- Email: [email protected]
Lactoferrin effects on the interaction of blood forms of Trypanosoma cruzi with mononuclear phagocytes
RESEARCH NOTE
LACTOFERFUN EFFECTS ON THE INTERACTION OF BLOOD FORMS OF TR YPANOSOMA CR UZZ WITH MONONUCLEAR PHAGOCYTES MARIA Department
of Microbiology
F. LIMA
and Public Health,
and
Michigan
(Received
F. KIERSZENBAUM* State University,
13 January
East Lansing,
MI 48824-l
101, U.S.A.
1987)
Abstract-Mouse macrophages and human monocytes displayed increased capacities to take up blood trypomastigotes of Ttypanosoma cruzi after a 24-h and 2-h lactoferrin (LF) pretreatment, respectively. Lactoferrin binding to trypomastigotes was not detectable by indirect immunofluorescence and pretreatment of the parasite with LF did not affect its capacity to interact with macrophages. Macrophages treated with LF also displayed a greater capacity to kill T. cruzi, whether the treatment was applied before or after parasite internalization. Since serum levels of LF increase during T. cruzi infection, the noted effects might play a role in host defense. INDEX
KEY WORDS:
Lactoferrin;
Trypanosoma cruzi; phagocytes.
Previous work from our laboratory has shown that lactoferrin (LF)-an iron-binding glycoprotein produced by neutrophils in increased amounts during inflammatory conditions (Pryswansky, Macrae, Spitznagel & Cooney, 1979)-can significantly enhance the capacities of macrophages and monocytes to take up and destroy Trypanosoma crud amastigotes (Lima & Kierszenbaum, 1985). The present work was designed to establish the effects of LF on macrophage or monocyte interaction with the invasive, trypomastigote (TRY) form of the parasite. Four to six-week-old Crl :CD-l(ICR)BR Swiss mice (Charles River Laboratory, Portage, Michigan), sacrificed by excess ether anesthesia, were the source of peritoneal macrophages (MPM) (Zenian & Kierszenbaum, 1982) and were also used to produce T. cruzi TRY (Tulahuen strain). The purified flagellates (Budzko & Kierszenbaum, 1974; Villalta & Leon, 1979) were suspended at 1 X 10’ organisms ml-’ in Dulbecco’s modified minimal essential medium containing penicillin (100 units/ml) and streptomycin (100 y g/ml) (DMEM), supplemented with either 1% bovine serum albumin (Sigma Chemical Co., St. Louis, Missouri) (DMEM + BSA) or 10% fetal bovine serum (Gibco, Grand Island, New York) (DMEM + FBS). Parasite viability was always >99.8%. The MPM cultures consisted of >98% nonspecific-esterase-positive cells with typical macrophage morphology. Human blood monocytes (HBM) were purified from the blood of healthy donors and cultured as described (Villalta & Kierszenbaum, 1984). These cultures consisted of >99% nonspecific-esterase-positive monocytes; cell viability, determined by trypan blue exclusion, was >99%. Monolayers of MPM or HBM were incubated at 37 “C for the appropriate periods of time with 0.3 ml of DMEM + FBS alone or containing varying concentrations of LF (described below). Unless otherwise
* Correspondence: Dr. F. Kierszenbaum, Department of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824, U.S.A. 1205
stated, LF was removed from the cultures by three washings with DMEM prior to incorporating TRY into the cultures. The human LF was 10% iron saturated (Sigma). To pretreat TRY, a mixture of 9 volumes of parasite suspension and 1 volume of DMEM + BSA alone or containing 10 times the desired final LF concentration was incubated at 37°C for 2 h. The TRY were then washed twice with DMEM (800 g, 4”C, 20 min) and resuspended in DMEM + BSA at 1 X 10’ organisms ml-‘. To measureTRY uptake by MPM or HBM, monolayers of LF-treated or mock-treated MPM or HBM received 0.3 ml of the appropriate parasite suspension and were incubated at 37 ‘C and 5% CO, for 2 h. After removing the free TRY, the cultures were fixed with methanol and stained with Giemsa. Not less than 200 cells were screened microscopically (X 1000) in each culture and the percentage of MPM or HBM with parasites and the average number of organisms per 100 cells were determined. All tests were performed in triplicate. To measure parasite killing by MPM or HBM, TRY were first co-cultured with MPM or HBM at 37 ‘C and 5% CO? for 2 h; after removing the free parasites, 0.3 ml of fresh DMEM + FBS was added and the cultures were further incubated for various periods of time. The cultures were terminated and processed as described above. In some experiments, MPM were treated with LF after they had internalized untreated TRY. In these cases, MPM monolayers co-cultured with the TRY were washed to remove the free parasites and 0.3 ml of DMEM + FBS alone or containing 1Opg LF ml-’ was added. After incubation at 37 “C for various periods of time, the cultures were washed and terminated. To examine the involvement of HzOz in parasite killing, catalase (recrystallized beef liver catalase, Sigma) was added in 0.4 ml DMEM + FBS to attain a final concentration in the cultures of 160,000 units/ ml and remained present in the culture medium during the entire experiment. Heat-inactivated (lOO”C, 20 min) catalase was used in control assays. Binding of LF by TRY was tested by incubating parasites fixed with 0.25% formaldehyde in phosphate-buffered saline pH 7.0 (PBS) and smeared on microscope slides with 10 or 100 pg LF/ml
24
Absent Present
21.1 t 2.0 42.1 54.6 (100)
23.3 t 4.2 24.6 t 0.3
24.1 + 1.4 19.0 f 0.9
% MPM with parasites (XC)t
26.3 -t 3.7 54.6 + 6.8 (108)
28.8 k 3.4 35.5 + 1.8 (23)
30.5 + 1.3 23.0 z!z2.5
No. parasites per 100 MPM (“XC)
21.6 t: 1.5 16.0 ir 2.8 (62)
21.3+ 1.4 16.8 + 2.0 132)
21.6 It 2.0 24.8 It 1.O
% MPM with parasites rhR)$
27.1 k 0.6 lY.1 k3.2 (55)
27.5 + 2.2 18.6 + 2.7 (47)
29.0 k 4.3 28.3 k 0.7
No. parasites per IO0 MPM (%R)
IOh*
18.7 k 1.2 13.5 + 2.5 (68)
20.1 i 2.2 12.8 + 1.5 148)
20.0 + 1.9 20.1 F 3.6
9/oMPM with parasites (‘%R)
23.2 k 0.7 15.8 k 2.7 (71)
24.0 dz2.1 15.3 i 2.3 (57)
26.5 i 5.6 26.1 14.3
No. parasites per 100 MPM p&R)
22 h*
The MPM were incubated with LF for 6, 12 or 24 h, washed and cultured with the TRY for 2 h. After removing the free TRY, one third of the MPM cultures were fixed. The received fresh medium and were incubated for an additional 10 or 22 h, (XC), per cent increasein parasite uptake (calculated with respect to the value obtained in the absence of LF); shown where statistically significant ( PS 0.05). (%RJ, per cent reduction in parasite load with respect to the O-h value; shown where statistically significant ( PS 0.05).
12
Absent Present
* rest t $
6
Time* fh)
0 h*
I-MINIMAI. PKETREATMENT TIMEOFMPM WITHLF KEQU~RED FORPRODW~~ON OFENHA~~CED UPTAKE ANDK~L~KGOFmooo foaMs OF ‘f. cruzi
Absent Present
LF (lOpg/ml)
TABLE
s
‘2
Research in DMEM + BSA for 2 h. The smears were then washed three times with PBS and incubated with fluorescein-labeled rabbit anti-human LF IgG (Cappel Laboratories, West Chester, Pennsylvania) at 37°C for 30 min. After washing again with PBS, the slides were air dried and examined by fluorescence microscopy. Data in this paper are expressed as the mean + 1 so. The sets of results are typically representative of two to four repeat experiments. Parasite loads measured immediatelv after removing the non-bound TRY were always greater-in MPM pretreated with 10 fig LF ml-’ for 24 h than in mock-treated MPM (Table l), although in some repeat experiments (e.g., the one represented in Table l), a relatively small but nevertheless significant increase was also seen after a 12-h LF pretreatment. A similar effect was seen with HBM but a 2-h LF pretreatment with either 10 or 100 pg LF ml-’ sufficed to elicit it (Table 2, results obtained at 0 h). The average number of receptors for LF per cell has been determined to be 2 X 10’ for MPM (van Snick & Masson, 1976) and 2 X lox for HBM (Bennett & Davis, 1981). This difference could explain why HBM required a shorter LF treatment. No significant change in the extent of MPM-TRY interaction was seen when the parasites were pretreated with up to 100 pg LF ml-l for 2 h (data not shown). In view of this result, we tested whether the TRY would bind LF. Indirect immunofluorescence tests failed to evidence such binding even after parasite incubation with up to 100 pg LF ml-‘. Thus, the LF effect appeared to be on the phagocytes. When MPM were pretreated with 10 pug LF ml-’ for 2 h, washed, exposed to TRY for 2 h in the absence of LF and then incubated with DMEM + FBS for 70 h, the number of parasites per 100 MPM was significantly smaller (45.1 f 3.8) than that of mock-treated MPM (105.5 f 5.6). Similar results were obtained when MPM and TRY were cocultured for 2 h in the presence of 10 pug LF ml-’ and measurements were made 70 h after removing LF and the free TRY (data not shown). However, the percentages of infected MPM were not affected by LF over the 70-h period (data not shown). The reduction in parasite load without a significant change in the proportion of infected cells implied that LF-treated MPM were clearing the parasites. This notion found support in the observation that pretreatment of MPM with 10 p g LF ml-’ for 24 h and of HMB with 10 or 100 ,u g LF ml-’ for 2 h increased their cytotoxic capacities (Table 1, results obtained after 10 or 22 h; Table 2, results obtained after 10 h). LF-treated phagocytes could have destroyed more TRY simply because more had been taken up initially or because they had been activated. To test these possibilities, we measured TRY killing by MPM receiving the LF treatment after parasite internalization. In the various
Note
1207
repeat experiments, addition of 10 ,u g LF ml-’ to the culture medium caused the MPM to destroy over 54% of the internalized organisms over a 72 h period whereas substantial parasite growth occurred in the mock-treated MPM 100 MPM to (e.g., from 47.3 rf- 6.4 parasites per 127.5 & 11.3 during the same period of time). Thus, LF enhanced cytotoxicitv via MPM activation. That H,d, played a role in TRY killing by LF-stimulated MPM was inferred from the observation that MPM which had been pretreated with 10 ,ug LF ml-’ for 24 h and then incubated with TRY failed to clear significant numbers of organisms when catalase, a scavenger of H>O,, was present (data not shown). This was not the case when catalase was absent or when heat-inactivated catalase was used. Neither catalase nor heated catalase affected the level of TRY internalization by mediumor LF-treated MPM. The involvement of H,OZ in parasite killing by LF-treated MPM was also an additional indication that LF had activated the MPM. It is noteworthy that catalase also inhibits TRY destruction by MPM stimulated with interferon gamma mirth. Kierszenbaum. Sonnenfeld & Zlotnik. 1985). ’ The’ concentrations of LF that enhanced phagocyte interaction with T. cruzi were within the range found in the plasma of patients with inflammatory conditions (Hansen, Karle, Andersen, Malmquist & Hoff, 1976). Inflammation occurs in Chagas’ disease and serum levels of LF were found to be increased in mice during acute T. cruzi infection (Lima & Kierszenbaum, unpublished results). Thus, the conditions producing enhanced amastigote (Lima & Kierszenbaum, 1985) and TRY disposal by mononuclear phagocytes in vitro are given in viva and might enhance host resistance. This work was supported by United States Public Health Service grants AI 14848 and AI 1704 1.
REFERENCES BENNETT R. M. & DAVIS J. 1981. Journal of Immunolo~-, 127: 1211-1216. BIJDZKO D. B. & KIERSZENBAUMF. 1974. Journal of Parasitology 60: 1037-1038. HANSEN N. E., KARLE H., ANDERSEN V., MALMQUIST J. & HOFF G. E. 1976. Clinical and Experimental Immuno-
logy 26: 463-468. LIMA M. F. & KIERSZENBAUM F. 1985. Journal of Immunology 134:4176-4183. PRYSWANSKY K.B., MACRAE E. K., SPITZNAGEL J.K. & Coons M.H. 1979. Cell 18: 1025-1033. VAN SNICK J. R. & MASSON P. L. 1976. Journal of Experimental Medicine 144: 1568-l S80.
TABLE ~--EFFECTS OF PRETREATMENTOF HBM WITH LF ON THEIR CAPACITYTO T. cruzi
TAKE
UP AND
Oh Treatment of HBM* DMEM + FBS LF, lOO~gg/ml 10 p g/ml
KlLL BLOOD
FORMS
OF
10h
% HBM with parasites (XC)?
No. parasites per 100 HBM (%C)
% HBM with parasites rhR]$
No. parasites per 100 HBM [%R)
18.2 f 3.2 40.2 26.0 f 2.1 3.9 (121) (43)
19.4 +_ 2.8 53.0 + 30.5 f 6.4 2.8 (57) (173)
15.0 f 0.7 10.5 6.0 Jr f 0.7 1.4 (60) [85)
19.5 f 2.1 13.7 7.5 + f 5.3 3.5 (55) (87)
* The HBM were incubated with LF for 1 h, washed and cultured with the TRY for 2 h. After removing the free TRY, half of the cultures were fixed (0 h). The rest received fresh medium and were incubated for an additional 10 h. t$ See footnotes under Table 1.
1208
M. F. LIMA and F. KIERSZENBAUM
VILLALTA F. & KIERSZENBAUM F. 1984. Journal of Immunology 133,3338-3343. VILLALTAF. & LEON W. 1979. Journal of Parasitology 65: 188-189.
WIRTH .I. J., KIERSZENBAUMF., SONNENFELDG. & ZLOTNIK A. 1985. Infection and Immunity 49: 6 I-66. ZENIAN A. & KIERSZENB~UM F. 1982. Journul of Parasitology 68: 408-4 IS.
LACTOFERFUN EFFECTS ON THE INTERACTION OF BLOOD FORMS OF TR YPANOSOMA CR UZZ WITH MONONUCLEAR PHAGOCYTES MARIA Department
of Microbiology
F. LIMA
and Public Health,
and
Michigan
(Received
F. KIERSZENBAUM* State University,
13 January
East Lansing,
MI 48824-l
101, U.S.A.
1987)
Abstract-Mouse macrophages and human monocytes displayed increased capacities to take up blood trypomastigotes of Ttypanosoma cruzi after a 24-h and 2-h lactoferrin (LF) pretreatment, respectively. Lactoferrin binding to trypomastigotes was not detectable by indirect immunofluorescence and pretreatment of the parasite with LF did not affect its capacity to interact with macrophages. Macrophages treated with LF also displayed a greater capacity to kill T. cruzi, whether the treatment was applied before or after parasite internalization. Since serum levels of LF increase during T. cruzi infection, the noted effects might play a role in host defense. INDEX
KEY WORDS:
Lactoferrin;
Trypanosoma cruzi; phagocytes.
Previous work from our laboratory has shown that lactoferrin (LF)-an iron-binding glycoprotein produced by neutrophils in increased amounts during inflammatory conditions (Pryswansky, Macrae, Spitznagel & Cooney, 1979)-can significantly enhance the capacities of macrophages and monocytes to take up and destroy Trypanosoma crud amastigotes (Lima & Kierszenbaum, 1985). The present work was designed to establish the effects of LF on macrophage or monocyte interaction with the invasive, trypomastigote (TRY) form of the parasite. Four to six-week-old Crl :CD-l(ICR)BR Swiss mice (Charles River Laboratory, Portage, Michigan), sacrificed by excess ether anesthesia, were the source of peritoneal macrophages (MPM) (Zenian & Kierszenbaum, 1982) and were also used to produce T. cruzi TRY (Tulahuen strain). The purified flagellates (Budzko & Kierszenbaum, 1974; Villalta & Leon, 1979) were suspended at 1 X 10’ organisms ml-’ in Dulbecco’s modified minimal essential medium containing penicillin (100 units/ml) and streptomycin (100 y g/ml) (DMEM), supplemented with either 1% bovine serum albumin (Sigma Chemical Co., St. Louis, Missouri) (DMEM + BSA) or 10% fetal bovine serum (Gibco, Grand Island, New York) (DMEM + FBS). Parasite viability was always >99.8%. The MPM cultures consisted of >98% nonspecific-esterase-positive cells with typical macrophage morphology. Human blood monocytes (HBM) were purified from the blood of healthy donors and cultured as described (Villalta & Kierszenbaum, 1984). These cultures consisted of >99% nonspecific-esterase-positive monocytes; cell viability, determined by trypan blue exclusion, was >99%. Monolayers of MPM or HBM were incubated at 37 “C for the appropriate periods of time with 0.3 ml of DMEM + FBS alone or containing varying concentrations of LF (described below). Unless otherwise
* Correspondence: Dr. F. Kierszenbaum, Department of Microbiology and Public Health, Michigan State University, East Lansing, MI 48824, U.S.A. 1205
stated, LF was removed from the cultures by three washings with DMEM prior to incorporating TRY into the cultures. The human LF was 10% iron saturated (Sigma). To pretreat TRY, a mixture of 9 volumes of parasite suspension and 1 volume of DMEM + BSA alone or containing 10 times the desired final LF concentration was incubated at 37°C for 2 h. The TRY were then washed twice with DMEM (800 g, 4”C, 20 min) and resuspended in DMEM + BSA at 1 X 10’ organisms ml-‘. To measureTRY uptake by MPM or HBM, monolayers of LF-treated or mock-treated MPM or HBM received 0.3 ml of the appropriate parasite suspension and were incubated at 37 ‘C and 5% CO, for 2 h. After removing the free TRY, the cultures were fixed with methanol and stained with Giemsa. Not less than 200 cells were screened microscopically (X 1000) in each culture and the percentage of MPM or HBM with parasites and the average number of organisms per 100 cells were determined. All tests were performed in triplicate. To measure parasite killing by MPM or HBM, TRY were first co-cultured with MPM or HBM at 37 ‘C and 5% CO? for 2 h; after removing the free parasites, 0.3 ml of fresh DMEM + FBS was added and the cultures were further incubated for various periods of time. The cultures were terminated and processed as described above. In some experiments, MPM were treated with LF after they had internalized untreated TRY. In these cases, MPM monolayers co-cultured with the TRY were washed to remove the free parasites and 0.3 ml of DMEM + FBS alone or containing 1Opg LF ml-’ was added. After incubation at 37 “C for various periods of time, the cultures were washed and terminated. To examine the involvement of HzOz in parasite killing, catalase (recrystallized beef liver catalase, Sigma) was added in 0.4 ml DMEM + FBS to attain a final concentration in the cultures of 160,000 units/ ml and remained present in the culture medium during the entire experiment. Heat-inactivated (lOO”C, 20 min) catalase was used in control assays. Binding of LF by TRY was tested by incubating parasites fixed with 0.25% formaldehyde in phosphate-buffered saline pH 7.0 (PBS) and smeared on microscope slides with 10 or 100 pg LF/ml
24
Absent Present
21.1 t 2.0 42.1 54.6 (100)
23.3 t 4.2 24.6 t 0.3
24.1 + 1.4 19.0 f 0.9
% MPM with parasites (XC)t
26.3 -t 3.7 54.6 + 6.8 (108)
28.8 k 3.4 35.5 + 1.8 (23)
30.5 + 1.3 23.0 z!z2.5
No. parasites per 100 MPM (“XC)
21.6 t: 1.5 16.0 ir 2.8 (62)
21.3+ 1.4 16.8 + 2.0 132)
21.6 It 2.0 24.8 It 1.O
% MPM with parasites rhR)$
27.1 k 0.6 lY.1 k3.2 (55)
27.5 + 2.2 18.6 + 2.7 (47)
29.0 k 4.3 28.3 k 0.7
No. parasites per IO0 MPM (%R)
IOh*
18.7 k 1.2 13.5 + 2.5 (68)
20.1 i 2.2 12.8 + 1.5 148)
20.0 + 1.9 20.1 F 3.6
9/oMPM with parasites (‘%R)
23.2 k 0.7 15.8 k 2.7 (71)
24.0 dz2.1 15.3 i 2.3 (57)
26.5 i 5.6 26.1 14.3
No. parasites per 100 MPM p&R)
22 h*
The MPM were incubated with LF for 6, 12 or 24 h, washed and cultured with the TRY for 2 h. After removing the free TRY, one third of the MPM cultures were fixed. The received fresh medium and were incubated for an additional 10 or 22 h, (XC), per cent increasein parasite uptake (calculated with respect to the value obtained in the absence of LF); shown where statistically significant ( PS 0.05). (%RJ, per cent reduction in parasite load with respect to the O-h value; shown where statistically significant ( PS 0.05).
12
Absent Present
* rest t $
6
Time* fh)
0 h*
I-MINIMAI. PKETREATMENT TIMEOFMPM WITHLF KEQU~RED FORPRODW~~ON OFENHA~~CED UPTAKE ANDK~L~KGOFmooo foaMs OF ‘f. cruzi
Absent Present
LF (lOpg/ml)
TABLE
s
‘2
Research in DMEM + BSA for 2 h. The smears were then washed three times with PBS and incubated with fluorescein-labeled rabbit anti-human LF IgG (Cappel Laboratories, West Chester, Pennsylvania) at 37°C for 30 min. After washing again with PBS, the slides were air dried and examined by fluorescence microscopy. Data in this paper are expressed as the mean + 1 so. The sets of results are typically representative of two to four repeat experiments. Parasite loads measured immediatelv after removing the non-bound TRY were always greater-in MPM pretreated with 10 fig LF ml-’ for 24 h than in mock-treated MPM (Table l), although in some repeat experiments (e.g., the one represented in Table l), a relatively small but nevertheless significant increase was also seen after a 12-h LF pretreatment. A similar effect was seen with HBM but a 2-h LF pretreatment with either 10 or 100 pg LF ml-’ sufficed to elicit it (Table 2, results obtained at 0 h). The average number of receptors for LF per cell has been determined to be 2 X 10’ for MPM (van Snick & Masson, 1976) and 2 X lox for HBM (Bennett & Davis, 1981). This difference could explain why HBM required a shorter LF treatment. No significant change in the extent of MPM-TRY interaction was seen when the parasites were pretreated with up to 100 pg LF ml-l for 2 h (data not shown). In view of this result, we tested whether the TRY would bind LF. Indirect immunofluorescence tests failed to evidence such binding even after parasite incubation with up to 100 pg LF ml-‘. Thus, the LF effect appeared to be on the phagocytes. When MPM were pretreated with 10 pug LF ml-’ for 2 h, washed, exposed to TRY for 2 h in the absence of LF and then incubated with DMEM + FBS for 70 h, the number of parasites per 100 MPM was significantly smaller (45.1 f 3.8) than that of mock-treated MPM (105.5 f 5.6). Similar results were obtained when MPM and TRY were cocultured for 2 h in the presence of 10 pug LF ml-’ and measurements were made 70 h after removing LF and the free TRY (data not shown). However, the percentages of infected MPM were not affected by LF over the 70-h period (data not shown). The reduction in parasite load without a significant change in the proportion of infected cells implied that LF-treated MPM were clearing the parasites. This notion found support in the observation that pretreatment of MPM with 10 p g LF ml-’ for 24 h and of HMB with 10 or 100 ,u g LF ml-’ for 2 h increased their cytotoxic capacities (Table 1, results obtained after 10 or 22 h; Table 2, results obtained after 10 h). LF-treated phagocytes could have destroyed more TRY simply because more had been taken up initially or because they had been activated. To test these possibilities, we measured TRY killing by MPM receiving the LF treatment after parasite internalization. In the various
Note
1207
repeat experiments, addition of 10 ,u g LF ml-’ to the culture medium caused the MPM to destroy over 54% of the internalized organisms over a 72 h period whereas substantial parasite growth occurred in the mock-treated MPM 100 MPM to (e.g., from 47.3 rf- 6.4 parasites per 127.5 & 11.3 during the same period of time). Thus, LF enhanced cytotoxicitv via MPM activation. That H,d, played a role in TRY killing by LF-stimulated MPM was inferred from the observation that MPM which had been pretreated with 10 ,ug LF ml-’ for 24 h and then incubated with TRY failed to clear significant numbers of organisms when catalase, a scavenger of H>O,, was present (data not shown). This was not the case when catalase was absent or when heat-inactivated catalase was used. Neither catalase nor heated catalase affected the level of TRY internalization by mediumor LF-treated MPM. The involvement of H,OZ in parasite killing by LF-treated MPM was also an additional indication that LF had activated the MPM. It is noteworthy that catalase also inhibits TRY destruction by MPM stimulated with interferon gamma mirth. Kierszenbaum. Sonnenfeld & Zlotnik. 1985). ’ The’ concentrations of LF that enhanced phagocyte interaction with T. cruzi were within the range found in the plasma of patients with inflammatory conditions (Hansen, Karle, Andersen, Malmquist & Hoff, 1976). Inflammation occurs in Chagas’ disease and serum levels of LF were found to be increased in mice during acute T. cruzi infection (Lima & Kierszenbaum, unpublished results). Thus, the conditions producing enhanced amastigote (Lima & Kierszenbaum, 1985) and TRY disposal by mononuclear phagocytes in vitro are given in viva and might enhance host resistance. This work was supported by United States Public Health Service grants AI 14848 and AI 1704 1.
REFERENCES BENNETT R. M. & DAVIS J. 1981. Journal of Immunolo~-, 127: 1211-1216. BIJDZKO D. B. & KIERSZENBAUMF. 1974. Journal of Parasitology 60: 1037-1038. HANSEN N. E., KARLE H., ANDERSEN V., MALMQUIST J. & HOFF G. E. 1976. Clinical and Experimental Immuno-
logy 26: 463-468. LIMA M. F. & KIERSZENBAUM F. 1985. Journal of Immunology 134:4176-4183. PRYSWANSKY K.B., MACRAE E. K., SPITZNAGEL J.K. & Coons M.H. 1979. Cell 18: 1025-1033. VAN SNICK J. R. & MASSON P. L. 1976. Journal of Experimental Medicine 144: 1568-l S80.
TABLE ~--EFFECTS OF PRETREATMENTOF HBM WITH LF ON THEIR CAPACITYTO T. cruzi
TAKE
UP AND
Oh Treatment of HBM* DMEM + FBS LF, lOO~gg/ml 10 p g/ml
KlLL BLOOD
FORMS
OF
10h
% HBM with parasites (XC)?
No. parasites per 100 HBM (%C)
% HBM with parasites rhR]$
No. parasites per 100 HBM [%R)
18.2 f 3.2 40.2 26.0 f 2.1 3.9 (121) (43)
19.4 +_ 2.8 53.0 + 30.5 f 6.4 2.8 (57) (173)
15.0 f 0.7 10.5 6.0 Jr f 0.7 1.4 (60) [85)
19.5 f 2.1 13.7 7.5 + f 5.3 3.5 (55) (87)
* The HBM were incubated with LF for 1 h, washed and cultured with the TRY for 2 h. After removing the free TRY, half of the cultures were fixed (0 h). The rest received fresh medium and were incubated for an additional 10 h. t$ See footnotes under Table 1.
1208
M. F. LIMA and F. KIERSZENBAUM
VILLALTA F. & KIERSZENBAUM F. 1984. Journal of Immunology 133,3338-3343. VILLALTAF. & LEON W. 1979. Journal of Parasitology 65: 188-189.
WIRTH .I. J., KIERSZENBAUMF., SONNENFELDG. & ZLOTNIK A. 1985. Infection and Immunity 49: 6 I-66. ZENIAN A. & KIERSZENB~UM F. 1982. Journul of Parasitology 68: 408-4 IS.