INFECTION AND IMMUNITY, Mar. 1975, p. 598-600 Copyright © 1975 American Society for Microbiology

Vol. 11, No. 3 Printed in U.S.A.

Reactivity of Mast Cells to Mycoplasma salivarium CLAYTON F. PARKINSON' Department of Biology, Utah State University, Logan, Utah 84322 Received for publication 12 September 1974

Mice were sensitized to Mycoplasma salivarium and then challenged with heterologous and homologous antigens. The number of degranulating tissue mast cells was used to assess the degree of local tissue hyperactivity after antigenic challenge. Signif'icant differences existed between homologous and heterologous antigenic challenges. Microscopic studies have shown that mast cells are present in the connective tissue of human gingiva (19). Electron microscopic observations by Barnett (2) of human marginal gingiva revealed mast cells within the epithelium. This suggests that these cells cannot be considered exclusively as connective tissue cells since they seem to migrate from the lamina propria into the epithelium. Uvnas (18) implicated mast cells in inflammation and discussed the factors influencing their degranulation. The granular content of mast cells can be released either by complete disruption and loss of granules (16) or with fragmentation of' only the granular constituents (17). Mycoplasma salivarium was identified by Engel and Kenny (6) as the predominant human gingival sulcular mycoplasmal species. It occurred in a significantly higher percentage of diseased than healthy subjects-86.7 versus 31.8% (14). Specifically, individuals with periodontal disease had a fourf'old greater incidence of sera positive to M. salivarium than did the control group. Because of' these observations, it was decided to investigate the response of connective tissue mast cells to local injections of homologous and heterologous mycoplasmal antigens in previously sensitized tissue. 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 (BBL) and the National 'Present address: University of Connecticut Health Center, Department of General Dentistry, Farmington, Conn. 06032. 5.)98

Institutes of Health. The growth precipitation test described by Krogsgaard-Jensen (13), which has a high degree of specificity, was periodically used to test culture purity. The basic medium used for culturing mycoplasms contained 80% PPLO broth or agar (BBL), 10% agamma horse serum (BBL), and 10% of 1% Albimi Laboratories yeast autolysate solution with 500 U of penicillin per ml and 250 mg of thallium acetate per liter. From the original culture, 10-ml aliquots were stored at -90 C for reinoculation and use. 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). Enumeration and viability of mycoplasmas were determined from broth and phosphate-buffered saline suspensions, which were serially diluted in PPLO broth free of additives and supplements and then plated on agar. The inoculated agar was incubated anaerobically at 37 C for 48 h and then inspected for colony-forming units of M. salivarium, using a Unitron inverted microscope at x75 magnif'ication. Six-week-old Swiss White mice of mixed sexes weighing approximately 20 g each were used for sensitization and controls throughout the experiment. The harvest of logarithmically growing M. salilarium organisms from 50 ml of' broth (approximately 5 x 108 to 109 organisms) was reconstituted into 0.5 ml of phosphatebuffered saline. This suspension read between 3 and 4 on the MacFarland scale and was introduced intraperitoneally by a series of' five injections of 0.5 ml each on alternate days. Eighteen days after sensitization, the sensitized and nonsensitized animals were injected subcutaneously on the dorsal surface with 1 ml of air to form an air pouch in the manner

VOL. 11, 1975

599

NOTES

described by Higginbotham et al. (9). The harvest of 25 ml of broth containing logarithmically growing mycoplasmas was reconstituted with the phosphate-buffered saline to form a 0.25-ml suspension for both whole-cell and homogenized cellular material for injection into the air enclave. Cells of the mycoplasmas were homogenized by adding glass beads of 0.11-mm diameter (Glasperlen) to the mycoplasmal suspensions. A ratio of 60% (wt/vol) beads to cells was placed in a 60-ml metal bottle, which was inserted in the socket of a Bronwill homogenizer. The homogenizer was run twice with liquid carbon dioxide for 2 min at 4,000 cycles. The suspension containing ruptured cells was poured into centrifuge tubes and centrifuged at 180 x g for 6 min to sediment the glass beads differentially from membranes or membraneous fractions. To insure absence of viable cells in the supernatant fluids used for challenge, serial dilutions were placed on agar disks, which were examined microscopically for absence of colony-forming units of M. salivarium. After killing of the animals, the enclosed bubble of connective tissue was dissected from the surrounding tissue and a portion of it was spread on a glass microscope slide (9). The tissue spread was air-dried and stained with May-Gruenwald-Giesma in the usual manner (4). The slides were examined with a ReichertZetopan microscope using x 100, x 250, and x400 magnification. Counts of degranulated and intact mast cells of the loose connective tissue of the 10 treatment groups were made. Mast cells within the loose connective tissue occurred in groups or aggregates and stained intensely with the dye used. Only cells occurring in groups and those showing a marked degree of degranulation were counted. A minimum of 200 mast cells was counted on each tissue spread and the percentage of degranulation calculated. Tissue reactions often obscured the degranulation of mast cells in sensitized animals after a few minutes. Therefore, tissue samples were obtained from each group of 10 mice 5 min after antigenic challenge. Intact mast cells predominated in control groups and in those groups challenged with heterologous antigenic preparations. The tissue mast cells of sensitized animals demonstrated widespread degranulation after injection of homologous antigen. The mean percentage of degranulation and standard errors of each of the treatment groups are recorded in Fig. 1. The sensitized group receiving whole-cell homologous antigen showed a

100 0

90

11

80 I '

70

° 60

50

C' X 40

i c 3030-

i 0

20

i

T

1

T

T

T

i

i

I

i

I

10

IC

2 IS

3 SC

4 SS

5 6 HSC HSS

7

a

mC

aS

9 10 HBC HIBS

Treatment

FIG. 1. Tissue mast cell degranulation of the 10 treatment groups. The first letter(s) refers to the challenging preparation (B, buffer; S, M. salivarium; HS, homogenized M. salivarium; HB, homogenized M. bovimastitis; the "B" in treatments 7 and 8 indicates M. bovimastitis). The last letter indicates the mouse group tested (C, control; S, sensitized).

marked degranulation of 74 ± 1.81%. This group demonstrated explosive degranulation and concurrent dispersion of granules into the adjacent ground substance, with few remaining intact mast cells. The disparity of the mean differences encountered between treatment 4 (viable whole-cell M. salivarium) and each of the other nine treatments is evident. The lowest mean percentage difference of 38.7 between treatments 4 and 6 (homogenized M. salivarium cells) suggested similar antigenic determinant groups. Most other mean differences to treatment 4 were near 50%. Although mast cell degranulation occurred in response to each stimulus, it was greater after homologous antigenic challenge. Mycoplasmal membrane-associated antigens appear to be important in the host response since the intact microbe produced more intense degranulation than other challenges. The second most marked degranulation occurred with homogenized M. salivarium. In this case the supernatant fluids lacked the capacity to develop colony-forming units on agar, indicating dissolution of membranes. These results tend to confirm earlier observations (1, 11) that the antigenic properties of mycoplasmas are located in the cell membrane and its fractions. Correlations between mast cell degranulation and periodontal disease have been made (7, 8,

600

NOTES

10, 12). This presumptive evidence in association with the observation that the immediate inflammatory response after an antigen-antibody reaction depended on the presence of tissue mast cells and histamine (5, 12) suggests that connective tissue and epithelial mast cells may function in the pathogenesis of periodontal disease. This pathosis would depend upon a sensitized host's response to the oral microbe. This investigation was supported by Public Health Service Fellowship Grants 1 F02 DE53063-01 and 5 F03 DE53063-02 from the National Institute of Dental Research. LITERATURE CITED 1. Argaman, M., and S. Razin. 1969. Antigenic properties of mycoplasma organisms and membranes. J. Gen. Microbiol. 55:45-58. 2. Barnett, M. L. 1973. Mast cells in the epithelial layer of human gingiva. J. Ultrastruct. Res. 43:247-255. 3. Clyde, W. A. 1964. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol. 92:958-965. 4. Dougherty, T. F., and G. L. Schneebeli. 1955. Use of steroids as anti-inflammatory agents. Ann. N.Y. Acad. Sci. 61:328-348. 5. Draper, L. R., and D. E. Smith. 1961. A function for tissue mast cells. Science 134:842. 6. Engel, D. L., and G. E. Kenny. 1970. Mycoplasma salivarium in human gingival sulci. J. Periodontal Res. 5:163-171. 7. Goldhaber, P. 1965. Heparin enhancement of factors stimulating bone resorption in tissue culture. Science 147:407-409.

INFECT. IMMUN.

8. Goldhaber, P. 1971. Tissue culture studies of bone as a model system for periodontal research. J. Dent. Res. 50:278-285. 9. Higginbotham, R. D., T. F. Dougherty, and W. S. S. Jee. 1956. Fate of shed mast cell granules. Proc. Soc. Exp. Biol. Med. 92:256-261. 10. Hook, W. A., R. Snyderman, and S. E. Mergenhagen. 1972. Further characterization of a factor from endotoxin-treated serum which releases histamine and heparin from mast cells. Infect. Immun. 5:909-814. 11. Kahane, I., and S. Razin. 1969. Immunological analysis of mycoplasma membranes. J. Bacteriol. 100:187-194. 12. Kaley, G., and R. Weiner. 1971. Prostaglandin E,: a potential mediator of the inflammatory response. Ann. N.Y. Acad. Sci. 180:338-350. 13. Krogsgaard-Jensen, A. 1972. Growth precipitation as a serodiagnostic method. J. Appl. Microbiol. 23:553-558. 14. Kumagai, K., T. Iwabuchi, Y. Hinuma, K. Yuri, and N. Ishida. 1971. Incidence, species, and significance of mycoplasma species in the mouth. J. Infect. Dis. 123:16-21. 15. Mergenhagen, S. E., and R. Snyderman. 1971. Periodontal disease: a model for the study of inflammation. J. Infect. Dis. 123:676-677. 16. Rohlich, P., P. Anderson, and B. Uvnas. 1971. Electron microscopic observations on compound 48/80-indirect degranulation in rat mast cells. J. Cell Biol. 51:465-483. 17. Taichman, N. S. 1971. Ultrastructural alterations in guinea pig mast cells during anaphylaxis. Int. Arch. Allergy Appl. Immunol. 40:934-942. 18. Uvnas, B. 1964. Release processes in mast cells and their activation by injury. Ann. N.Y. Acad. Sci. 161:880-890. 19. Weinstock, A., and J. T. Albright. 1967. The fine structure of mast cells in normal human gingiva. J. Ultrastruct. Res. 17:245-256.

Reactivity of mast cells to Mycoplasma salivarium.

Mice were sensitized to Mycoplasma salivarium and then challenged with heterologous and homologous antigens. The number of degranulating tissue mast c...
396KB Sizes 0 Downloads 0 Views