Brief Communication: Chemotactic Response of Leukocytes to Cord Factor (Trehalose-6 ,6'dimycolate) 1, 2 I. Ofek and A. Bekierkunst 3, 4 ABSTRACT-Cord factor (trehalose-6,6' -dimycolate), a glycolipid extractable from mycobacteria, was chemotactic for peritoneal cells of mice at concentrations from 5 to 25 "g/ml medium, as well as for peripheral white blood cells of mice and humans. At higher concentrations, presumably only macrophages were attracted.-J Natl Cancer Inst 57: 1379-1381, 1976.
Intravenous injections of cord factor (trehalose-6,6'dimycolate), a glycolipid extractable from mycobacteria (1, 2), can induce in the lungs of mice a typical granulomatous response indistinguishable in its cellular composition from that caused by living BCG. In both instances, the predominant cells were macrophages, lymphocytes, and epithelioid cells (3). Injected into the footpads of mice, cord factor induced in the draining lymph nodes histologic changes similar to those following administration of living BCG by the same route (4). The adjuvant and antitumor activity of living BCG, killed BCG, cord factor, and other mycobacterial fractions are apparently related to the inflammatory chronic granulomatous reaction of the host. This reaction, which develops at the site where these agents lodge, may take place in the skin (5), lymph nodes (4, 6-8), lungs, or peritoneal cavity (9,10). However, to achieve a strong stimulation of the immune response against unrelated antigens, among them weak tumor-specific antigens, the latter have to be at the same site where large numbers of macrophages and lymphocytes are attracted and acted upon by the BCG cells (5). Our results showed that cord factor strongly attracted peritoneal cells of mice and white blood cells of mice and humans. MATERIALS AND METHODS
Animals.-Albino mice about 10 weeks old, used as a source for peritoneal cells, were supplied by the Animal Production Section, Hebrew University-Hadassah Medical School. Preparation of peritoneal cell suspensions from mice . -The cells were collected from the peritoneal cavities of mice killed by ether after ip injection of 5 ml Hanks' solution or medium 199 (Microbiological Associates be., Bethesda, Md.) containing per ml: 8 U heparin, 100 U penicillin, and 100 j.tg streptomycin. The cells, pooled from several mice, were centrifuged at 1,000 rpm for 10 minutes at room temperature and were washed twice with media without heparin. The number was adjusted to 14x 106 cells/ml, after having been counted in a hemacytometer. The differential counts of cells, stained by Giemsa, showed about 90% mononuclear cells (lymphocytes and macrophages) and about 10% polymorphonuclear leukocytes. Cell viability, revealed by the try pan blue dye exclusion test, was usually about 98%. Chamber for chemotaxis and evaluation of chemotactic activVOL. 57, NO.6, DECEMBER 1976
ity.-The chemotactic activity was tested in a chamber similar to that used by Boyden (11). The lower compartment consisted of the lower part of a small plastic cup with the following dimensions: inner diameter of the bottom, 10 mm; open top part, 11.6 mm; and height, 23 mm. The upper compartment consisted of a thickwalled glass tube 22 mm in length and 11 mm in its outer diameter. The upper compartment was adjusted to the lower compartment and separated from the lower compartment by a 5-j.t polycarbon membrane filter (Nucleopore Corp., Pleasanton, Calif.) fixed to the thick edges of the tube with stopcock grease (Dow Corning Corp., Midland, Mich.). The chemotactic agent in the lower com partment and the cell suspension in the upper compartment were incubated at 37° C in an atmosphere of 95% air and 5% CO 2 for 3 hours. After incubation andremoval of the fluid from the upper compartment, the membrane was placed in a petri dish with methanol for 5 minutes. After fixation of the cells and drying of the membranes, the migrated cells were stained with Giemsa. The stained filters were dipped in xylene and fixed to microscope slides with a drop of Cover Bond synthetic resin (Hartman-Leddon Co., Philadelphia, Pa.). On top of the filter another drop of resin was added and a cover slide was attached. The migration of cells was evaluated by counting under oil immersion the cells in 10 fields on the site of the filter that faced the lower compartment of the chamber. Preparation of suspension of human and mouse white blood cells .-The white blood cells were separated from heparinized blood of a normal human donor by sedimentation of whole blood with 6% dextran, as previously described (12). The separated cells were washed three times in medium 199 and adjusted to a suspension containing 7 X 106 cells/ml. White blood cells of mice were prepared in the same way; the suspension contained 3 X 106 cells/ml. Preparation of emulsions of cord factors.-Chromatographically pure cord factors from Mycobacterium tuberculosis strain Peurois (a human virulent strain), M. bovis AN5, and M. smegmatis were prepared as described by Bekierkunst et al. (3). The cord factors were weighed, placed in a glass tube, and homogenized in saline containing 0.2% Tween 80 and with a Teflon pestle. UsuReceived March 30, 1976; accepted May 10, 1976. Supported by a grant from the Joint Research Fund of the Hebrew University and Hadassah and by the Cure Research Fellowship in memory of Roda and Henry Diamond. 3 Laboratory of Medical Bacteriology, Hebrew University-Hadassah Medical School, Jerusalem, Israel. 4 We thank Dr. E. Lederer (Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France) for the preparation and generous supply of cord factors.
1379
1
2
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NATL CANCER INST
1380
OFEK AND BEKIERKUNST 70
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TEXT-FIGURE 2.-Chemotaxis of peripheral white blood cells of humans ( _ e ) and mice (0-----0). See legend of text-fig. 1 for explanation.
lymphocyte and complement-derived stimuli were recently observed (13). If cord factor displays in vivo an activity similar to that shown in vitro, explainable would be its role in the granulomagenic, adjuvant, and antitumor activities, all of which are interrelated. REFERENCES (1) BLOCH H: Studies on the virulence of tubercle bacilli. Isolation
and biological properties of a constituent of virulent organisms. j Exp Med 91:197-218,1950 (2) NOLL H, BLOCH H, ASSELINEAU F, et al: The chemical structure of the cord factor of Mycobacterium tuberculosis. Biochim Biophys Acta 20:299-309, 1956 (3) BEKIERKUNST A, LEVI] ]S, YARKONI E, et al: Granuloma formation induced in mice by chemically defined mycobacterial fractions.] Bacteriol 100:95-102, 1969 (4) BEKIERKUNST A, LEVI] jS, YARKONI E, et al: Cellular reaction in the footpad and draining lymph nodes of mice induced by mycobacterial fractions and BCG bacilli. Infect Immun 4:245255, 1971 (5) BEKIERKUNST A, WANG L, TOUBIANA R, et al: Immunotherapy of cancer with nonliving BCG and fractions derived from mycobacteria: Role of cord factor (trehalose-6,6' -dimycolate) in tumor regression. Infect Immun 10: 1044-1050, 1974 (6) BEKIERKUNST A, YARKONI E, FLECHNER j, et al: Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions. Infect Immun 4:256-263, 1971 (7) MACKANESS GB, AUCLAIR Dj, LAGRANGE PH: Immunopotentiation with BCG. I. Immune response to different strains and preparations. j Nat! Cancer Inst 51: 1655-1667, 1973 (8) MILLER FE, MACKANESS GB, LAGRANGE PH: Immunopotentiation with BCG. II. Modulation of the response to sheep red blood cells. j Nat! Cancer Inst 51:1669-1676,1973 (9) BEKIERKUNST A, LEVI] ]S, YARKONI E, et al: Suppression of urethan-induced lung adenomas in mice treated with trehalose-6,6'-dimycolate (cord factor) and living bacillus CalmetteGuerin. Science 174:1270-1272, 1971 (10) YARKONI E, WANG L, BEKIERKUNST A: Suppression of growth of Ehrlich's ascites tumor cells in mice by trehalose-6,6'-dimycolate (cord factor) and BCG. Infect Immun 9:977-984, 1974 (11) BOYDEN SV: The chemotactic effect of mixtures of antibody and
VOL. 57, NO.6, DECEMBER 1976
1381
CORD FACTOR-A CHEMOTACTIC AGENT
antigen on polymorphonuclear leucocytes. J Exp Med 115:453, 1962
(12) OFEK I, BEACHEY EH, BISNO AL: Resistance of Neisseria gonorrhoeae to phagocytosis: Relationship to colonial morphology
VOL. 57, NO.6, DECEMBER 1976
and surface pili. J Infect Dis 129:310, 1974 (13) BOUMSELL L, MELTZER MS: Mouse mononuclear cell chemotaxis. I. Differential response of monocytes and macrophages. J Immunol 115:1746, 1976
J
NATL CANCER INST
Intravenous injections of cord factor (trehalose-6,6'dimycolate), a glycolipid extractable from mycobacteria (1, 2), can induce in the lungs of mice a typical granulomatous response indistinguishable in its cellular composition from that caused by living BCG. In both instances, the predominant cells were macrophages, lymphocytes, and epithelioid cells (3). Injected into the footpads of mice, cord factor induced in the draining lymph nodes histologic changes similar to those following administration of living BCG by the same route (4). The adjuvant and antitumor activity of living BCG, killed BCG, cord factor, and other mycobacterial fractions are apparently related to the inflammatory chronic granulomatous reaction of the host. This reaction, which develops at the site where these agents lodge, may take place in the skin (5), lymph nodes (4, 6-8), lungs, or peritoneal cavity (9,10). However, to achieve a strong stimulation of the immune response against unrelated antigens, among them weak tumor-specific antigens, the latter have to be at the same site where large numbers of macrophages and lymphocytes are attracted and acted upon by the BCG cells (5). Our results showed that cord factor strongly attracted peritoneal cells of mice and white blood cells of mice and humans. MATERIALS AND METHODS
Animals.-Albino mice about 10 weeks old, used as a source for peritoneal cells, were supplied by the Animal Production Section, Hebrew University-Hadassah Medical School. Preparation of peritoneal cell suspensions from mice . -The cells were collected from the peritoneal cavities of mice killed by ether after ip injection of 5 ml Hanks' solution or medium 199 (Microbiological Associates be., Bethesda, Md.) containing per ml: 8 U heparin, 100 U penicillin, and 100 j.tg streptomycin. The cells, pooled from several mice, were centrifuged at 1,000 rpm for 10 minutes at room temperature and were washed twice with media without heparin. The number was adjusted to 14x 106 cells/ml, after having been counted in a hemacytometer. The differential counts of cells, stained by Giemsa, showed about 90% mononuclear cells (lymphocytes and macrophages) and about 10% polymorphonuclear leukocytes. Cell viability, revealed by the try pan blue dye exclusion test, was usually about 98%. Chamber for chemotaxis and evaluation of chemotactic activVOL. 57, NO.6, DECEMBER 1976
ity.-The chemotactic activity was tested in a chamber similar to that used by Boyden (11). The lower compartment consisted of the lower part of a small plastic cup with the following dimensions: inner diameter of the bottom, 10 mm; open top part, 11.6 mm; and height, 23 mm. The upper compartment consisted of a thickwalled glass tube 22 mm in length and 11 mm in its outer diameter. The upper compartment was adjusted to the lower compartment and separated from the lower compartment by a 5-j.t polycarbon membrane filter (Nucleopore Corp., Pleasanton, Calif.) fixed to the thick edges of the tube with stopcock grease (Dow Corning Corp., Midland, Mich.). The chemotactic agent in the lower com partment and the cell suspension in the upper compartment were incubated at 37° C in an atmosphere of 95% air and 5% CO 2 for 3 hours. After incubation andremoval of the fluid from the upper compartment, the membrane was placed in a petri dish with methanol for 5 minutes. After fixation of the cells and drying of the membranes, the migrated cells were stained with Giemsa. The stained filters were dipped in xylene and fixed to microscope slides with a drop of Cover Bond synthetic resin (Hartman-Leddon Co., Philadelphia, Pa.). On top of the filter another drop of resin was added and a cover slide was attached. The migration of cells was evaluated by counting under oil immersion the cells in 10 fields on the site of the filter that faced the lower compartment of the chamber. Preparation of suspension of human and mouse white blood cells .-The white blood cells were separated from heparinized blood of a normal human donor by sedimentation of whole blood with 6% dextran, as previously described (12). The separated cells were washed three times in medium 199 and adjusted to a suspension containing 7 X 106 cells/ml. White blood cells of mice were prepared in the same way; the suspension contained 3 X 106 cells/ml. Preparation of emulsions of cord factors.-Chromatographically pure cord factors from Mycobacterium tuberculosis strain Peurois (a human virulent strain), M. bovis AN5, and M. smegmatis were prepared as described by Bekierkunst et al. (3). The cord factors were weighed, placed in a glass tube, and homogenized in saline containing 0.2% Tween 80 and with a Teflon pestle. UsuReceived March 30, 1976; accepted May 10, 1976. Supported by a grant from the Joint Research Fund of the Hebrew University and Hadassah and by the Cure Research Fellowship in memory of Roda and Henry Diamond. 3 Laboratory of Medical Bacteriology, Hebrew University-Hadassah Medical School, Jerusalem, Israel. 4 We thank Dr. E. Lederer (Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France) for the preparation and generous supply of cord factors.
1379
1
2
J
NATL CANCER INST
1380
OFEK AND BEKIERKUNST 70
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TEXT-FIGURE 2.-Chemotaxis of peripheral white blood cells of humans ( _ e ) and mice (0-----0). See legend of text-fig. 1 for explanation.
lymphocyte and complement-derived stimuli were recently observed (13). If cord factor displays in vivo an activity similar to that shown in vitro, explainable would be its role in the granulomagenic, adjuvant, and antitumor activities, all of which are interrelated. REFERENCES (1) BLOCH H: Studies on the virulence of tubercle bacilli. Isolation
and biological properties of a constituent of virulent organisms. j Exp Med 91:197-218,1950 (2) NOLL H, BLOCH H, ASSELINEAU F, et al: The chemical structure of the cord factor of Mycobacterium tuberculosis. Biochim Biophys Acta 20:299-309, 1956 (3) BEKIERKUNST A, LEVI] ]S, YARKONI E, et al: Granuloma formation induced in mice by chemically defined mycobacterial fractions.] Bacteriol 100:95-102, 1969 (4) BEKIERKUNST A, LEVI] jS, YARKONI E, et al: Cellular reaction in the footpad and draining lymph nodes of mice induced by mycobacterial fractions and BCG bacilli. Infect Immun 4:245255, 1971 (5) BEKIERKUNST A, WANG L, TOUBIANA R, et al: Immunotherapy of cancer with nonliving BCG and fractions derived from mycobacteria: Role of cord factor (trehalose-6,6' -dimycolate) in tumor regression. Infect Immun 10: 1044-1050, 1974 (6) BEKIERKUNST A, YARKONI E, FLECHNER j, et al: Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions. Infect Immun 4:256-263, 1971 (7) MACKANESS GB, AUCLAIR Dj, LAGRANGE PH: Immunopotentiation with BCG. I. Immune response to different strains and preparations. j Nat! Cancer Inst 51: 1655-1667, 1973 (8) MILLER FE, MACKANESS GB, LAGRANGE PH: Immunopotentiation with BCG. II. Modulation of the response to sheep red blood cells. j Nat! Cancer Inst 51:1669-1676,1973 (9) BEKIERKUNST A, LEVI] ]S, YARKONI E, et al: Suppression of urethan-induced lung adenomas in mice treated with trehalose-6,6'-dimycolate (cord factor) and living bacillus CalmetteGuerin. Science 174:1270-1272, 1971 (10) YARKONI E, WANG L, BEKIERKUNST A: Suppression of growth of Ehrlich's ascites tumor cells in mice by trehalose-6,6'-dimycolate (cord factor) and BCG. Infect Immun 9:977-984, 1974 (11) BOYDEN SV: The chemotactic effect of mixtures of antibody and
VOL. 57, NO.6, DECEMBER 1976
1381
CORD FACTOR-A CHEMOTACTIC AGENT
antigen on polymorphonuclear leucocytes. J Exp Med 115:453, 1962
(12) OFEK I, BEACHEY EH, BISNO AL: Resistance of Neisseria gonorrhoeae to phagocytosis: Relationship to colonial morphology
VOL. 57, NO.6, DECEMBER 1976
and surface pili. J Infect Dis 129:310, 1974 (13) BOUMSELL L, MELTZER MS: Mouse mononuclear cell chemotaxis. I. Differential response of monocytes and macrophages. J Immunol 115:1746, 1976
J
NATL CANCER INST
