Vol. 58, No. 8
INFECTION AND IMMUNITY, Aug. 1990, p. 2675-2677 0019-9567/90/082675-03$02.00/0 Copyright © 1990, American Society for Microbiology
Activation of Tuberculostatic Macrophage Functions by Gamma Interferon, Interleukin-4, and Tumor Necrosis Factor E. A. FLESCH AND S. H. E. KAUFMANN* Department of Medical Microbiology and Immunology, University of Ulm, Albert-Einstein-Allee 11, D-7900 Ulm, Federal Republic of Germany I.
Received 27 December 1989/Accepted 8 May 1990
The capacity of the cytokines gamma interferon (IFN-'y), interleukin-4 (IL-4), and tumor necrosis factor (TNF) to activate tuberculostatic functions in murine bone marrow-derived macrophages (BMMo) was investigated. In confirmation of earlier findings, IFN--y rendered BMM+ capable of inhibiting subsequent infection with Mycobacterium bovis. In contrast, IL-4 and TNF failed to inhibit mycobacterial growth. However, in already infected BMM+, tuberculostasis was induced by subsequent stimulation with IL-4. Although TNF alone was ineffective, it showed synergy with IFN-y in the stimulation of tuberculostasis. Our data suggest that not only IFN-y but also IL-4 and TNF participate in the control of mycobacterial growth.
bacteria was determined by [3H]uracil uptake. It is our experience that an 80 to 95% reduction of [3H]uracil uptake reflects a 10- to 100-fold decrease in the number of intracellular bacteria as determined by CFU (7). In another approach, BMM4X (105 per well) were infected with M. bovis (106 per well) for 18 h, washed thoroughly to remove nonphagocytized bacteria, and subsequently stimulated with lymphokines. After 40 h of incubation, BMM4 were lysed with 0.1% saponin and the viability of intracellular bacteria was assessed by [3H]uracil uptake (8). Since the cells were
Mononuclear phagocytes represent the major habitat of the etiologic agents of tuberculosis, Mycobacterium tuberculosis and Mycobacterium bovis. According to current concepts, acquired resistance against tuberculosis primarily depends on macrophage activation by cytokines from specific T lymphocytes (2), and it is assumed that gamma interferon (IFN-y) is of particular relevance (10). However, cytokines other than IFN--y have recently been shown to activate certain macrophage functions (1, 3, 9, 11). We compared the roles of interleukin-4 (IL-4), tumor necrosis factor (TNF), and IFN--y in the activation of tuberculostasis in murine bone marrow-derived macrophages (BMM4O). In all experiments, serum-free and antibiotic-free media were used for cultivation as well as stimulation and infection of the cells. Bone marrow cells were prepared from the femora of 6- to 8-week-old C57BL/6 male or female mice which were bred under specific-pathogen-free conditions. BMMX were obtained in Teflon film bags (Heraeus, Hanau, Federal Republic of Germany) and harvested after 9 to 10 days of culture as described previously (6, 7). Viability as assessed by trypan blue exclusion was >90%. M. bovis BCG Phipps was grown with shaking in Dubos broth (Difco Laboratories, Detroit, Mich.) supplemented with bovine serum albumin and Tween 20. Numbers of viable organisms were determined by plating 1/10 dilutions on Middlebrook Dubos agar plates (Difco). Murine recombinant IFN--y (rIFN--y) was produced by Genentech Inc. and kindly supplied by Boehringer Ingelheim (Ingelheim, Federal Republic of Germany). Murine rIL-4 was purchased from Genzyme Corp. (Boston, Mass.). rTNF-a was a kind gift from BASF (Ludwigshafen, Federal Republic of Germany). Cytokines were free of detectable endotoxin contamination as determined by the Limulus amoebocyte lysate assay (Sigma Chemical Co., Munich, Federal Republic of Germany). As described in detail previously (7), BMM4X (105 per well) were placed in flat-bottomed microdilution plates (Nunc, Roskilde, Denmark) in Iscove modified Dulbecco medium without supplements together with lymphokines. After 24 h of incubation at 37°C in humidified air-10% CO2, medium was removed and BMM4~were infected with 106 live mycobacteria per well. After 4 days, the viability of intracellular *
TABLE 1. Effect of recombinant cytokines on the tuberculostatic activity of BMM+a
Cytokine Expt 1 None IFN-,y
TNF
Expt 2 None IFN--y
Concn
(U/ml) 10 100 500 2,500
of mycoInhibitiongrowth (%)b bacterial
± 511 ± 871 ± 788 ± 574 531 ± 118
0 6 16 87 97
15,515 14,521 13,060 2,069
10 100 500 2,500
14,164 16,193 16,029 15,490
± 1,530 ± 600 ± 2,132 ± 3,067
10 100 500
13,509 15,722 14,529 1,478 527
± 306 ± 456 ± 536
± 190 ± 90
13,081 13,535 14,876 12,922
± ± ± ±
2,500 IL-4
[3H]uracil uptake (cpm)
10 100 500
2,500
957 2,134 636 2,122
9 0 0 0 0 0 0 89 96
3 0 0 4
a BMM4X were stimulated with cytokines for 24 h, washed, and infected with viable M. bovis organisms. After 4 days, cells were lysed with saponin, and the viability of intracellular bacteria was determined after 4 days by [3H]uracil incorporation. Percent inhibition = {(1-[3H]uracil uptake after culture with interleukins)/[3H]uracil uptake after culture with nonactivated BMM4} x 100. [3H]uracil uptake is shown as means + standard deviations from triplicate cultures. b Zeros indicate values equal to or lower than zero.
Corresponding author. 2675
INFECT. IMMUN.
NOTES
2676
TABLE 2. Effect of recombinant cytokines on the antimycobacterial activity of infected BMM4a Cytokine
Expt 1 None IFN--y
TNF
Expt 2 None IFN--y
IL-4
Concn (U/ml)
[3H]uracil uptake (cpm) 1,895 737 492 632 467
10 100 500 2,500
17,917 11,725 5,506 5,544 6,520
± ± ± ± ±
10 100 500 2,500
17,588 15,487 15,785 16,834
± 764 ± 884 ± 434
± 268
Inhibitiongrowth of myco(%)b bacterial
0 35 69 69 64 2 14 12 6
500 2,500
27,954 ± 1,694 16,975 ± 3,700 14,379 ± 2,144 11,814 ± 496 12,968 ± 735
0 39 49 58 54
10 100 500 2,500
26,352 19,769 15,791 15,853
6 29 44 43
10 100
± ± ± ±
1,429 1,355 373 182
a BMM4 were infected with live M. bovis organisms. Nonphagocytized bacteria were washed off, and cells were stimulated with cytokines. Intracellular survival was measured after 40 h by [3H]uracil uptake after lysis of the cells. Percent inhibition was calculated as indicated in Table 1, footnote a. [3H]uracil uptake is shown as means + standard deviations from triplicate cultures. b Zeros indicate values equal to or lower than zero.
lysed before the addition of [3H]uracil, influences of the phagocyte itself on [3H]uracil availability for mycobacteria seem extremely unlikely (7). As shown previously, BMM4 stimulated with rIFN--y for 24 h and subsequently infected with M. bovis organisms for 4 to 5 days were able to markedly inhibit intracellular growth of mycobacteria (7). Under these conditions, namely, activation of BMM4 and subsequent infection with mycobacteria, rIL-4 and rTNF failed to stimulate antimycobacterial activities in BMM4 (Table 1). On the other hand, in BMM4. which had first been infected with M. bovis and were afterward stimulated with cytokines for 40 h, rIL-4 induced inhibition of mycobacterial growth (Table 2). rIFN-y was capable of activating already infected BMM4, although its activity was reduced when compared with activation of uninfected BMMX (Tables 1 and 2) (9). rTNF induced negligible tuberculostasis in infected BMM4. The data presented show that rIL-4 can activate tuberculostatic functions in macrophages already infected with mycobacteria, whereas rIFN--y preferentially acts on noninfected macrophages. rTNF which alone was totally ineffective showed marked synergy with rIFN--y in the activation of tuberculostasis in BMM4o which were infected afterward. Under these conditions, minute doses of TNF and IFN--y were sufficient for significant inhibition of mycobacterial growth (Table 3). In a previous study, we showed that IFN-y activates tuberculostasis in BMMX which are subsequently infected with tubercle bacilli (7). In humans the situation might be far more complex, since IFN--y has been shown to stimulate mycobacterial growth in human monocytes, while 1,25dihydroxyvitamin D3 seems to inhibit mycobacterial growth
TABLE 3. TNF acts synergistically with IFN-y to activate
tuberculostatic macrophage functionsa Inhibition of myco[3H]uracil
Cytokine (U/ml)
IFN--y
TNF
uptake (cpm)
bacterial growth (%)b
± ± ± ±
0 16 49 73
11,109 9,294 5,701 3,050
766 401 168 151
0 10 100 500
0 0 0 0
0 0 0
10 100 500
11,393 + 849 12,069 ± 1,077 11,003 ± 1,670
0 0 1
10 10 10
10 100 500
4,404 + 306
3,877 ± 454 3,368 ± 583
60 65 70
100 100 100
10 100 500
4,170 ± 117 3,615 ± 343 3,502 ± 662
63 68 69
a BMMO were stimulated with cytokines for 24 h. Cells were washed and infected with M. bovis organisms. The viability of intracellular bacteria was determined after 4 days by [3H]uracil incorporation. Percent inhibition was calculated as indicated in Table 1, footnote a. [3H]uracil uptake is shown as means ± standard deviations from triplicate cultures. b Zeros indicate values equal to or lower than zero.
in these cells (4, 5). The present study reveals participation of other cytokines. Furthermore, it indicates a remarkable influence of the sequence of cytokine stimulation and infection. Activation of already infected BMM4 by IFN--y was reproducibly reduced. In some instances, reduction was partial (Table 2); in others, the activity was totally abolished (8). These findings suggest a negative effect of M. bovis infection on the responsiveness of BMM4 to IFN--y. Interestingly, infection had just the opposite effect on IL-4 responsiveness of BMMO, since IL-4 failed to stimulate noninfected macrophages but induced remarkable tuberculostasis in already infected BMM4). Thus, M. bovis infection itself must have rendered these macrophages responsive to IL-4. Killed mycobacteria could not induce IL-4 responsiveness (data not shown). Although TNF alone failed to activate tuberculostasis when given before or after infection, it showed marked synergy with IFN-y. Thus, low concentrations of TNF and IFN--y which by themselves had no or only negligible effects together induced notable tuberculostasis. We have shown recently that the B-cell-stimulating factor IL-6 can also activate tuberculostasis in BMMo (8). Taken together, these findings show that in vitro several cytokines contribute to antimycobacterial resistance. Further work will be required to determine whether these observations indeed reflect the in vivo situation. We thank R. Mahmoudi for excellent secretarial help. rIFN--y was
produced by Genentech and kindly provided by G. R. Adolph, Boehringer Ingelheim; rTNF-a was a kind gift from F. H. Hillen, BASF, Ludwigshafen.
This work received financial support from the World Health
Organization as part of its Program for Vaccine Development, Landesschwerpunkt 30, and the A. Krupp award for young professors to S.H.E.K.
LITERATURE CITED 1. Belosevic, M., C. E. Davis, M. S. Meltzer, and C. A. Nacy. 1988. Regulation of activated macrophage antimicrobial activities. Identification of lymphokines that cooperate with IFN--y for induction of resistance to infection. J. Immunol. 141:890-896.
NOTES
VOL. 58, 1990
2. Cohn, Z. A. 1978. The activation of mononuclear phagocytes: fact, fancy, and future. J. Immunol. 121:813-816. 3. Crawford, R. M., D. S. Finbloom, J. Ohara, W. E. Paul, and M. S. Meltzer. 1987. B-cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens. J. Immunol. 139:135-141. 4. Crowle, A. J., E. J. Ross, and M. H. May. 1987. Inhibition by 1,25(OH)2-vitamin D3 of the multiplication of virulent tubercle bacilli in cultured human macrophages. Infect. Immun. 55: 2945-2950.
5. Douvas, G. S., D. L. Looker, A. E. Vatter, and A. J. Crowle. 1985. Gamma interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria. Infect. Immun. 50:1-8. 6. Flesch, I., and E. Ferber. 1986. Growth requirements of murine bone marrow macrophages in serum-free cell culture. Immunobiology 171:14-26. 7. Flesch, I., and S. H. E. Kaufmann. 1987. Mycobacterial growth
8. 9. 10.
11.
2677
inhibition by interferon--y activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138:4408-4413. Flesch, I. E. A., and S. H. E. Kaufmann. 1990. Stimulation of antibacterial macrophage activities by B-cell stimulatory factor 2 (interleukin-6). Infect. Immun. 58:269-271. McInnes, A., and D. M. Rennick. 1988. Interleukin-4 induces cultured monocytes/macrophages to form giant multinucleated cells. J. Exp. Med. 167:598-611. Nathan, C. F., H. W. Murray, M. W. Wiebe, and B. Y. Rubin. 1983. Identification of interferon as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670-689. Zlotnik, A., M. Fischer, N. Roehm, and D. Zipori. 1987. Evidence for effects of interleukin 4 (B-cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages. J. Immunol. 138:4275-4279.
INFECTION AND IMMUNITY, Aug. 1990, p. 2675-2677 0019-9567/90/082675-03$02.00/0 Copyright © 1990, American Society for Microbiology
Activation of Tuberculostatic Macrophage Functions by Gamma Interferon, Interleukin-4, and Tumor Necrosis Factor E. A. FLESCH AND S. H. E. KAUFMANN* Department of Medical Microbiology and Immunology, University of Ulm, Albert-Einstein-Allee 11, D-7900 Ulm, Federal Republic of Germany I.
Received 27 December 1989/Accepted 8 May 1990
The capacity of the cytokines gamma interferon (IFN-'y), interleukin-4 (IL-4), and tumor necrosis factor (TNF) to activate tuberculostatic functions in murine bone marrow-derived macrophages (BMMo) was investigated. In confirmation of earlier findings, IFN--y rendered BMM+ capable of inhibiting subsequent infection with Mycobacterium bovis. In contrast, IL-4 and TNF failed to inhibit mycobacterial growth. However, in already infected BMM+, tuberculostasis was induced by subsequent stimulation with IL-4. Although TNF alone was ineffective, it showed synergy with IFN-y in the stimulation of tuberculostasis. Our data suggest that not only IFN-y but also IL-4 and TNF participate in the control of mycobacterial growth.
bacteria was determined by [3H]uracil uptake. It is our experience that an 80 to 95% reduction of [3H]uracil uptake reflects a 10- to 100-fold decrease in the number of intracellular bacteria as determined by CFU (7). In another approach, BMM4X (105 per well) were infected with M. bovis (106 per well) for 18 h, washed thoroughly to remove nonphagocytized bacteria, and subsequently stimulated with lymphokines. After 40 h of incubation, BMM4 were lysed with 0.1% saponin and the viability of intracellular bacteria was assessed by [3H]uracil uptake (8). Since the cells were
Mononuclear phagocytes represent the major habitat of the etiologic agents of tuberculosis, Mycobacterium tuberculosis and Mycobacterium bovis. According to current concepts, acquired resistance against tuberculosis primarily depends on macrophage activation by cytokines from specific T lymphocytes (2), and it is assumed that gamma interferon (IFN-y) is of particular relevance (10). However, cytokines other than IFN--y have recently been shown to activate certain macrophage functions (1, 3, 9, 11). We compared the roles of interleukin-4 (IL-4), tumor necrosis factor (TNF), and IFN--y in the activation of tuberculostasis in murine bone marrow-derived macrophages (BMM4O). In all experiments, serum-free and antibiotic-free media were used for cultivation as well as stimulation and infection of the cells. Bone marrow cells were prepared from the femora of 6- to 8-week-old C57BL/6 male or female mice which were bred under specific-pathogen-free conditions. BMMX were obtained in Teflon film bags (Heraeus, Hanau, Federal Republic of Germany) and harvested after 9 to 10 days of culture as described previously (6, 7). Viability as assessed by trypan blue exclusion was >90%. M. bovis BCG Phipps was grown with shaking in Dubos broth (Difco Laboratories, Detroit, Mich.) supplemented with bovine serum albumin and Tween 20. Numbers of viable organisms were determined by plating 1/10 dilutions on Middlebrook Dubos agar plates (Difco). Murine recombinant IFN--y (rIFN--y) was produced by Genentech Inc. and kindly supplied by Boehringer Ingelheim (Ingelheim, Federal Republic of Germany). Murine rIL-4 was purchased from Genzyme Corp. (Boston, Mass.). rTNF-a was a kind gift from BASF (Ludwigshafen, Federal Republic of Germany). Cytokines were free of detectable endotoxin contamination as determined by the Limulus amoebocyte lysate assay (Sigma Chemical Co., Munich, Federal Republic of Germany). As described in detail previously (7), BMM4X (105 per well) were placed in flat-bottomed microdilution plates (Nunc, Roskilde, Denmark) in Iscove modified Dulbecco medium without supplements together with lymphokines. After 24 h of incubation at 37°C in humidified air-10% CO2, medium was removed and BMM4~were infected with 106 live mycobacteria per well. After 4 days, the viability of intracellular *
TABLE 1. Effect of recombinant cytokines on the tuberculostatic activity of BMM+a
Cytokine Expt 1 None IFN-,y
TNF
Expt 2 None IFN--y
Concn
(U/ml) 10 100 500 2,500
of mycoInhibitiongrowth (%)b bacterial
± 511 ± 871 ± 788 ± 574 531 ± 118
0 6 16 87 97
15,515 14,521 13,060 2,069
10 100 500 2,500
14,164 16,193 16,029 15,490
± 1,530 ± 600 ± 2,132 ± 3,067
10 100 500
13,509 15,722 14,529 1,478 527
± 306 ± 456 ± 536
± 190 ± 90
13,081 13,535 14,876 12,922
± ± ± ±
2,500 IL-4
[3H]uracil uptake (cpm)
10 100 500
2,500
957 2,134 636 2,122
9 0 0 0 0 0 0 89 96
3 0 0 4
a BMM4X were stimulated with cytokines for 24 h, washed, and infected with viable M. bovis organisms. After 4 days, cells were lysed with saponin, and the viability of intracellular bacteria was determined after 4 days by [3H]uracil incorporation. Percent inhibition = {(1-[3H]uracil uptake after culture with interleukins)/[3H]uracil uptake after culture with nonactivated BMM4} x 100. [3H]uracil uptake is shown as means + standard deviations from triplicate cultures. b Zeros indicate values equal to or lower than zero.
Corresponding author. 2675
INFECT. IMMUN.
NOTES
2676
TABLE 2. Effect of recombinant cytokines on the antimycobacterial activity of infected BMM4a Cytokine
Expt 1 None IFN--y
TNF
Expt 2 None IFN--y
IL-4
Concn (U/ml)
[3H]uracil uptake (cpm) 1,895 737 492 632 467
10 100 500 2,500
17,917 11,725 5,506 5,544 6,520
± ± ± ± ±
10 100 500 2,500
17,588 15,487 15,785 16,834
± 764 ± 884 ± 434
± 268
Inhibitiongrowth of myco(%)b bacterial
0 35 69 69 64 2 14 12 6
500 2,500
27,954 ± 1,694 16,975 ± 3,700 14,379 ± 2,144 11,814 ± 496 12,968 ± 735
0 39 49 58 54
10 100 500 2,500
26,352 19,769 15,791 15,853
6 29 44 43
10 100
± ± ± ±
1,429 1,355 373 182
a BMM4 were infected with live M. bovis organisms. Nonphagocytized bacteria were washed off, and cells were stimulated with cytokines. Intracellular survival was measured after 40 h by [3H]uracil uptake after lysis of the cells. Percent inhibition was calculated as indicated in Table 1, footnote a. [3H]uracil uptake is shown as means + standard deviations from triplicate cultures. b Zeros indicate values equal to or lower than zero.
lysed before the addition of [3H]uracil, influences of the phagocyte itself on [3H]uracil availability for mycobacteria seem extremely unlikely (7). As shown previously, BMM4 stimulated with rIFN--y for 24 h and subsequently infected with M. bovis organisms for 4 to 5 days were able to markedly inhibit intracellular growth of mycobacteria (7). Under these conditions, namely, activation of BMM4 and subsequent infection with mycobacteria, rIL-4 and rTNF failed to stimulate antimycobacterial activities in BMM4 (Table 1). On the other hand, in BMM4. which had first been infected with M. bovis and were afterward stimulated with cytokines for 40 h, rIL-4 induced inhibition of mycobacterial growth (Table 2). rIFN-y was capable of activating already infected BMM4, although its activity was reduced when compared with activation of uninfected BMMX (Tables 1 and 2) (9). rTNF induced negligible tuberculostasis in infected BMM4. The data presented show that rIL-4 can activate tuberculostatic functions in macrophages already infected with mycobacteria, whereas rIFN--y preferentially acts on noninfected macrophages. rTNF which alone was totally ineffective showed marked synergy with rIFN--y in the activation of tuberculostasis in BMM4o which were infected afterward. Under these conditions, minute doses of TNF and IFN--y were sufficient for significant inhibition of mycobacterial growth (Table 3). In a previous study, we showed that IFN-y activates tuberculostasis in BMMX which are subsequently infected with tubercle bacilli (7). In humans the situation might be far more complex, since IFN--y has been shown to stimulate mycobacterial growth in human monocytes, while 1,25dihydroxyvitamin D3 seems to inhibit mycobacterial growth
TABLE 3. TNF acts synergistically with IFN-y to activate
tuberculostatic macrophage functionsa Inhibition of myco[3H]uracil
Cytokine (U/ml)
IFN--y
TNF
uptake (cpm)
bacterial growth (%)b
± ± ± ±
0 16 49 73
11,109 9,294 5,701 3,050
766 401 168 151
0 10 100 500
0 0 0 0
0 0 0
10 100 500
11,393 + 849 12,069 ± 1,077 11,003 ± 1,670
0 0 1
10 10 10
10 100 500
4,404 + 306
3,877 ± 454 3,368 ± 583
60 65 70
100 100 100
10 100 500
4,170 ± 117 3,615 ± 343 3,502 ± 662
63 68 69
a BMMO were stimulated with cytokines for 24 h. Cells were washed and infected with M. bovis organisms. The viability of intracellular bacteria was determined after 4 days by [3H]uracil incorporation. Percent inhibition was calculated as indicated in Table 1, footnote a. [3H]uracil uptake is shown as means ± standard deviations from triplicate cultures. b Zeros indicate values equal to or lower than zero.
in these cells (4, 5). The present study reveals participation of other cytokines. Furthermore, it indicates a remarkable influence of the sequence of cytokine stimulation and infection. Activation of already infected BMM4 by IFN--y was reproducibly reduced. In some instances, reduction was partial (Table 2); in others, the activity was totally abolished (8). These findings suggest a negative effect of M. bovis infection on the responsiveness of BMM4 to IFN--y. Interestingly, infection had just the opposite effect on IL-4 responsiveness of BMMO, since IL-4 failed to stimulate noninfected macrophages but induced remarkable tuberculostasis in already infected BMM4). Thus, M. bovis infection itself must have rendered these macrophages responsive to IL-4. Killed mycobacteria could not induce IL-4 responsiveness (data not shown). Although TNF alone failed to activate tuberculostasis when given before or after infection, it showed marked synergy with IFN-y. Thus, low concentrations of TNF and IFN--y which by themselves had no or only negligible effects together induced notable tuberculostasis. We have shown recently that the B-cell-stimulating factor IL-6 can also activate tuberculostasis in BMMo (8). Taken together, these findings show that in vitro several cytokines contribute to antimycobacterial resistance. Further work will be required to determine whether these observations indeed reflect the in vivo situation. We thank R. Mahmoudi for excellent secretarial help. rIFN--y was
produced by Genentech and kindly provided by G. R. Adolph, Boehringer Ingelheim; rTNF-a was a kind gift from F. H. Hillen, BASF, Ludwigshafen.
This work received financial support from the World Health
Organization as part of its Program for Vaccine Development, Landesschwerpunkt 30, and the A. Krupp award for young professors to S.H.E.K.
LITERATURE CITED 1. Belosevic, M., C. E. Davis, M. S. Meltzer, and C. A. Nacy. 1988. Regulation of activated macrophage antimicrobial activities. Identification of lymphokines that cooperate with IFN--y for induction of resistance to infection. J. Immunol. 141:890-896.
NOTES
VOL. 58, 1990
2. Cohn, Z. A. 1978. The activation of mononuclear phagocytes: fact, fancy, and future. J. Immunol. 121:813-816. 3. Crawford, R. M., D. S. Finbloom, J. Ohara, W. E. Paul, and M. S. Meltzer. 1987. B-cell stimulatory factor-1 (interleukin 4) activates macrophages for increased tumoricidal activity and expression of Ia antigens. J. Immunol. 139:135-141. 4. Crowle, A. J., E. J. Ross, and M. H. May. 1987. Inhibition by 1,25(OH)2-vitamin D3 of the multiplication of virulent tubercle bacilli in cultured human macrophages. Infect. Immun. 55: 2945-2950.
5. Douvas, G. S., D. L. Looker, A. E. Vatter, and A. J. Crowle. 1985. Gamma interferon activates human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobacteria. Infect. Immun. 50:1-8. 6. Flesch, I., and E. Ferber. 1986. Growth requirements of murine bone marrow macrophages in serum-free cell culture. Immunobiology 171:14-26. 7. Flesch, I., and S. H. E. Kaufmann. 1987. Mycobacterial growth
8. 9. 10.
11.
2677
inhibition by interferon--y activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138:4408-4413. Flesch, I. E. A., and S. H. E. Kaufmann. 1990. Stimulation of antibacterial macrophage activities by B-cell stimulatory factor 2 (interleukin-6). Infect. Immun. 58:269-271. McInnes, A., and D. M. Rennick. 1988. Interleukin-4 induces cultured monocytes/macrophages to form giant multinucleated cells. J. Exp. Med. 167:598-611. Nathan, C. F., H. W. Murray, M. W. Wiebe, and B. Y. Rubin. 1983. Identification of interferon as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158:670-689. Zlotnik, A., M. Fischer, N. Roehm, and D. Zipori. 1987. Evidence for effects of interleukin 4 (B-cell stimulatory factor 1) on macrophages: enhancement of antigen presenting ability of bone marrow-derived macrophages. J. Immunol. 138:4275-4279.
