Vol. 59, No. 11
INFECTION AND IMMUNITY, Nov. 1991, p. 4227-4229
0019-9567/91/114227-03$02.00/0
Copyright C 1991, American Society for Microbiology
Effects of Recombinant Gamma Interferon and Tumor Necrosis Factor on In Vitro Interactions of Human Mononuclear Phagocytes with Coccidioides immitis LoVELLE BEAMAN Medical Microbiology and Immunology, University of California School of Medicine, Davis, California 95616 Received 5 August 1991/Accepted 14 August 1991
Human peripheral blood monocytes readily phagocytized Coccidioides immitis endospores (2 to 5 ,um) in vitro. Within 24 to 30 h at 37°C, the phagocytized endospores started developing into immature spherules. However, when the monocytes were incubated with recombinant human gamma interferon (rIFN-'y) or recombinant human tumor necrosis factor alpha (rTNF-a) and then infected, fewer endospores developed into spherules. Treatment with rIFN-'y or rTNF-aL activated the fungicidal capabilities of the monocytes as evidenced by the significant reduction in CFU that could be recovered from rIFN-y- or rTNF-a-activated monocytes compared with nontreated controls. Human infections with the fungus Coccidioides immitis occur when soil containing the fungus is disturbed and aerosols containing infectious arthroconidia are inhaled. Within human tissue, the arthroconidia develop into multinucleate spherules which later release uninucleate endospores. Alveolar macrophages from normal nonhuman primates or from mice will phagocytize arthroconidia and endospores; however, the phagocytized fungus develops into hyphae or spherules without apparent loss of viability (2-4). Within alveolar macrophages, C. immitis appears to inhibit the fusion of phagosomes containing arthroconidia or endospores with lysosomes (4). Human peripheral blood monocytes also phagocytize C. immitis without affecting the survival of the fungus in vitro (5). When monocytes were incubated in the presence of lymphocytes from immune donors, a significant increase in phagosome-lysosome fusion was observed. This increase in fusion correlated with the ability of the monocytes to kill C. immitis (5). In murine alveolar macrophages treated with recombinant gamma interferon (rIFN--y) during infection with C. immitis, phagocytized endospores did not develop into spherules and there was a 50% reduction in CFU recovered compared with nontreated macrophages (1). rIFN--y alone was not toxic to C. immitis, and the reduced CFU could not be explained by interferon-induced clumping (1). Since it had been previously shown in this laboratory that rIFN-,y activated murine macrophage fungicidal activity, this study was done to see whether human monocytes could be activated by cytokines such as rIFN-y or recombinant tumor necrosis factor alpha
(rTNF-ao). MATERIALS AND METHODS Cultures. The spherule-endospore phase of C. immitis was grown in modified Converse medium (8), and the endospores were harvested by differential centrifugation after 120 h of incubation. This process resulted in a preparation of which more than 99% comprised small endospores (2 to 5 ,um) as counted in a hemacytometer. Monocytes. Peripheral blood monocytes were obtained from healthy individuals who did not have a positive skin test for C. immitis coccidioidin. The mononuclear cells were
separated from neutrophils and erythrocytes by using a modification of a Ficoll-Hypaque gradient (Neutrophil Isolation Media; Los Alamos Diagnostics) and allowed to adhere to Lab-Tek slide chambers (Miles Scientific Div., Miles Laboratories, Inc., Naperville, Ill.) at a concentration of 4 x 106 mononuclear cells per well for 90 min at 37°C in an atmosphere of 5% CO2. The nonadherent cells (primarily lymphocytes) were then removed. It was estimated that 4 x 105 cells remained per well. There were no neutrophils observed in the adherent monolayers. In addition, 106 mononuclear cells in 0.1 ml were allowed to adhere to 96-well tissue culture plates for 90 min. Then, the nonadherent cells were removed. It was estimated that 105 cells per well remained in the 96-well tissue culture plates. Cytokines. Stock solutions of rIFN--y (gift of Genentech, Inc., South San Francisco, Calif.) were prepared in serumfree medium (Neuman and Tytell; GIBCO Laboratories, Grand Island, N.Y.) with 1 mg of bovine serum albumin per ml. The final dilution of rIFN-y or rTNF-a (gift of Genentech, Inc.) in serum-free medium was less than 0.8 ng per ml of endotoxin as measured by E-toxate assay (Sigma Chemical Co., St. Louis, Mo.). Infection of monocytes in slide chambers. The monocytes in slide chambers were incubated with 1,000 U of rIFN--y or 1,000 U of rTNF-a or with serum-free medium for 24 h. After 24 h, the monocytes were washed, fresh medium was added, and the monocytes were divided into three groups: those incubated with rIFN--y or rTNF-a for 24 h and washed (pretreated), those incubated with rIFN-y or rTNF-a during infection (coincubation), and those incubated with medium during infection with C. immitis for 1 h. The infection ratio was approximately 1 endospore per 10 monocytes. Then, the monocytes were washed free of the nonphagocytized fungi and incubated in medium (pretreated and normal) or rIFN-y and rTNF-a (coincubation) for a total of 27 h prior to staining with Giemsa. The total number of intracellular endospores, small developing spherules, or hyphae was counted in at least 400 monocytes per group. Infection of monocytes in 96-well plates. Monocytes in 96-well plates were incubated with 1,000 U of rIFN-y, rTNF-a, or serum-free medium for 24 h prior to infection with C. immitis endospores for 1 h. After 1 h, the monocytes 4227
4228
INFECT. IMMUN.
BEAMAN *
TREATMENT
*
rIFN--y, 82%
ENDOSPORE SPHERULE HYPHAE
NORMAL
+ 1% of the phagocytized fungi remained as endospores after 27 h of incubation. In monocytes preincubated with rTNF-a, 76% + 1% of the phagocytized fungi remained as endospores and only 24% 1% developed into spherules or hyphae (Fig. 1). There was a significant decrease in the number of spherules seen in monocytes pretreated with rIFN-y or rTNF-oa compared with normal monocytes (P < 0.01). These results indicate that rIFN--y or rTNF-oa can activate monocytes to inhibit the development of endospores into spherules. Since it is difficult to determine from the slides whether the monocytes were being activated to kill the fungus or were simply inhibiting spherule development, further experiments were done to determine the number of CFU which could be recovered from monocyte cultures within an 8-h incubation before the maturation of spherules and the eventual release of endospores (24 to 48 h). Recovery of C. immitis from infected monocytes. As shown in Table 1, there was not a significant difference in the CFU recovered at 1 h from nontreated monocytes compared with monocytes pretreated or infected in the presence of rIFN--y. This indicates that rIFN-y did not enhance phagocytosis or induce fungal clumping. As shown in Table 2, with nontreated monocytes there was a small loss of CFU recovered 8 h postinfection compared with the CFU recovered at 1 h postinfection (11% 4%). However, in monocytes pretreated with rIFN--y, approximately half of the endospores were not recovered (41, 68, or 44%). Pretreatment with rIFN-y or rTNF-a resulted in a significant loss of C. immitis CFU recovered 8 h postinfection (P < 0.01). The combination of rIFN--y and rTNF-a was effective in activating monocytes when added prior to infection (57, 35, or 29% reduction in CFU) or when added during infection (37, 48, or 75% reduction in CFU). ±
PRE-IFN
CO-IFN PRE-TNF
Hi
CO-TNF
0
10
20
30
40
50
60
70
80
90
PERCENT OF TOTAL FUNGAL CELLS
FIG. 1. Slide cultures of monocytes that had been incubated 24 h with rIFN--y or rTNF-cx (PRE-) and then infected with endospores or of monocytes that were infected with endospores in the presence of rTNF-ax or rIFN--y (CO-) or media (NORMAL). Bars indicate the mean percent standard deviation of total fungal cells within monocytes at 27 h postinfection that were endospores, spherules, or hyphae (germ tubes) in 400 monocytes obtained in each of three experiments. ±
washed free of nonphagocytized fungi, serum-free medium was added, and the monocytes were incubated for another 7 h. Then, the monocytes were lysed in 2.5% sodium deoxycholate, diluted in distilled water, and plated on glustandard error cose-yeast extract agar. The mean CFU was determined from four samples in two experiments. The significance was determined by Student's t test. were
±
±
RESULTS Inhibition of spherule formation by monocytes treated with rIFN-y or rTNF-a. In nontreated (normal) monocytes in slide chambers as shown in Fig. 1, 49% 6% of the total fungi phagocytized developed into spherules and 51% remained endospores. However in monocytes pretreated with ±
DISCUSSION IFN--y has been shown to suppress the growth of another pathogenic fungus, Histoplasma capsulatum, in murine macrophages (11) and to enhance the fungicidal activity of murine macrophages for Candida albicans and Blastomyces
TABLE 1. Effect of treatment of monocytes with rIFN-y or rTNF-ax on phagocytosis of C. immitis endospores % Phagocytosis' CFU of monocytes at 1 h Total CFU at 1 hb
Donor and treatmenta
1
None
Pre-rIFN-y Co-rIFN--y Co-rTNF-a Co-rIFN--y+rTNF-a
± 0.3) x
103
(3.18 (2.6 (3.33 (2.63 (3.6
± ± ± ±
(2.2 (2.4 (2.6 (2.5 (2.25
± 0.1) ± 0.6) ± 0.1) ± 1.1) ± 1.2)
x x x x x
104 104 104 104 104
(2.2 (2 (1.88 (2.9 (2.24
± 0.1) ± 0.5) ± 0.2) ± 1.5) ± 1.2)
x 104 x 104 x 104
0.3) 0.4) 0.2) 0.7)
x 103 x 103
x 103 x 103
103 103 103 103 103
23 20 23 17 23
x 104
35 37 38 38 35
(13.8 ± 0.8) (13.2 ± 1.2) (14.2 ± 0.6) (15.9 ± 4) (15.9 ± 2)
x x x x
(6.2 (6.5 (6.7 (6.6 (6.35
± ± ± ± ±
x x x x
(6.2 (5.9 (5.78 (6.8 (6.1
± ± ± ±
x
2 None
Pre-rIFN--y Co-rIFN--y Co-TNF-ot Co-rIFN--y+rTNF-a
0.1) 0.6) 0.1) 1.1) 1.2)
104 104
104 104
3
None
Pre-rIFN-y Co-rIFN--y Co-rTNF-a Co-rIFN-y+rTNF-a
x 104
x 104
0.1) 0.1) 0.1) 0.1) ± 0.1)
x 104 x 104 x 104 x 104 x 104
35 33 33 43 37
a Treatments are defined in the legend to Fig. 1. b The mean CFU recovered from monocytes after washing twice was determined from duplicate samples obtained in five separate experiments involving monocytes from three individuals. Total CFU = CFU associated with monocytes plus CFU in culture supernatants and cell washings at 1 h after infection. I % Phagocytosis = (CFU monocytes/total CFU) x 100.
VOL. 59, 1991
EFFECT OF IFN-y ON C. IMMITIS
TABLE 2. CFU of C. immitis recovered from monocytes treated with rIFN-y or rTNF-ot Treatmenta and donor donor
None 1 2 3
CFU
(104)b
Reduction" ~~~~~~~~~~%
1h
8h
2.6 ± 0.4 2.2 ± 0.1 0.7 ± 0.4
2.4 ± 0.2 2.2 ±0.1 0.78 ± 0.4
11 ± 4 0 0
1.74 ± 0.5 2.39 ± 0.5 1.95 ± 0.5
0.8 ± 0.1 1.06 ± 0.7 1.08 ± 0.4
41 ± 5 68 ± 5 44 ± 6
3.1 ± 0.1 2.28 ± 0.1 1.5 ± 0.7
1.74 ± 0.1 1.1 ± 0.3 1.1 ± 0.4
41 ± 5 53 ± 3 28 ± 8
Pre-rIFN--y 1 2 3 Pre-rTNF-a 1 2 3
Pre-rINF--y + 2.35 ± 0.3 1.75±0.1 2.5 ± 0.6
1.14 ± 0.2 1.1±0.2 1.7 ± 0.7
57 ± 5 35±3 29 ± 6
2.15 ± 0.1 2.2 ± 1 2.5 ± 1
1.34 ± 0.3 0.96 0.3 0.87 ± 0.1
37 ± 5 48 ± 8 75 ± 5
Co-rINF-y + rTNF-a 1 2 3
study confirms that rIFN-y and rTNF-a activate the fungicidal capabilities of human monocytes for C. immitis endospores in vitro. This is an indication that rIFN--y and rTNF-a may play a role in host resistance to C. immitis infection. ACKNOWLEDGMENTS The gift of rIFN--y and rTNF-a from Genentech, South San Francisco, Calif., is gratefully acknowledged. The assistance of D. Pappagianis and B. Zimmer in providing cultures of C. immitis and the typing of this manuscript by Theresa Andreozzi and Lynn Diaz were greatly appreciated. REFERENCES 1. Beaman, L. 1987. Fungicidal activation of murine macrophages by recombinant gamma interferon. Infect. Immun. 55:29512955. 2. Beaman, L., E. Benjamini, and D. Pappagianis. 1981. Role of
lymphocytes in macrophage-induced killing of Coccidioides
rTNF-a 1 2 3
4229
a Treatments are defined in the legend to Fig. 1 b Monocytes were obtained from three donors. A total of 6.2 x 104 endospores was added to 1 x 10i monocytes per well. The mean CFU + standard deviation was determined from 8 samples in 2 different experiments at 1 and 8 h postinfection. I % reduction in CFU = [(CFU at 1 h - CFU at 8 h)/CFU at 1 h] x 100.
dermatitidis (6). However, human alveolar, peritoneal, and cultured macrophages exposed to rIFN--y for 72 h were not activated to inhibit the growth of H. capsulatum yeast cells (7), indicating that other factors may be necessary to activate fungicidal activity for H. capsulatum in human phagocytes. Candidacidal activity in murine macrophages was observed after incubation with rIFN--y for 24 h but not after incubation with rIFN-y for 72 h (10). The candidacidal activity correlated with enhanced acidification of macrophage phagolysosomes but not with production of reactive oxygen molecules. There is evidence that spherules of C. immitis may stimulate the release of TNF by murine macrophages (9). The interaction of cytokines such as IFN--y and rTNF-a and their relative importance during the immune response to C. immitis in infected individuals remains to be determined. This
immitis in vitro. Infect. Immun. 34:347-353. 3. Beaman, L., E. Benjamini, and D. Pappagianis. 1983. Activation of macrophages by lymphokines: enhancement of phagosomelysosome fusion and killing of Coccidioides immitis. Infect. Immun. 39:1201-1207. 4. Beaman, L., and C. A. Holmberg. 1980. In vitro response of alveolar macrophages to infection with Coccidioides immitis.
Infect. Immun. 28:594-600. 5. Beaman, L., and D. Pappagianis. 1985. Fate of Coccidioides immitis arthroconidia in human peripheral blood monocyte cultures in vitro, p. 170-180. In H. E. Einstein and A. Catanzaro (ed.), Coccidioidomycosis. National Foundation for Infectious Diseases, Washington, D.C. 6. Brummer, E., C. J. Morrison, and D. A. Stevens. 1985. Recombinant and natural gamma-interferon activation of macrophages in vitro: different dose requirements for induction of killing activity against phagocytizable and nonphagocytizable fungi. Infect. Immun. 49:724-730. 7. Fleischmann, J., B. Wu-Hsieh, and D. H. Howard. 1990. The intracellular fate of Histoplasma capsulatum in human macrophages is unaffected by recombinant interferon--y. J. Infect. Dis.,
161:143-145. 8. Levine, H. B., J. M. Cobb, and C. E. Smith. 1960. Immunity to coccidioidomycosis induced in mice by purified spherule, arthrospore and mycelial vaccines. Trans. N.Y. Acad. Sci. 22: 436-449. 9. Slagle, D. C., R. A. Cox, and U. Kuraganti. 1989. Induction of tumor necrosis factor alpha by spherules of Coccidioides immitis. Infect. Immun. 57:1916-1921. 10. Watanabe, K., K. Kagaya, T. Yamada, and Y. Fukazawa. 1991. Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon. Infect. Immun. 59:521-528. 11. Wu-Hsieh, B., and D. H. Howard. 1987. Inhibition of the intracellular growth of Histoplasma capsulatum by recombinant murine gamma interferon. Infect. Immun. 55:1014-1016.
INFECTION AND IMMUNITY, Nov. 1991, p. 4227-4229
0019-9567/91/114227-03$02.00/0
Copyright C 1991, American Society for Microbiology
Effects of Recombinant Gamma Interferon and Tumor Necrosis Factor on In Vitro Interactions of Human Mononuclear Phagocytes with Coccidioides immitis LoVELLE BEAMAN Medical Microbiology and Immunology, University of California School of Medicine, Davis, California 95616 Received 5 August 1991/Accepted 14 August 1991
Human peripheral blood monocytes readily phagocytized Coccidioides immitis endospores (2 to 5 ,um) in vitro. Within 24 to 30 h at 37°C, the phagocytized endospores started developing into immature spherules. However, when the monocytes were incubated with recombinant human gamma interferon (rIFN-'y) or recombinant human tumor necrosis factor alpha (rTNF-a) and then infected, fewer endospores developed into spherules. Treatment with rIFN-'y or rTNF-aL activated the fungicidal capabilities of the monocytes as evidenced by the significant reduction in CFU that could be recovered from rIFN-y- or rTNF-a-activated monocytes compared with nontreated controls. Human infections with the fungus Coccidioides immitis occur when soil containing the fungus is disturbed and aerosols containing infectious arthroconidia are inhaled. Within human tissue, the arthroconidia develop into multinucleate spherules which later release uninucleate endospores. Alveolar macrophages from normal nonhuman primates or from mice will phagocytize arthroconidia and endospores; however, the phagocytized fungus develops into hyphae or spherules without apparent loss of viability (2-4). Within alveolar macrophages, C. immitis appears to inhibit the fusion of phagosomes containing arthroconidia or endospores with lysosomes (4). Human peripheral blood monocytes also phagocytize C. immitis without affecting the survival of the fungus in vitro (5). When monocytes were incubated in the presence of lymphocytes from immune donors, a significant increase in phagosome-lysosome fusion was observed. This increase in fusion correlated with the ability of the monocytes to kill C. immitis (5). In murine alveolar macrophages treated with recombinant gamma interferon (rIFN--y) during infection with C. immitis, phagocytized endospores did not develop into spherules and there was a 50% reduction in CFU recovered compared with nontreated macrophages (1). rIFN--y alone was not toxic to C. immitis, and the reduced CFU could not be explained by interferon-induced clumping (1). Since it had been previously shown in this laboratory that rIFN-,y activated murine macrophage fungicidal activity, this study was done to see whether human monocytes could be activated by cytokines such as rIFN-y or recombinant tumor necrosis factor alpha
(rTNF-ao). MATERIALS AND METHODS Cultures. The spherule-endospore phase of C. immitis was grown in modified Converse medium (8), and the endospores were harvested by differential centrifugation after 120 h of incubation. This process resulted in a preparation of which more than 99% comprised small endospores (2 to 5 ,um) as counted in a hemacytometer. Monocytes. Peripheral blood monocytes were obtained from healthy individuals who did not have a positive skin test for C. immitis coccidioidin. The mononuclear cells were
separated from neutrophils and erythrocytes by using a modification of a Ficoll-Hypaque gradient (Neutrophil Isolation Media; Los Alamos Diagnostics) and allowed to adhere to Lab-Tek slide chambers (Miles Scientific Div., Miles Laboratories, Inc., Naperville, Ill.) at a concentration of 4 x 106 mononuclear cells per well for 90 min at 37°C in an atmosphere of 5% CO2. The nonadherent cells (primarily lymphocytes) were then removed. It was estimated that 4 x 105 cells remained per well. There were no neutrophils observed in the adherent monolayers. In addition, 106 mononuclear cells in 0.1 ml were allowed to adhere to 96-well tissue culture plates for 90 min. Then, the nonadherent cells were removed. It was estimated that 105 cells per well remained in the 96-well tissue culture plates. Cytokines. Stock solutions of rIFN--y (gift of Genentech, Inc., South San Francisco, Calif.) were prepared in serumfree medium (Neuman and Tytell; GIBCO Laboratories, Grand Island, N.Y.) with 1 mg of bovine serum albumin per ml. The final dilution of rIFN-y or rTNF-a (gift of Genentech, Inc.) in serum-free medium was less than 0.8 ng per ml of endotoxin as measured by E-toxate assay (Sigma Chemical Co., St. Louis, Mo.). Infection of monocytes in slide chambers. The monocytes in slide chambers were incubated with 1,000 U of rIFN--y or 1,000 U of rTNF-a or with serum-free medium for 24 h. After 24 h, the monocytes were washed, fresh medium was added, and the monocytes were divided into three groups: those incubated with rIFN--y or rTNF-a for 24 h and washed (pretreated), those incubated with rIFN-y or rTNF-a during infection (coincubation), and those incubated with medium during infection with C. immitis for 1 h. The infection ratio was approximately 1 endospore per 10 monocytes. Then, the monocytes were washed free of the nonphagocytized fungi and incubated in medium (pretreated and normal) or rIFN-y and rTNF-a (coincubation) for a total of 27 h prior to staining with Giemsa. The total number of intracellular endospores, small developing spherules, or hyphae was counted in at least 400 monocytes per group. Infection of monocytes in 96-well plates. Monocytes in 96-well plates were incubated with 1,000 U of rIFN-y, rTNF-a, or serum-free medium for 24 h prior to infection with C. immitis endospores for 1 h. After 1 h, the monocytes 4227
4228
INFECT. IMMUN.
BEAMAN *
TREATMENT
*
rIFN--y, 82%
ENDOSPORE SPHERULE HYPHAE
NORMAL
+ 1% of the phagocytized fungi remained as endospores after 27 h of incubation. In monocytes preincubated with rTNF-a, 76% + 1% of the phagocytized fungi remained as endospores and only 24% 1% developed into spherules or hyphae (Fig. 1). There was a significant decrease in the number of spherules seen in monocytes pretreated with rIFN-y or rTNF-oa compared with normal monocytes (P < 0.01). These results indicate that rIFN--y or rTNF-oa can activate monocytes to inhibit the development of endospores into spherules. Since it is difficult to determine from the slides whether the monocytes were being activated to kill the fungus or were simply inhibiting spherule development, further experiments were done to determine the number of CFU which could be recovered from monocyte cultures within an 8-h incubation before the maturation of spherules and the eventual release of endospores (24 to 48 h). Recovery of C. immitis from infected monocytes. As shown in Table 1, there was not a significant difference in the CFU recovered at 1 h from nontreated monocytes compared with monocytes pretreated or infected in the presence of rIFN--y. This indicates that rIFN-y did not enhance phagocytosis or induce fungal clumping. As shown in Table 2, with nontreated monocytes there was a small loss of CFU recovered 8 h postinfection compared with the CFU recovered at 1 h postinfection (11% 4%). However, in monocytes pretreated with rIFN--y, approximately half of the endospores were not recovered (41, 68, or 44%). Pretreatment with rIFN-y or rTNF-a resulted in a significant loss of C. immitis CFU recovered 8 h postinfection (P < 0.01). The combination of rIFN--y and rTNF-a was effective in activating monocytes when added prior to infection (57, 35, or 29% reduction in CFU) or when added during infection (37, 48, or 75% reduction in CFU). ±
PRE-IFN
CO-IFN PRE-TNF
Hi
CO-TNF
0
10
20
30
40
50
60
70
80
90
PERCENT OF TOTAL FUNGAL CELLS
FIG. 1. Slide cultures of monocytes that had been incubated 24 h with rIFN--y or rTNF-cx (PRE-) and then infected with endospores or of monocytes that were infected with endospores in the presence of rTNF-ax or rIFN--y (CO-) or media (NORMAL). Bars indicate the mean percent standard deviation of total fungal cells within monocytes at 27 h postinfection that were endospores, spherules, or hyphae (germ tubes) in 400 monocytes obtained in each of three experiments. ±
washed free of nonphagocytized fungi, serum-free medium was added, and the monocytes were incubated for another 7 h. Then, the monocytes were lysed in 2.5% sodium deoxycholate, diluted in distilled water, and plated on glustandard error cose-yeast extract agar. The mean CFU was determined from four samples in two experiments. The significance was determined by Student's t test. were
±
±
RESULTS Inhibition of spherule formation by monocytes treated with rIFN-y or rTNF-a. In nontreated (normal) monocytes in slide chambers as shown in Fig. 1, 49% 6% of the total fungi phagocytized developed into spherules and 51% remained endospores. However in monocytes pretreated with ±
DISCUSSION IFN--y has been shown to suppress the growth of another pathogenic fungus, Histoplasma capsulatum, in murine macrophages (11) and to enhance the fungicidal activity of murine macrophages for Candida albicans and Blastomyces
TABLE 1. Effect of treatment of monocytes with rIFN-y or rTNF-ax on phagocytosis of C. immitis endospores % Phagocytosis' CFU of monocytes at 1 h Total CFU at 1 hb
Donor and treatmenta
1
None
Pre-rIFN-y Co-rIFN--y Co-rTNF-a Co-rIFN--y+rTNF-a
± 0.3) x
103
(3.18 (2.6 (3.33 (2.63 (3.6
± ± ± ±
(2.2 (2.4 (2.6 (2.5 (2.25
± 0.1) ± 0.6) ± 0.1) ± 1.1) ± 1.2)
x x x x x
104 104 104 104 104
(2.2 (2 (1.88 (2.9 (2.24
± 0.1) ± 0.5) ± 0.2) ± 1.5) ± 1.2)
x 104 x 104 x 104
0.3) 0.4) 0.2) 0.7)
x 103 x 103
x 103 x 103
103 103 103 103 103
23 20 23 17 23
x 104
35 37 38 38 35
(13.8 ± 0.8) (13.2 ± 1.2) (14.2 ± 0.6) (15.9 ± 4) (15.9 ± 2)
x x x x
(6.2 (6.5 (6.7 (6.6 (6.35
± ± ± ± ±
x x x x
(6.2 (5.9 (5.78 (6.8 (6.1
± ± ± ±
x
2 None
Pre-rIFN--y Co-rIFN--y Co-TNF-ot Co-rIFN--y+rTNF-a
0.1) 0.6) 0.1) 1.1) 1.2)
104 104
104 104
3
None
Pre-rIFN-y Co-rIFN--y Co-rTNF-a Co-rIFN-y+rTNF-a
x 104
x 104
0.1) 0.1) 0.1) 0.1) ± 0.1)
x 104 x 104 x 104 x 104 x 104
35 33 33 43 37
a Treatments are defined in the legend to Fig. 1. b The mean CFU recovered from monocytes after washing twice was determined from duplicate samples obtained in five separate experiments involving monocytes from three individuals. Total CFU = CFU associated with monocytes plus CFU in culture supernatants and cell washings at 1 h after infection. I % Phagocytosis = (CFU monocytes/total CFU) x 100.
VOL. 59, 1991
EFFECT OF IFN-y ON C. IMMITIS
TABLE 2. CFU of C. immitis recovered from monocytes treated with rIFN-y or rTNF-ot Treatmenta and donor donor
None 1 2 3
CFU
(104)b
Reduction" ~~~~~~~~~~%
1h
8h
2.6 ± 0.4 2.2 ± 0.1 0.7 ± 0.4
2.4 ± 0.2 2.2 ±0.1 0.78 ± 0.4
11 ± 4 0 0
1.74 ± 0.5 2.39 ± 0.5 1.95 ± 0.5
0.8 ± 0.1 1.06 ± 0.7 1.08 ± 0.4
41 ± 5 68 ± 5 44 ± 6
3.1 ± 0.1 2.28 ± 0.1 1.5 ± 0.7
1.74 ± 0.1 1.1 ± 0.3 1.1 ± 0.4
41 ± 5 53 ± 3 28 ± 8
Pre-rIFN--y 1 2 3 Pre-rTNF-a 1 2 3
Pre-rINF--y + 2.35 ± 0.3 1.75±0.1 2.5 ± 0.6
1.14 ± 0.2 1.1±0.2 1.7 ± 0.7
57 ± 5 35±3 29 ± 6
2.15 ± 0.1 2.2 ± 1 2.5 ± 1
1.34 ± 0.3 0.96 0.3 0.87 ± 0.1
37 ± 5 48 ± 8 75 ± 5
Co-rINF-y + rTNF-a 1 2 3
study confirms that rIFN-y and rTNF-a activate the fungicidal capabilities of human monocytes for C. immitis endospores in vitro. This is an indication that rIFN--y and rTNF-a may play a role in host resistance to C. immitis infection. ACKNOWLEDGMENTS The gift of rIFN--y and rTNF-a from Genentech, South San Francisco, Calif., is gratefully acknowledged. The assistance of D. Pappagianis and B. Zimmer in providing cultures of C. immitis and the typing of this manuscript by Theresa Andreozzi and Lynn Diaz were greatly appreciated. REFERENCES 1. Beaman, L. 1987. Fungicidal activation of murine macrophages by recombinant gamma interferon. Infect. Immun. 55:29512955. 2. Beaman, L., E. Benjamini, and D. Pappagianis. 1981. Role of
lymphocytes in macrophage-induced killing of Coccidioides
rTNF-a 1 2 3
4229
a Treatments are defined in the legend to Fig. 1 b Monocytes were obtained from three donors. A total of 6.2 x 104 endospores was added to 1 x 10i monocytes per well. The mean CFU + standard deviation was determined from 8 samples in 2 different experiments at 1 and 8 h postinfection. I % reduction in CFU = [(CFU at 1 h - CFU at 8 h)/CFU at 1 h] x 100.
dermatitidis (6). However, human alveolar, peritoneal, and cultured macrophages exposed to rIFN--y for 72 h were not activated to inhibit the growth of H. capsulatum yeast cells (7), indicating that other factors may be necessary to activate fungicidal activity for H. capsulatum in human phagocytes. Candidacidal activity in murine macrophages was observed after incubation with rIFN--y for 24 h but not after incubation with rIFN-y for 72 h (10). The candidacidal activity correlated with enhanced acidification of macrophage phagolysosomes but not with production of reactive oxygen molecules. There is evidence that spherules of C. immitis may stimulate the release of TNF by murine macrophages (9). The interaction of cytokines such as IFN--y and rTNF-a and their relative importance during the immune response to C. immitis in infected individuals remains to be determined. This
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