colony-forming units (CFU) of M. salivarium per ml using a Unitron inverted microscope at x 75 magnification. CFU could be seen without magnification if incubation was extended for 5 to 7 days. The microorganisms were harvested during logarithmic growth phase by centrifugation at 27,000 x g for 20 min at 4 C. The mycoplasmas were washed twice and resuspended in phosphate-buffered saline (pH 7.2). The buffer was prepared in two parts. The first part consisted of 80 g of NaCl, 2 g of KCl, 1 g of CaCl2, 1 g of MgCl, .6H2O, and triple distilled water to make 1 liter; the second part contained 11.4 g of NaHPO4, 2 g of anhydrous KH2PO4, and triple distilled water to make 1 liter. Each solution was autoclaved, and then 100 ml of each was added to 800 ml of triple distilled water. Appropriate dilutions enabled quantitation of the microorganisms. Separation of leukocytes. Human blood without serum growth-inhibiting antibody to M. salivarium was collected in test tubes containing 10 U of heparin per ml. Three normal males having an average age of 23.7 years and without clinical evidence of periodontal disease were the blood donors. Tests of growth inhibition were performed on these sera by a modified method of Bailey et al. (2). All sera were heated at 56 C for 30 min before use. One-half milliliter of a 1:5 dilution of sera in phosphate-buffered saline (pH 7.2) and 0.5 ml of broth suspension of logarithmically growing mycoplasmas were mixed and added to agar. After incubation, the agar was examined for inhibition of growth. The tested serum was considered without antibody when CFU per milliliter were greater than nine-tenths of the control. Plastic or polycarbonate tubes and bottles were used to avoid contact between leukocytes and glassware. Sedimentation of erythrocytes occurred after 60 to 90 min at room temperature and resulted in the formation of a sharp interface between the erythrocytes and the platelet-leukocyte-rich buffy coat. The buffy coat and plasma were aspirated with a plastic pipette and centrifuged at 180 x g at 4 C for 8 min. Most of the platelets remained in the plasma supernatant fluid, which was discarded. The button of cells was gently

resuspended and washed twice in Hanks balanced saline. The number of leukocytes used in these experiments was adjusted to a final concentration of 107 to 3 x 107 cells per ml. The characteristics of the mixed leukocytic populations were checked by microscopy, and polymorphonuclear leukocytes comprised 60 to 70% of the cells. Incubation mixtures. Mycoplasmas of various concentrations of viable organisms resuspended in a broth base were added to the leukocytic suspension to bring the total volume to 10 ml. Incubation was in a G24 environmental shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.) at 37 C for periods of time to 4 h while being shaken at 150 rpm. Fixation and electron microscopy. A ratio of 50 mycoplasmal organisms to each human leukocyte was incubated for varying time periods and then fixed in suspension with an equal volume of Karnovsky's fixative (10). Centrifugation at 180 x g for 8 min formed a pellet of cells, and the supernatant fluid was discarded. The pellets were fixed in Karnovsky's fixative for 1.5 to 2 h, rinsed for 12 h in cacodylate buffer (pH 7.4), postfixed in 2.0% osmium tetroxide for 1.5 h, and again rinsed in cacodylate buffer. The pellets were cut to appropriate size with a razor blade and dehydrated through an ethanol series. Further dehydration was accomplished with a graded series of ethanol and two to three changes of propylene oxide. The fixed specimens were embedded in Epon, sectioned with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 9A electron microscope.

RESULTS Viability of M. salivarium in the incubation mixtures. In this study, the total number of viable mycoplasmas in incubation suspensions was based on the capacity of the organism to form colonies on agar. Decline in CFU per milliliter and apparent mycoplasmacidal activity in the presence of human peripheral blood leukocytes is presented in Fig. 1. PPLO broth free of additives and Hanks balanced saline permitted survival of the mycoplasmas during the incubation experiments. When the mean values for CFU per milliliter from the incubation mixtures were recorded, there was evidence of some igcrease of initial mycoplasmal growth in those incubation suspensions containing M. salivarium. This was postulated as a result of dissociation of microbial aggregates. With the concentration of mycoplasmas to leukocyte adjusted to 100 to 1, colony counts after 90 min of incubation were virtually the same as control suspensions of M. salivarium. CFU per milliliter within the incubation mixture showed a reduction of viable organisms after 2 h. At no time during the 4-h incubation period was the mycoplasmal decrease greater than fourfold. With both lower

VOL. 11, 1975

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FIG. 1. Viability of M. salivarium after incubation with human peripheral blood leukocytes at varying ratios of microorganisms to cells. Mean values of three experiments are recorded. Continuous lines represent CFU of mycoplasmas per milliliter after incubation in the suspending medium without leukocytes. Dashed lines represent CFU of mycoplasmas per milliliter after incubation with leukocytes.

ratios of mycoplasmas to leukocyte, the incubation mixtures exhibited fewer CFU per milliliter than the controls as early as 30 min. At the lowest ratio, a greater than fivefold decrease occurred after 90 min of incubation, and after 4 h a tenfold decrease in viable mycoplasmas was evident. With this method of determining mycoplasmacidal activity and/or phagocytosis, the lag time before decline in the number of viable mycoplasmas decreased as the ratio of mycoplasmas to leukocyte decreased. Prior differential centrifugation to sediment only the leukocytes and then plating of supernatant fluids did not markedly alter the mycoplasmal counts. Such sedimentation and count determination would assess mycoplasmal disappearance from the supernatant fluids but would not necessarily measure microorganisms adsorbed to the leukocytes. The leukocytes were not lysed to release intracellular mycoplasmas in order to assess viable count changes. The addition of antiserum during the present study resulted in slightly lower mycoplasmal CFU per milliliter. However, the effect could be attributed to agglutination of the microorganisms by the

407

serum rather than phagocytosis, and therefore these studies were not pursued. Electron microscopy of incubation mixtures. An additional means to determine the accuracy of the conclusion that M. salivarium is phagocytized by human leukocytes seemed essential. Electron microscopy was employed to show the phagocytic process in vitro to be independent of specific antiserum. Various morphological forms of M. salivarium which were phagocytized are presented in Fig. 2, 3, and 4. It was observed that this mycoplasmal species demonstrated polymorphism during its exponential growth phase. Each form presented a trilaminar membrane surrounding cytoplasm which contained loosely arranged ribosomes, fibrillar deoxyribonucleic acid, one or more electron-dense areas, and occasionally empty vesicles. Anderson and Barile (1) suggested the empty vesicles were more prevalent in aging cells and may represent deterioration. The observed loss of limiting membrane continuity may be mycoplasmal dissociation. Elongated coccoid cells and a dividing cell connected by a membrane tubule were seen. Phagocytosis of M. salivarium was initiated when the microorganisms contacted the plasma membrane of either monocytes or neutrophils. Free mycoplasmas appeared to be engulfed by monocytes (Fig. 5) after initial membrane contact with pseudopods exhibiting a minor endocytic role. Active pseudopods of neutrophils surrounded the mycoplasmas (Fig. 6). Closure of neutrophil pseudopodia (Fig. 7) completed the early phase of phagocytosis. Identifiable mycoplasmas were observed within the phagocytic vacuoles of monocytes (Fig. 8) and of neutrophils (Fig. 9). Neutrophils were observed to contain phagocytized M. salivarium which were both identifiable and degraded (Fig. 10). Two morphological forms of engulfed mycoplasmas were observed within a single phagocytic vacuole (Fig. 11). Incubation of M. salivarium with peripheral blood leukocytes at 37 C for up to 4 h under the described experimental conditions with a ratio of mycoplasmas to leukocyte of 50 to 1 had little detrimental effect on the ultrastructure of either the leukocyte or the uningested mycoplasma. The attraction of mycoplasmas to the surface membranes of peripheral blood leukocytes was very evident. Phagocytized microorganisms were altered and became unidentifiable except for density similarities, whereas those surrounding the phagocyte showed wellpreserved ultrastructure. This indicated that phagocytosis was a continual process. It was

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