Vol. 13, No. 4 Printed in U.SA.
INFECTION AND IMMUNrrY, Apr. 1976, P. 1296-1298 Copyright C 1976 American Society for Microbiology
Acquired Immunity to Aspergillus fumigatus PAUL F. LEHMANNI* AND LES 0. WHITE Department of Microbiology, University of Birmingham, Birmingham B15 2TT, United Kingdom
Received for publication 11 November 1975
In mice preinfected with Aspergillus fumigatus for -10 days, administration of cortisone failed to stimulate mycelial growth. The liver and heart of mice preinfected for 14 days resisted infection after a cortisone + conidia challenge. To date, there has been no clear evidence of acquired immunity to Aspergillus fumigatus (8), a fungus which has caused a troublesome, often fatal infection of patients treated with immunosuppressive drugs (1). We have shown that immunosuppression with cortisone increases the susceptibility of mice to infection by A. fumigatus and that this is associated with an increased mycelial growth rate in the kidneys (5). We now present evidence that these effects can no longer be demonstrated if a localized kidney infection has been present for some time; i.e., infected mice can aquire a systemic immunity against fungal infection and growth which is not affected by cortisone. We use the term immunity as meaning the suppression of fungal multiplication as defined by Kong and Levine (4). When untreated mice were injected intravenously with 105 conidia, the infection localized in the kidneys where conidia germinated and mycelium developed (5). In the spleen, lungs, heart, and liver, no viable fungal material could be found after 4 days. Figure 1 shows the effect of cortisone on the growth of mycelium established in the kidneys. The magnitude of the cortisone effect decreased with time after infection, and cortisone brought about no significant increase in fungal growth when administered after 10 days. We wished to see whether this phenomenon was associated with the development of a cortisone-insensitive immunity to a second conidial challenge. Treatment of mice with cortisone before intravenous administration of 105 conidia rendered the liver and heart susceptible to infection, whereas in untreated mice these organs resisted infection (5). We reasoned that a cortisone-insensitive systemic immunity, developing in mice with established kidney infections, would protect the liver and heart against a second conidial challenge given after cortisone ' Present address: Department of Experimental Pathology, University of Birmingham, The Medical School, Birmingham B15 2TJ, U.K.
treatment. Table 1 shows the levels of fungus developing in the heart, liver, and kidneys of preinfected and normal mice, 4 days after being given cortisone and conidia. Compared with the normal mice, significantly greater resistance to infection was shown in the organs from mice preinfected 14 days before the cortisone plus conidia challenge. Protection reflected an acquired immunity and not a failure of conidia to reach the liver and heart of preinfected animals, as similar numbers were found 2 h postchallenge in the organs of both these and the controls (Table 1). Experimentally induced immunity to pathogenic yeasts, causing deep-seated mycoses, has been reported (4, 7). However, these organisms are small enough to be taken up by phagocytes, so that enhanced uptake or intracellular killing could explain the immunity. Such intracellular killing of fungal cells has been demonstrated in vitro (6), and the immunity to conidia (Table 1), which are small enough to be phagocytosed, could be due to such a mechanism. However, this cannot apply to the suppression of mycelial growth because mycelium is too bulky to be taken up by phagocytes. Injected conidia did not bring about massive mycelial growth in previously infected kidneys (Table 1). It therefore seems unlikely that the decrease of cortisone's effect on mycelial growth in the kidney, which was observed at increasing times after infection (Fig. 1), is due to a change in the innate capacity of established mycelium to respond to the cortisone treatment. Rather, it appears that an extracellular resistance mechanism, insensitive to cortisone, develops in infected mice and restricts mycelial proliferation. This mechanism is unknown; cortisone affects a large number of reactions involved in the immune response of animals (2). There was no difference in the conidial germination in the sera of normal and infected mice, and antibody-containing sera from A. fumigatus-infected rabbits did not hinder the germination of conidia (unpublished observations), an
1296
VOL. 13, 1976
NOTES
V IABLE COUNT x103
+g
+
20[
-
__
T~~~~~ J~~~~~
10[
l CITI -
I
Glucosamine
(p9)
1297
observation which agrees with others (4) that humoral factors alone do not prevent fungal growth. Destruction of A. fumigatus hyphae seems to occur, for mycelium was not found in 50% of the kidneys from mice sacrificed at 29 days after infection (Fig. 1) though only 2 of 12 mice had no fungal material in either kidney. Diamond (3) has demonstrated in vitro antibody-dependent cell-mediated killing of the yeast Cryptococcus neoformans, and a similar mechanism could be involved in our system in vivo, both with resistance to mycelial expansion (Fig. 1) and with immunity to conidial challenge (Table 1). An understanding of the mechanisms responsible for the cortisone-insensitive acquired immunity to conidial challenge and mycelial growth may lead to the development of means of preventing the onset of invasive fungal infections in patients at risk and of treating established infections.
10 .'
'
o~--
5
afterwards
groups
of mice
were
sacrificed, and the
amount of mycelium in individual kidneys was determined by viable counts and chitin assay (5). Symbols: Mycelium content of the kidneys of mice given cortisone acetate (5 mg, subcutaneously) 3 days before sacrifice; 0, kidney mycelium content of un0,
I 4 7 10 13 1629 Day after infection FIG. 1. Influence of cortisone on the growth of A. fumigatus in mouse kidneys. Male, 3-week-old LACA mice were injected intravenously with 5 x 105 conidia of A. fumigatus strain 2085. At intervals
treated mice. Points are the means of 12 infected kidneys except those at 29 days, which are the means of six infected kidneys since the others were free of mycelium. No significant difference; +, a statistically signifcant difference (P < 0.05) between the results from cortisone-treated and untreated mice sacrificed on a particular day. ,
TABLE 1. Mycelium developing in organs ofpreinfected and normal mice 4 days after administration of cortisone and challenge with A. fumigatus conidiaa Fungal material present in organ 4 days Organ
Liver
Heart
Whether mice were preinfected 14 days before challenge
No. of conidia present in organ 2 h postchallenge (mean of 2)
Yes No
114,000
Yes No
600 570
106,000
postchallenge Chitin content (J.g of glucosamine)
Viable count
80
6,700
40
350 130 5,000+ 1,000
0.04 0.2J
0.06 1.1
1,800 b
c c
11.1 ± 3.0 ) ND' Yes 12,100 + 3,300 1b 35.8 ± 3.1 ) ND' No 87,700 +t 12,0001 a Primary infection of mice was as described in Fig. 1. After 14 days groups of preinfected and uninfected mice were given 5 mg of cortisone and challenged 6 h later with 1.5 x 105 conidia. Fungal mycelium was assayed 4 days later as in Fig. 1. (Six mice per group. Mean and standard error shown. Left and right kidneys were assayed independently.) b Difference between levels developed in preinfected and normal statistically significant (P < 0.01). *, Chitin present below minimum measurable level since uninfected hearts gave relatively high blanks. d ND, Not determined due to presence of mycelium in kidneys of preinfected mice making comparison impossible.
Kidney
1298
NOTES
We wish to thank the Medical Research Council for its financial support. LITERATURE CITED 1. Bach, M. C., A. Sahyoun, J. L. Adler, R. M. Schlesinger, J. Breman, P. Madras, F. P'eng, and A. P. Monaco. 1973. High incidence of fungus infections in renal transplantation patients treated with antilymphocyte and conventional immunosuppression. Transplant. Proc. 5:549-553. 2. Claman, H. N. 1975. How corticosteroids work. J. Allergy Clin. Immunol. 55:145-151. 3. Diamond, R. D. 1974. Antibody-dependent killing of Cryptococcus neoformans by human peripheral blood mononuclear cells. Nature (London) 247:148-150.
INFECT. IMMUN. 4. Kong, Y. M., and H. B. Levine. 1967. Experimentally induced immunity in the mycoses. Bacteriol. Rev. 31:35-53. 5. Lehmann, P. F., and L. 0. White. 1975. Chitin assay used to demonstrate renal localisation and cortisone enhanced growth of Aspergillus fumigatus mycelium in mice. Infect. Immun. 12:987-992. 6. Lehrer, R. I., and M. J. Cline. 1969. Interaction of Candida albicans with human leukocytes and serum. J. Bacteriol. 98:996-1004. 7. Marra, S., and E. Balish. 1974. Immunity to Candida albicans induced by Listeria monocytogenes. Infect. Immun. 10:72-82. 8. Richard, J. L. 1975. Aspergillosis, p. 529-532. In W. T. Hubbert, W. F. McCulloch, and P. R. Schnurrenberger (ed.), Diseases transmitted from animals to man, 6th ed. Charles C Thomas, Springfield.
INFECTION AND IMMUNrrY, Apr. 1976, P. 1296-1298 Copyright C 1976 American Society for Microbiology
Acquired Immunity to Aspergillus fumigatus PAUL F. LEHMANNI* AND LES 0. WHITE Department of Microbiology, University of Birmingham, Birmingham B15 2TT, United Kingdom
Received for publication 11 November 1975
In mice preinfected with Aspergillus fumigatus for -10 days, administration of cortisone failed to stimulate mycelial growth. The liver and heart of mice preinfected for 14 days resisted infection after a cortisone + conidia challenge. To date, there has been no clear evidence of acquired immunity to Aspergillus fumigatus (8), a fungus which has caused a troublesome, often fatal infection of patients treated with immunosuppressive drugs (1). We have shown that immunosuppression with cortisone increases the susceptibility of mice to infection by A. fumigatus and that this is associated with an increased mycelial growth rate in the kidneys (5). We now present evidence that these effects can no longer be demonstrated if a localized kidney infection has been present for some time; i.e., infected mice can aquire a systemic immunity against fungal infection and growth which is not affected by cortisone. We use the term immunity as meaning the suppression of fungal multiplication as defined by Kong and Levine (4). When untreated mice were injected intravenously with 105 conidia, the infection localized in the kidneys where conidia germinated and mycelium developed (5). In the spleen, lungs, heart, and liver, no viable fungal material could be found after 4 days. Figure 1 shows the effect of cortisone on the growth of mycelium established in the kidneys. The magnitude of the cortisone effect decreased with time after infection, and cortisone brought about no significant increase in fungal growth when administered after 10 days. We wished to see whether this phenomenon was associated with the development of a cortisone-insensitive immunity to a second conidial challenge. Treatment of mice with cortisone before intravenous administration of 105 conidia rendered the liver and heart susceptible to infection, whereas in untreated mice these organs resisted infection (5). We reasoned that a cortisone-insensitive systemic immunity, developing in mice with established kidney infections, would protect the liver and heart against a second conidial challenge given after cortisone ' Present address: Department of Experimental Pathology, University of Birmingham, The Medical School, Birmingham B15 2TJ, U.K.
treatment. Table 1 shows the levels of fungus developing in the heart, liver, and kidneys of preinfected and normal mice, 4 days after being given cortisone and conidia. Compared with the normal mice, significantly greater resistance to infection was shown in the organs from mice preinfected 14 days before the cortisone plus conidia challenge. Protection reflected an acquired immunity and not a failure of conidia to reach the liver and heart of preinfected animals, as similar numbers were found 2 h postchallenge in the organs of both these and the controls (Table 1). Experimentally induced immunity to pathogenic yeasts, causing deep-seated mycoses, has been reported (4, 7). However, these organisms are small enough to be taken up by phagocytes, so that enhanced uptake or intracellular killing could explain the immunity. Such intracellular killing of fungal cells has been demonstrated in vitro (6), and the immunity to conidia (Table 1), which are small enough to be phagocytosed, could be due to such a mechanism. However, this cannot apply to the suppression of mycelial growth because mycelium is too bulky to be taken up by phagocytes. Injected conidia did not bring about massive mycelial growth in previously infected kidneys (Table 1). It therefore seems unlikely that the decrease of cortisone's effect on mycelial growth in the kidney, which was observed at increasing times after infection (Fig. 1), is due to a change in the innate capacity of established mycelium to respond to the cortisone treatment. Rather, it appears that an extracellular resistance mechanism, insensitive to cortisone, develops in infected mice and restricts mycelial proliferation. This mechanism is unknown; cortisone affects a large number of reactions involved in the immune response of animals (2). There was no difference in the conidial germination in the sera of normal and infected mice, and antibody-containing sera from A. fumigatus-infected rabbits did not hinder the germination of conidia (unpublished observations), an
1296
VOL. 13, 1976
NOTES
V IABLE COUNT x103
+g
+
20[
-
__
T~~~~~ J~~~~~
10[
l CITI -
I
Glucosamine
(p9)
1297
observation which agrees with others (4) that humoral factors alone do not prevent fungal growth. Destruction of A. fumigatus hyphae seems to occur, for mycelium was not found in 50% of the kidneys from mice sacrificed at 29 days after infection (Fig. 1) though only 2 of 12 mice had no fungal material in either kidney. Diamond (3) has demonstrated in vitro antibody-dependent cell-mediated killing of the yeast Cryptococcus neoformans, and a similar mechanism could be involved in our system in vivo, both with resistance to mycelial expansion (Fig. 1) and with immunity to conidial challenge (Table 1). An understanding of the mechanisms responsible for the cortisone-insensitive acquired immunity to conidial challenge and mycelial growth may lead to the development of means of preventing the onset of invasive fungal infections in patients at risk and of treating established infections.
10 .'
'
o~--
5
afterwards
groups
of mice
were
sacrificed, and the
amount of mycelium in individual kidneys was determined by viable counts and chitin assay (5). Symbols: Mycelium content of the kidneys of mice given cortisone acetate (5 mg, subcutaneously) 3 days before sacrifice; 0, kidney mycelium content of un0,
I 4 7 10 13 1629 Day after infection FIG. 1. Influence of cortisone on the growth of A. fumigatus in mouse kidneys. Male, 3-week-old LACA mice were injected intravenously with 5 x 105 conidia of A. fumigatus strain 2085. At intervals
treated mice. Points are the means of 12 infected kidneys except those at 29 days, which are the means of six infected kidneys since the others were free of mycelium. No significant difference; +, a statistically signifcant difference (P < 0.05) between the results from cortisone-treated and untreated mice sacrificed on a particular day. ,
TABLE 1. Mycelium developing in organs ofpreinfected and normal mice 4 days after administration of cortisone and challenge with A. fumigatus conidiaa Fungal material present in organ 4 days Organ
Liver
Heart
Whether mice were preinfected 14 days before challenge
No. of conidia present in organ 2 h postchallenge (mean of 2)
Yes No
114,000
Yes No
600 570
106,000
postchallenge Chitin content (J.g of glucosamine)
Viable count
80
6,700
40
350 130 5,000+ 1,000
0.04 0.2J
0.06 1.1
1,800 b
c c
11.1 ± 3.0 ) ND' Yes 12,100 + 3,300 1b 35.8 ± 3.1 ) ND' No 87,700 +t 12,0001 a Primary infection of mice was as described in Fig. 1. After 14 days groups of preinfected and uninfected mice were given 5 mg of cortisone and challenged 6 h later with 1.5 x 105 conidia. Fungal mycelium was assayed 4 days later as in Fig. 1. (Six mice per group. Mean and standard error shown. Left and right kidneys were assayed independently.) b Difference between levels developed in preinfected and normal statistically significant (P < 0.01). *, Chitin present below minimum measurable level since uninfected hearts gave relatively high blanks. d ND, Not determined due to presence of mycelium in kidneys of preinfected mice making comparison impossible.
Kidney
1298
NOTES
We wish to thank the Medical Research Council for its financial support. LITERATURE CITED 1. Bach, M. C., A. Sahyoun, J. L. Adler, R. M. Schlesinger, J. Breman, P. Madras, F. P'eng, and A. P. Monaco. 1973. High incidence of fungus infections in renal transplantation patients treated with antilymphocyte and conventional immunosuppression. Transplant. Proc. 5:549-553. 2. Claman, H. N. 1975. How corticosteroids work. J. Allergy Clin. Immunol. 55:145-151. 3. Diamond, R. D. 1974. Antibody-dependent killing of Cryptococcus neoformans by human peripheral blood mononuclear cells. Nature (London) 247:148-150.
INFECT. IMMUN. 4. Kong, Y. M., and H. B. Levine. 1967. Experimentally induced immunity in the mycoses. Bacteriol. Rev. 31:35-53. 5. Lehmann, P. F., and L. 0. White. 1975. Chitin assay used to demonstrate renal localisation and cortisone enhanced growth of Aspergillus fumigatus mycelium in mice. Infect. Immun. 12:987-992. 6. Lehrer, R. I., and M. J. Cline. 1969. Interaction of Candida albicans with human leukocytes and serum. J. Bacteriol. 98:996-1004. 7. Marra, S., and E. Balish. 1974. Immunity to Candida albicans induced by Listeria monocytogenes. Infect. Immun. 10:72-82. 8. Richard, J. L. 1975. Aspergillosis, p. 529-532. In W. T. Hubbert, W. F. McCulloch, and P. R. Schnurrenberger (ed.), Diseases transmitted from animals to man, 6th ed. Charles C Thomas, Springfield.
