Effects of antimacrophage serum on the experimental tuberculosis infection and skin allograft
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. Cell-mediated immunity 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 made 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 intracellular bacterial pathogens is cell-mediated and not determind by humoral antibodies (Suter and Ramsier, 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 an increased capacity to destroy tubercle bacilli.
Through studies on experimental tuberculosis in rabbits inoculated with BCG intracutaneously, Ando et al. (1972) and Dannenberg et al. (1972) demonstrated that most of the mononuclear cells in tuberculous lesions ware 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 mononuclear cell entry and a high rate of mononuclear cell activation.
Antimacrophage serum (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 and Argyris, 1969; kirsch et al., 1969; Loewi et al, 1969).
Though immunosuppressive effects 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 and 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 Warrren, 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 on both experimental tuberculosis infection and skin allograft survival.
Materials and Methods
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^^37 Rv):
Bacillary harvest of 3 weeks-culture on Ogawa media was used for experimental infection of mice.
C. Macrophage suspension:
By the method of Dyminski and Argyris (1969), 0.2 ml of thioglycollate broth was inoculated intraperitoneally into old, multipara female mice 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 ½hr at 37℃ under 5% CO^^2 atmosphere to facilitate adsorption of macrophages to glass surface. 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 sarum:
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**7, 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 100 mm petri dishes covered tilth 0.85% purified agar(Difco) in phosphate buffered saline (pH 7.2). A concentrated macrophage suspension was sonicated (Ultrasonic probe, Model BP-2, Fisher, U.S.A.) and used as macrophage antigen.
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^^2 atmosphere. The macrophase 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 complement, the macrophage cells were stained with 1% trypan blue and numbers of dead cells (trepan blue stained) were counted among 200 macrophage cells and the cytoxicity of AMS was expressed in the percentage of dead cells.
G. White cell count of 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 counts were made at 0,4,8,24 and 48 hrs 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.
I. 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 counting a total number of acid -fast bacilli in the smear(1㎠) made with one loopful(ca. 0.02m1) 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 (250 Kv, 15mA), ranging 400 to 900 r, and LD^^50 by 4 weeks was calculated. Mice which received total body irradiation of the dose of LD^^50 by 4 wee17s were used forchallenge 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 after transplantation, and survival time was measured by the time of complete rejection of transplanted skin.
To establish the effects of antimacrophage serum (AMS) on tuberculosis infection and skin allograft in mice, a saries 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 afar gel diffusion Among them, only precipitation line appeared to be macrophage-specific and the other two shared common antigenicity 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 organs (spleen, 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 and intensified the extent of tuberculosis infection.
4. X_ray total body irradiation (458r=LD^^50 by 4 weeks), both before and after challenge with tubercle bacilli, of mice failed to affect the development and progress of tuberculosis infection.
5. Treatment with AMS significantly prolonged survival time of skin allograft and this effect was similar to those by X-ray total body irradiation.