SCOPUS
KCI등재
항 거식세포(Macrophage)혈청이 결핵균 감염 및 동종 피부이식에 미치는 영향 = EFFECTS OF ANTIMACROPHAGE SERUM ON THE EXPERIMENTAL TUBERCULOSIS INFECTION AND SKIN ALLOGRAFT
저자
李英浩 (연세대학교 의과대학 성형외과학교실)
발행기관
大韓成形外科學會(The Korean Society of Plastic and Reconstructive Surgeons)
학술지명
권호사항
발행연도
1992
작성언어
Korean
KDC
514.251
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SCOPUS,KCI등재,ESCI
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학술저널
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896-916(21쪽)
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소장기관
In addition to the classic humoral immunity, increasing emphasis has been recently placed on the significance of cellular or cell-mediated immunity as the specific immunological responses of the host in a number of microbial infections and transplantation procedures. Cellmediatedimmunity can be divided into two subdivisions, i.e., antimicrobial cellular immunity and anti-tissue cellular immunity (Pearsall and Weiser, 1970) and extensive reviews on antimicrobial cellular immunity have been made by others(Mackaness and Blanden, 1967; Turk, 1967; Dannenberg, 1968).
It is generally accepted that macrophages are the effector cells in the antimicrobial cellular immunity of the host (Pearsall and Weiser, 1970). Intensive studies have been nade on the role of macrophages in virus infections(Mims, 1964 ; Johnson, 1964 ; Zisman et al., 1970 ; Hirsch et al., 1970;Stevens and Cook, 1971;Porter et al., 1969;Dabrowski et al., 1972;Zisman et al., 1971). The macrophages are also known to be the host cells for facultative and obligate intracellular bacteria (tuberculosis, listeria, brucella and leprosy) (Godal et al, 1971), and immunity to such infection with most intracelular bacterial pathogens is cellmediated and not determind by humoral antibodies(Suter and Ramisier, 1964).
Experimental evidences have been provided to support that macrophages play a major role in determining the course of tuberculosis infection of the host(Rich, 1951 ; Canetti, 1955 ; Lurie, 1964 ; Lurie and Dannenberg, 1965). In reviews on macrophage function in tuberculosis infection, Lurie and Dannenberg, 1965) considered resistance to be primarily a function of host macrophages, i.e., phagocytosis and early intracellular inactivation of the organisms, and that capability of macrophage determines the progress of tuberculosis infection.
A "multiple response" theory of immunity in tuberculosis was proposed by Youmans and Youmans(1969), i.e., 1) a nonspecific activation of RES by endotoxin-like component of bacilli, 2) specific immune response, mediated by an antibody, 3) granulomatous response caused mainly by macrophage and 4) each of this contributes to the total immunity of host. Dannenberg(1968)defined cellular immunity of the host in tuberculosis as a state in which macrophages have been activated, have proliferated and do possess and increased capacity to destroy tubercle bacilli.
through studies on experimental tuberculosis in rabbits inoculated with GCG intracutaneously, Ando et al.(1972) and Dannenberg et al. (1972) demonstrated that most of the mononuclear cells in tuberculous lesions were relatively recent immigrants and macrophages constantly enter and die in tuberculous lesions, and proposed that effective cellular immunity in tuberculosis might be accompanied by a high rate of minonuclear cell entry and a high rate of minonuclear cell activation.
Antimacrophage serun(AMS) was first prepared by Unanue (1968), and thereafter AMS has been widely utilized in studying the role of macrophage in immunological responses of the host (Panijel and Cayeux, 1968 ; Argyris and Plotkin, 1969 : Dyminski nad Argyris, 1969 ; Hirsch et al. 1969 ; Loewi et al, 1969).
Though immunosuppressive efects of antilymphocyte serum (ALS) and antithymocyte serum (ATS) have been rather well established, certain disagreements are noted in the immunosuppressive effects of AMS (Unanue, 1968;Loewi et al., 1969;Panijel and Cayeux, 1968;Argyris and Plotkin, 1969). At the same time a clear discrepancy does exist in the effect of AMS on skin allograft which represents a well-established model of cellular immunity of the host, particularly of anti-tissue cellular immunity(Gallily, 1971; Dyminski nad Argyris, 1969).
In spite of the fact that considerable numbers of studies have been made on in vivo and in vitro effects of AMS, reports of the effects of AMS on the establishment and the progress of microbial infection in experimental animal hosts remain markedly scanty(Hirsch et al, 1969 ; Boros and Warren, 1971).
Based on the facts that 1) macrophages are host cells for tubercle bacilli, 2) macrophages are the major defense cells of the host in the process of tuberculosis infection and 3) cellular immunity represents a principal immunologic response of the host in tuberculosis infection, studies were carried out in the mouse system to establish the effect of AMS an both experimental tuberculosis infection and skin allograft survival.
Materials and Methods
A. Animals :
Mice of A strain were used for the preparation of macrophage suspension, experimental tuberculosis infection and skin allograft experiment. Albino rabbits were used for preparation of antimacrophage serum.
B. Mycobacterium tuberculosis var, Hominis(H?R?):
Bacillary harvest of 3 weeks-culture on Ogawa media was used for experimental infection of mice.
C. Macrophage suspension:
By the method of dyminskim and argyris (1969), 0.2ml of thioglyocllate broth was inoculated intraperitioneally into old, multipara female nice and peritoneal macrophages were harvested by washing with NCTC 135-heparin mixture. An aliquot of pooled peritoneal washings was inoculated into tissue culture bottle and incubated for 1 to 1 1/2hr at 37℃ under 5% CO? atmosphere to facilitate adsorption of macrophages to glass surpace. Then, non-adsorbed cells were washed out with PBS and macrophage medium(NCTC 135 ; 50%, calf serum ; 40% and 1 : 5 dilution of bovine embryo extract ; 10%) was inoculated into culture bottles. After 24 hrs of incubation macrophage cultures were washed 3 times with cold PBS, and the cells were gently detached from glass surface by rubber policeman and suspended in PBS-heparin solution.
D. Preparation of antimacrophage serum:
The method by marsman et al. (1970) was followed. Briefly, consecutive 4 injections of mixture of macrophage suspension and Freund's incomplete adjuvant were made intramuscularly into rabbit at 1 week interval, and 1 week later macrophage suspension alone was injected intravenously as a final immunization. A total number of macrophages inoculated per rabbit was 8.4×10?, and the serum(AMS) was prepared by total bleeding 1 week after the last immunization.
E. Double diffusion in agar gel :
Modified Ouchterlony's method(1958) was applied, using 100mm petri dishes covered with 0.85% purified agar(Difco) in phospate buffered saline (pH 7.2). a concentrated macrophage suspension was sonicated(Ultrasonic prolce, Model BP-2, Fisher, U.S.A) and used as macrophage andtigen.
F. cytotoxicity test of AMS :
Cytotoxicity of AMS was tested by the method of Gallily(1971), using macrophage cells grown on cover slip in Leighton tube for 24 hrs at 37℃ under 5% CO? atmospher. The macrophage cells were treated for 1 hr with NCTC 135 containing 10% AMS and then guinea pig complement(1:30) was added at a final concentration of 5%. One hour after addition of the omplement, the macrophage cells were stained with 1% trypan blue and numbers of dead cells(trypan blue stanined) were counted among 200 macrophage cells and the cytoxicity of AMS was expressed in the percentage of dead cells.
G. White cell count pf peripheral blood :
Effect of AMS on the white cell count of peripheral blood was measured by the method of Gray et al.(1966) One-half ml of AMS was inoculated intraperitoneally into mice, and white cell kcounts were made at 0, 4, 8, 24 and 48hrs following aMS inoculation.
H. Histopathological observation :
Mice were inoculated intraperitoneally with 0.5ml of AMS and at 4, 24 and 48 hrs spleen, liver, lung, lymph node and thymus were excised, fixed with buffered formalin, paraffin embedded, sectioned and stained with hematoxylin-eosin.
Following challenge with tubercle bacilli, the organs of mice were carefully examined macroscopically to identify tubercle formation, adhesion and swellings of lymph nodes at the time of sacrifice. A portion of the organs was used for histopathological observation and the other for bacteriological assessment.
1. Bacteriological assessment :
At 7, 14, 21, and 28 days following challenge with tubercle bacilli, a number of mice were sacrificed and bacteriological assessment of spleen, liver, lung and kidney was conducted by countion a total number of acid-fast bacilli in kthe smerat(1㎠)made with one ollpful(ca. 0.02ml) of the homogenate of respective organ.
J. X-ray total body irradiation :
A number of mice were caged in a Lucite box and total body irradiation was made by G-E, Maxima-Ⅲ(250Kv, 15mA), ranging 400 to 900r, and LD? by 4 weeks was calculated. Mice which received total body irradiation of the dose of LD? by 4 weeks were used for challenge with tubercle bacilli and for skin allograft experiment.
K. Skin allograft:
Skin transplantation was done by the method of Thoenes(1969). A protective bandage was removed 5 days transplantation, and survival time was meastred by the time of complete rejection of transplanted skin.
Conclusion
To establish the effects of antimacrophage serum(AMS) on tuberculosis infection and skin allograft in mice, a series of experiment was conducted, and the results are summarized as follows;
1. At least 3 precipitation lines were identified between AMS and macrophage antigen by agar gel diffusion test. Among them, only one precipitation line appeated to be macrophagespecific and the other two shared common antigenictiy with those of lymphoid cell antigen.
2. In AMS-treated mice, changes in white cell counts of peripheral blood and pathological findings of the spleens resembled closely other's obserations.
3. By bacteriological assessment of the infected organs9spleen, liver, lung and kidney) of mice inoculated with tubercle bacilli, it was observed that administration of AMS(day before the challenge with tubercle bacilli) accelerated the development an
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