Vol. 11, No. 3

INFECTION AND IMMUNrrY, Mar. 1975, p. 595-597 Copyright 0 1975 American Society for Microbiology

Printed in U.S.A.

Histamine Release from Human Leukocytes When Stimulated by Mycoplasma salivarium CLAYTON F. PARKINSON' Department of Biology, Utah State University, Logan, Utah 84322 Received for publication 11 September 1974

Incubated mixtures of Mycoplasma salivarium and nonsensitized mixed leukocytes released histamine. Spectrophotofluorometric detection of histamine occurred at physiological temperature and pH independent of complement and specific antiserum.

As early as the 1950s, it was postulated that the inflammatory response of tissue is mediated by histamine (5). Numerous antigenic substances have been shown to stimulate the release of histamine from leukocytes (7, 11-13, 15, 21). Each of these investigations found natural sensitization or preincubation of the leukocytes with sera from sensitive donors or anti-immunoglobulin E essential to cause leukocytic histamine release. Recently, lysates of mixed human leukocytic suspensions were found to release histamine from intact human leukocytes (8). Basophilic granulocytes are the major source of histamine in humans (6, 7, 17). Inflammatory reactions (9) and traumatic responses (19) cause extravascular migrations of basophils to sites of injury. These phenomena invite speculation that Mycoplasma salivarium, as it is phagocytized by human leukocytes (16), stimulates histamine release from leukocytes that are known to migrate to inflamed periodontal tissues (22). This is important since observations indicate M. salivarium to be the predominant mycoplasmal sulcular inhabitant in inflammatory gingivitis (4, 18). The present report indicates that prior leukocytic sensitization is not required for histamine release. In addition, explications for this observation are given. 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 (2), using paper disks saturated with homologous and heterologous antimycoplasmal sera (BBL). The medium used in culturing the mycoplasmas contained 8 volumes of PPLO broth or agar 'Present address: University of Connecticut Health Center, Department of General Dentistry, Farmington, Conn. 06032.

(BBL), 1 volume of agamma horse serum (BBL), and 1 volume of a 1% Albimi Laboratories yeast autolysate solution with 500 U of penicillin per ml and 250 mg of thallium acetate per liter. 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, phosphate-buffered saline suspensions, or incubation mixtures taken at various times, serially diluted in PPLO broth free of additives and supplements, and then plated on agar. The inoculated agar was incubated under CO2 at 37 C for 48 h and was then inspected for colony-forming units (CFU) of M. salivarium per milliliter by using a Unitron inverted microscope at x75 magnification. Human blood without serum growth-inhibiting antibody to M. salivarium was collected in plastic 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. (1) as reported by Kumagai et al. (10). The tested sera were considered without antibody when CFU of M. salivarium per milliliter were greater than nine-tenths of the control. The buffy coat and plasma were aspirated after sedimentation of erythrocytes with a plastic pipette and centrifuged at 180 x g at 4 C for 8 min. The button of cells containing the leukocytes was gently washed twice and resuspended in tris(hydroxymethyl)aminomethane buffer at pH 7.3 at 37 C to 1.5 ± 0.3 x 107 leukocytes per ml. Washed leukocytes were considered nonsensitized if they were derived from human blood in

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which the serum was unable to prevent colony formation of M. salivarium. The characteristics of cells were checked by microscopy. The preparation of incubation mixtures in polycarbonate tubes consisted of 1 ml of leukocytes, 1 ml of mycoplasmas of various concentrations, and 2 ml of buffer and essentially followed the protocol of Lichtenstein and Osler (13). The preparations and controls were incubated for 2 h at 37 C in an environmental shaker rotating at 150 rpm. After incubation, the reactive mixtures were centrifuged to sediment both mycoplasmas and leukocytes and each tube was deproteinized except a tube containing only leukocytes and buffer, which supplied the value for the total (complete) extractable histamine. Due to variations in the histamine content of cells derived from different donors, the results were plotted in terms of percentage of histamine released. The percentage of histamine released is equal to the specific relative fluorescent intensity less the spontaneous histamine release and reagent fluorescence from a control tube (blank) of leukocytes without mycoplasmas divided by the fluorescence from the "complete" less the "blank" times 100. Leukocytes remained viable for the duration of the experiments, but the CFU of M. salivarium per milliliter were fewer at the conclusion than at the beginning of experimentation. The leukocytes released part of their histamine content in the presence of 5, 50, or 100 mycoplasmas per leukocyte. Increased numbers of M. salivarium per leukocyte resulted in greater histamine release, and the dose response curve is presented in Fig. 1. The detected histamine release with 5 mycoplasmas per leukocyte was approximately one-half that detected when the ratio was increased to 100 mycoplasmas per leukocyte. This preliminary study shows that M. salivarium stimulated histamine release from mixed human nonsensitized leukocytes. Further investigation is needed and will be initiated to elucidate this phenomenon, but preliminary explanations are required at this time. This effect may be explained by nonspecific sensitivity and finds supportive evidence in the studies of May and Williams (14). Their investigations of unrelated allergens indicated nonspecific sensitivity by degranulating basophils in releasing histamine. Another explanation for the release of histamine may be caused by residual or undetected immunological reactions. The leukocytes used in these experiments were derived from donors whose sera did not

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FIG. 1. Percentage of histamine released by human leukocytes when incubated with increasing mycoplasmas for 2 h. Mean values of three determinations from different donors are recorded.

inhibit colony formation by M. salivarium on agar. This method, although used for confirmation of presence of mycoplasmal species (1, 2), may be insensitive to small amounts of antiserum. Reasonable explication for the detected histamine in this system may reside in factors associated with phagocytosis. The basis for this postulate is derived from ultrastructure studies by Parkinson and Carter (16), which show phagocytosis of M. salivarium with accompanying degranulation of leukocytes. During phagocytosis, Cohn and Hirsch (3) observed the release of lysosomal enzymes from cytoplasmic granules. Kelly et al. (8) demonstrated that lysosomal lysates from leukocytes contained significant histamine-releasing activity for leukocytes. The 2-h incubation of the reaction mixture allowed in this study provides adequate time for the release of lysosomal content (16). Therefore, it may be that the detected histamine occurred secondarily to factors associated with in vitro phagocytosis of M. salivarium. Since phagocytosis of M. salivarium decreases the number of mycoplasmas, all detectable levels of histamine would have to be considered with respect to the phagocytic process. The lower percentage of histamine released with fewer numbers of mycoplasmal organisms is presumably a dilution factor. At any concentration of mycoplasmas to leukocyte, histamine might be released but remain unavailable for

VOL. 11, 1975

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assay because of instability or destruction. This study is valuable because the in vitro system used to detect histamine release involved human leukocytes. This eliminated difficulties in crossing species and examined the effects of a known human oral microbial inhabitant on human cells. It is not untenable that released histamine from a few leukocytes in vivo might increase concentrations sufficient to mobilize additional histamine from neighboring blood leukocytes and potentiate the inflammatory process. 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. Bailey, J. S., H. W. Clark, W. R. Felts, R. C. Fowler, and T. M. Brown. 1961. Antigenic properties of pleuropneumonia-like organisms from tissue cell cultures and the human genital area. J. Bacteriol. 82:542-547. 2. Clyde, W. A. 1964. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol. 92:958-965. 3. Cohn, Z. A., and J. G. Hirsch. 1960. The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leukocytes. J. Exp. Med. 112:983-1004. 4. Engel, D. L., and G. E. Kenny. 1970. Mycoplasma salivarium in human gingival sulci. J. Periodontal Res. 5: 163-171. 5. Eyring, H., and T. F. Dougherty. 1955. Molecular mechanisms in inflammation and stress. Am. Sci. 43:457-467. 6. Graham, H. T., 0. H. Lowry, F. Wheelwright, M. A. Lenz, and H. H. Parish. 1955. Distribution of histamine among leukocytes and platelets. Blood 10:467-481. 7. Ishizaka, T., R. DeBernardo, H. Tomioka, L. M. Lichtenstein, and K. Ishizaka. 1972. Identification of basophil granulocytes as a site of allergic histamine release. J. Immunol. 108:1000-1008. 8. Kelly, M. T., R. R. Martin, and A. White. 1971. Mediators of histamine release from human platelets, lymphocytes, and granulocytes. J. Clin. Invest. 50:1044-1049.

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9. Kimura, I., M. Yoshiaki, T. Yoshiro, S. Katsuyashi, T. Kiyoshi, V. Nobuo, and S. Shuichi. 1973. Extravascular migration of basophils; particularly at the site of allergic reactions. Acta Haematol. Jap. 36:93-97. 10. 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. 11. Levy, D. A., and A. G. Osler. 1966. Studies on the mechanism of hypersensitivity phenomena. XIV. Passive sensitization in vitro of human leukocytes to ragweed pollen antigen. J. Immunol. 97:203-212. 12. Lichtenstein, L. M. 1971. The immediate allergic response in vitro separation of antigen activation, decay and histamine release. J. Immunol. 107:1122-1130. 13. Lichtenstein, L. M., and A. G. Osler. 1964. Studies on the mechanism of hypersensitivity phenomena. IX. Histamine release from human leukocytes by ragweed pollen antigen. J. Exp. Med. 120:507-530. 14. May, C. D., and C. S. Williams. 1973. Further studies on concordant fluctuation in sensitivity of peripheral leucocytes to unrelated allergens and the meaning of nonspecific desensitization. Clin. Allergy 3:319-337. 15. Middleton, E. 1960. In vitro passive transfer of atopic hypersensitivity. Proc. Soc. Exp. Biol. Med. 104:245-247. 16. Parkinson, C. F., and P. B. Carter. Phagocytosis of Mycoplasma salivarium by human polymorphonuclear leukocytes and monocytes. Infect. Immun. 11:405-414. 17. Pruzansky, J. J., and R. Patterson. 1970. Histamine in human leukocytes. Int. Arch. Allergy Appl. Immunol. 37:98-103. 18. Razin, S., J. Michmann, and Z. Shimshoni. 1964. The occurrence of mycoplasma (pleuropneumonia-like organisms) (PPLO) in the oral cavity of dentulous and edentulous subjects. J. Dent. Res. 43:402-405. 19. Reite, 0. B. 1973. Redistribution of tissue histamine stores (basophil leucocytes) of turtles in response to submersion and cold exposure. Acta Physiol. Scand. 88:62-66. 20. Shore, P. A., A. Burkhalter, and V. H. Cohn. 1959. A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther. 127:182-187. 21. Van Arsdel, P. P., and C. J. Sells. 1963. Antigenic histamine release from passively sensitized human leukocytes. Science 141:1190-1191. 22. Woolweaver, D. A., G. G. Koch, J. J. Crawford, and R. L. Lundblad. 1972. Relation of the orogranulocytic migratory rate to periodontal disease and blood leukocyte count. J. Dent. Res. 51:929-939.

Histamine release from human leukocytes when stimulated by Mycoplasma salivarium.

Vol. 11, No. 3 INFECTION AND IMMUNrrY, Mar. 1975, p. 595-597 Copyright 0 1975 American Society for Microbiology Printed in U.S.A. Histamine Release...
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