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RAISED SERUM LEVELS OF TUMOUR NECROSIS FACTOR IN PARASITIC INFECTIONS
1364
RAISED SERUM LEVELS OF TUMOUR NECROSIS FACTOR IN PARASITIC INFECTIONS PHILIP SCUDERI KIT S. LAM KENNETH J. RYAN ESKILD PETERSEN
KAREN E. STERLING PAUL R. FINLEY C. GEORGE RAY DONALD J. SLYMEN
SYDNEY F. SALMON
Departments of Microbiology and Immunology, Internal Medicine, and Pathology and Arizona Cancer Center, University of Arizona College of Medicine, Tucson, Arizona, USA
study of serum levels of endogenous necrosis factor (TNF) in healthy people and patients with neoplastic or infectious disease, only patients with kala-azar (visceral leishmaniasis) and malaria were found to have a strikingly increased frequency of raised TFN levels (66.6% and 70.0%, respectively). 7.9% of samples from both healthy subjects and patients with neoplastic disease contained measurable TNF. The discovery of elevated TNF levels in the sera of patients with parasitic diseases suggests that this cytokine may play a part in host defences against parasitic infections. Summary
In
a
tumour
,
Introduction
TUMOUR necrosis factor (TNF) is a cytokine, produced by macrophages, which can cause the destruction of tumour cells both in vitr01 and in vivo.2 TNF has a wide variety of biological actions, including the capacity to stimulate the proliferation of normal diploid fibroblasts3 and promote bone resorption.4 It might also have cytotoxic activity against malarial parasites.5,6 TNF has also been linked, because of its structure, to cachectin, a factor thought to have a role in weight-loss in parasite-infested animals.’ Little is known about the relation between TNF and human disease. The present study was conducted to determine whether serum TNF levels were associated with various neoplastic or infectious diseases. Methods
Enzyme Linked Immunosorbent Assay To detect TNF a monoclonal antibody specific for TNF and a set of standards made with human recombinant tumour necrosis factor (rTNF)8 (generously provided by Genentech Inc, San Francisco, CA) were used. The enzyme-linked immunosorbent assay (ELISA) produced with these reagents was sensitive to TNF concentrations in serum above 39 pg/ml. The ELISA was devised by coating 96-well plates with 6 25 ng/well of murine monoclonal antibody 6E with specificity for human TNF. Before use and between subsequent steps in the assay coated plates were washed twice with phosphate-buffered saline (PBS) containing 0-05% ’Tween-20’ (PBS-tween) and twice with PBS alone. All reagents used in this assay were incubated for 1 h at room temperature with coated wells. For the standard curve rTNF was added to serum previously determined to be negative for endogenous TNF. After exposure to serum, assay plates were sequentially exposed to rabbit anti-TNF, horseradish-peroxidase-conjugated goat anti-rabbit Ig, and 2,2’-azinobis (3-ethylbenz-thiazoline sulphonic acid substrate (Sigma Inc, St Louis, MO). Optical density readings were made within 10 min of addition of the substrate on a Titertek multiscan (Flow Labs Inc, McLean, VA) with a 405 nrn filter. Appropriate specificity controls were included. After addition of known amounts of rTNF to whole venous blood, equivalent cytokine levels were detected in either plasma or serum derived from such blood samples. In the current study only sera were tested.
Human Serum
Samples
Normal adult sera were obtained from blood-bank donors, sera from healthy pregnant women were collected during the first and
second trimesters, and neonatal samples came from cord blood obtained at the time of delivery. Sera from patients diagnosed as having neoplastic disease were collected in the course of testing for serum markers used as prognostic indicators of disease progress. Sera tested for brucella, treponema, entamoeba, toxoplasma, and viruses were assayed by the detection of serum antibodies to these agents. The remaining bacterial species and yeast were detected by the growth of organisms in cultures from the blood samples. In the cryptococcus sera antigen was detected with a latex agglutination assay. Leishmania donovani infection was diagnosed by detection of the parasites in biopsy tissue obtained from the lymph nodes and bone marrow of patients with kala-azar in the Sudan. Leishmania was also identified in some patients by the growth of organisms in culture. Plasmodium falciparum and P vivax were identified by morphological criteria in giemsa-stained blood smears from patients with malaria in Thailand.
Results Normal Sera Of the 88 sera from healthy subjects 7 (7-9%) contained measurable TNF. The positive results were reproducible on repeated testing. 8-7% of adult donors were positive, and 11-7% of pregnant women had detectable serum levels. None of the 14 neonatal cord blood samples examined were
positive. Sera from Patients with Infectious and Neoplastic Disease When sera from patients with a variety of infectious diseases were examined, only those from patients with visceral leishmaniasis or malaria showed a raised frequency of elevated TNF levels (table I) Ofthe 27 samples from TABLE I-TNF DETECTED IN SERA FROM PATIENTS WITH INFECTIOUS AND NEOPLASTIC DISEASES
1365
Distribution of TNF levels among the various groups of
samples.
serum
_
Serum samples listed under parasitic diseases were taken from patients with kala-azar (W) and malaria (+). Among the sera from patients with malaria, 4 were from individuals diagnosed as having been infected with Plasmodiumfalciparurra, 4 with Pvivax, and 2 with unidentified Plasmodium
species. TABLE II-GEOMETRIC MEAN TNF LEVELS IN PATIENTS WITH EITHER INFECTIOUS OR NEOPLASTIC DISEASES AND NORMAL
CONTROLS*
suggests that TNF may in some way serve as a host defence mechanism against Leishmania donovani and Plasmodium spp and perhaps other parasitic organisms. Malarial parasites are sensitive to the action of endotoxin-induced serum factors in vitro,s°6 which supports the idea that TNF may play an active part in parasitic infections. There is also evidence that serum factors may be active against Leishmania, at several stages in the parasite’s life cycle. L donovani enters the human host as a promastigote form and subsequently binds to macrophages. Once the parasite enters these cells it converts to the amastigote form.12 Both the promastigote and amastigote carry antigens which serve as targets for immune recognition. Normal human serum contains both IgG and IgM antibody capable of recognising promastigotes. 13 The parasite may, however, be able to circumvent humoral and cell-mediated immune responses by shedding factors that have antigenic cross-reactivity with parasite surface components.14 Host defences which do not rely on antigen recognition would be of benefit in such a situation. Heat-labile serum factors from both healthy and L donovani infected patients have been described which are able to mediate the lysis of amastigotes.15 TNF and other cytokines are reasonable candidates for additional host defences which do not involve immune mechanisms
requiring antigen recognition. This work was supported by grants CA-17094, CA-21839, CA-23074, and AI16312-07 and a basic research support grant from the National Institutes of Health, grant IN-110 from the American Cancer Society, and grant 3364-000000-1-1-AP-6621 from the Arizona Chronic Disease Research Commission.
*Samples below the detection level of 40pg/ml were assumed to have a value of 20-0 pg/ml, the midpoint of the interval between 0 and 40 0. Wilcoxon 2-sample tests were used to examine specific differences among the sample groups. A significance level of 0-01 was used for each comparison to accommodate multiple comparisons. The parasitic diseases were compared with the normal, neoplastic, and infectious disease groups and found to be significantly different (p <0’001). tKala-azar and malaria.
Correspondence should be addressed to P. S., University of Arizona Center, University Department of Microbiology and Immunology, Tucson, Arizona 85724, USA.
Health Sciences
REFERENCES
patients with kala-azar,
18
(66 6%) contained detectable
TNF. Of 44 additional sera from Sudanese donors in whom kala-azar was suspected but leishmania was not detected, only 5 (11 9%) were positive. Among the 10 samples from patients with malaria 7 (70-0%) contained measurable concentrations of TNF. Sera were examined from 176 patients diagnosed as having a variety of neoplastic diseases and representing a range of disease activity from clinical remission to progressive cancer. Among sera from this group a total of 14 (7-9%) samples were found to contain TNF (table I). Sera from three of the four largest subgroups within this category--colon, ovarian, and breast carcinoma-showed similar percentages of positive results. Samples from patients with multiple myeloma were all negative. Although elevated TNF values were encountered in the kala-azar and malaria samples, the maximum and minimum levels were similar in all sample groups (see figure). Samples taken from patients with kala-azar or malaria had geometric mean TNF levels nearly five times higher than any of the other sample groups (table n). In this respect TNF serum levels in samples from patients with parasitic diseases were significantly different from all others tested. Discussion Of the diseases studied, kala-azar and malaria alone were associated with substantially raised serum TNF levels. This
1. Matthews N. Anti-tumour
cytotoxin produced by human anti-tumour cytotoxin produced by human monocytes: Studies on its mode of action. Br J Cancer 1983;
48: 405-10. 2. Helson L, Helson C, Green S. Effects of murine tumour necrosis factor on heterotransplanted human tumors. Expl Cell Biol 1979, 47: 53-60 3. Sugarman BJ, Aggarwal BB, Hass PE, Palladino MA, Shepard HM. Recombinant human tumour necrosis factor-alpha: Effects on proliferation of normal and transformed cells in vitro. Science 1985; 230: 943-45. 4. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature 1986; 319: 516-18. 5. Taveme J, Depledge P, Playfair JHL. Differential sensitivity in vivo of lethal and nonlethal malarial parasites to endotoxin-induced serum factor. Infect Immun 1982, 37: 927-34.
6. Taveme J, Matthews N, Depledge P, Playfair HL. Malarial parasites and tumour cells are killed by the same component of tumour necrosis serum. Clin Exp Immunol 1984; 57: 293-300. 7. Beutler B, Mahoney J, Trang NL, Pekala P, Cerami A. Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264 7 cells. J Exp Med 1985; 161: 984-95. 8. Penica D, Nedwin GE, Hayflick JS, et al. Human tumour necrosis factor: Precursor structure, expression and homology to lymphotoxin. Nature 1984; 312: 724-29. 9. Blackwell JM. Receptors and recognition mechanisms of leishmania species. Roy Soc Trop Med Hyg 1985; 79: 606-12. 10. Beutler B, Greenwold D, Hulmes JD, et al. Identity of tumour necrosis factor and macrophage-secreted factor cachectin. Nature 1985; 316: 552-54. 11. Torti FM, Dieckmann B, Beutler B, Cerami A, Ringold GM. A macrophage factor inhibits adipocyte gene expression: An in vitro model of cachexia. Science 1985; 229: 867-71. 12. Chang KP, Dwyer DM. Multiplication of human parasite (Leishmama donovani) in phagolysosomes of hampster macrophages in vitro. Science 1976; 193: 678-80. 13. Pearson RD, Steigbigel RT. Mechanism of lethal effect of human serum upon Leishmania donovani. J Immunol 1980; 125: 2195-201. 14. El-On J, Schnur LF, Greenblatt CL. Leishmania donovani: Physiochemical, immunological, and biological characterisation of excreted factor from promastigotes. Exp Parasitol 1979; 47: 254-69. 15. Hoover DL, Berger M, Oppenheim MH, Hockmeyer WT, Meltzer MS Cytotoxicity of human serum for Leishmania donovani amastigotes: Antibody facilitation of alternate complement pathway-mediated killing. Infect Immun 1985; 47: 247-52.
RAISED SERUM LEVELS OF TUMOUR NECROSIS FACTOR IN PARASITIC INFECTIONS PHILIP SCUDERI KIT S. LAM KENNETH J. RYAN ESKILD PETERSEN
KAREN E. STERLING PAUL R. FINLEY C. GEORGE RAY DONALD J. SLYMEN
SYDNEY F. SALMON
Departments of Microbiology and Immunology, Internal Medicine, and Pathology and Arizona Cancer Center, University of Arizona College of Medicine, Tucson, Arizona, USA
study of serum levels of endogenous necrosis factor (TNF) in healthy people and patients with neoplastic or infectious disease, only patients with kala-azar (visceral leishmaniasis) and malaria were found to have a strikingly increased frequency of raised TFN levels (66.6% and 70.0%, respectively). 7.9% of samples from both healthy subjects and patients with neoplastic disease contained measurable TNF. The discovery of elevated TNF levels in the sera of patients with parasitic diseases suggests that this cytokine may play a part in host defences against parasitic infections. Summary
In
a
tumour
,
Introduction
TUMOUR necrosis factor (TNF) is a cytokine, produced by macrophages, which can cause the destruction of tumour cells both in vitr01 and in vivo.2 TNF has a wide variety of biological actions, including the capacity to stimulate the proliferation of normal diploid fibroblasts3 and promote bone resorption.4 It might also have cytotoxic activity against malarial parasites.5,6 TNF has also been linked, because of its structure, to cachectin, a factor thought to have a role in weight-loss in parasite-infested animals.’ Little is known about the relation between TNF and human disease. The present study was conducted to determine whether serum TNF levels were associated with various neoplastic or infectious diseases. Methods
Enzyme Linked Immunosorbent Assay To detect TNF a monoclonal antibody specific for TNF and a set of standards made with human recombinant tumour necrosis factor (rTNF)8 (generously provided by Genentech Inc, San Francisco, CA) were used. The enzyme-linked immunosorbent assay (ELISA) produced with these reagents was sensitive to TNF concentrations in serum above 39 pg/ml. The ELISA was devised by coating 96-well plates with 6 25 ng/well of murine monoclonal antibody 6E with specificity for human TNF. Before use and between subsequent steps in the assay coated plates were washed twice with phosphate-buffered saline (PBS) containing 0-05% ’Tween-20’ (PBS-tween) and twice with PBS alone. All reagents used in this assay were incubated for 1 h at room temperature with coated wells. For the standard curve rTNF was added to serum previously determined to be negative for endogenous TNF. After exposure to serum, assay plates were sequentially exposed to rabbit anti-TNF, horseradish-peroxidase-conjugated goat anti-rabbit Ig, and 2,2’-azinobis (3-ethylbenz-thiazoline sulphonic acid substrate (Sigma Inc, St Louis, MO). Optical density readings were made within 10 min of addition of the substrate on a Titertek multiscan (Flow Labs Inc, McLean, VA) with a 405 nrn filter. Appropriate specificity controls were included. After addition of known amounts of rTNF to whole venous blood, equivalent cytokine levels were detected in either plasma or serum derived from such blood samples. In the current study only sera were tested.
Human Serum
Samples
Normal adult sera were obtained from blood-bank donors, sera from healthy pregnant women were collected during the first and
second trimesters, and neonatal samples came from cord blood obtained at the time of delivery. Sera from patients diagnosed as having neoplastic disease were collected in the course of testing for serum markers used as prognostic indicators of disease progress. Sera tested for brucella, treponema, entamoeba, toxoplasma, and viruses were assayed by the detection of serum antibodies to these agents. The remaining bacterial species and yeast were detected by the growth of organisms in cultures from the blood samples. In the cryptococcus sera antigen was detected with a latex agglutination assay. Leishmania donovani infection was diagnosed by detection of the parasites in biopsy tissue obtained from the lymph nodes and bone marrow of patients with kala-azar in the Sudan. Leishmania was also identified in some patients by the growth of organisms in culture. Plasmodium falciparum and P vivax were identified by morphological criteria in giemsa-stained blood smears from patients with malaria in Thailand.
Results Normal Sera Of the 88 sera from healthy subjects 7 (7-9%) contained measurable TNF. The positive results were reproducible on repeated testing. 8-7% of adult donors were positive, and 11-7% of pregnant women had detectable serum levels. None of the 14 neonatal cord blood samples examined were
positive. Sera from Patients with Infectious and Neoplastic Disease When sera from patients with a variety of infectious diseases were examined, only those from patients with visceral leishmaniasis or malaria showed a raised frequency of elevated TNF levels (table I) Ofthe 27 samples from TABLE I-TNF DETECTED IN SERA FROM PATIENTS WITH INFECTIOUS AND NEOPLASTIC DISEASES
1365
Distribution of TNF levels among the various groups of
samples.
serum
_
Serum samples listed under parasitic diseases were taken from patients with kala-azar (W) and malaria (+). Among the sera from patients with malaria, 4 were from individuals diagnosed as having been infected with Plasmodiumfalciparurra, 4 with Pvivax, and 2 with unidentified Plasmodium
species. TABLE II-GEOMETRIC MEAN TNF LEVELS IN PATIENTS WITH EITHER INFECTIOUS OR NEOPLASTIC DISEASES AND NORMAL
CONTROLS*
suggests that TNF may in some way serve as a host defence mechanism against Leishmania donovani and Plasmodium spp and perhaps other parasitic organisms. Malarial parasites are sensitive to the action of endotoxin-induced serum factors in vitro,s°6 which supports the idea that TNF may play an active part in parasitic infections. There is also evidence that serum factors may be active against Leishmania, at several stages in the parasite’s life cycle. L donovani enters the human host as a promastigote form and subsequently binds to macrophages. Once the parasite enters these cells it converts to the amastigote form.12 Both the promastigote and amastigote carry antigens which serve as targets for immune recognition. Normal human serum contains both IgG and IgM antibody capable of recognising promastigotes. 13 The parasite may, however, be able to circumvent humoral and cell-mediated immune responses by shedding factors that have antigenic cross-reactivity with parasite surface components.14 Host defences which do not rely on antigen recognition would be of benefit in such a situation. Heat-labile serum factors from both healthy and L donovani infected patients have been described which are able to mediate the lysis of amastigotes.15 TNF and other cytokines are reasonable candidates for additional host defences which do not involve immune mechanisms
requiring antigen recognition. This work was supported by grants CA-17094, CA-21839, CA-23074, and AI16312-07 and a basic research support grant from the National Institutes of Health, grant IN-110 from the American Cancer Society, and grant 3364-000000-1-1-AP-6621 from the Arizona Chronic Disease Research Commission.
*Samples below the detection level of 40pg/ml were assumed to have a value of 20-0 pg/ml, the midpoint of the interval between 0 and 40 0. Wilcoxon 2-sample tests were used to examine specific differences among the sample groups. A significance level of 0-01 was used for each comparison to accommodate multiple comparisons. The parasitic diseases were compared with the normal, neoplastic, and infectious disease groups and found to be significantly different (p <0’001). tKala-azar and malaria.
Correspondence should be addressed to P. S., University of Arizona Center, University Department of Microbiology and Immunology, Tucson, Arizona 85724, USA.
Health Sciences
REFERENCES
patients with kala-azar,
18
(66 6%) contained detectable
TNF. Of 44 additional sera from Sudanese donors in whom kala-azar was suspected but leishmania was not detected, only 5 (11 9%) were positive. Among the 10 samples from patients with malaria 7 (70-0%) contained measurable concentrations of TNF. Sera were examined from 176 patients diagnosed as having a variety of neoplastic diseases and representing a range of disease activity from clinical remission to progressive cancer. Among sera from this group a total of 14 (7-9%) samples were found to contain TNF (table I). Sera from three of the four largest subgroups within this category--colon, ovarian, and breast carcinoma-showed similar percentages of positive results. Samples from patients with multiple myeloma were all negative. Although elevated TNF values were encountered in the kala-azar and malaria samples, the maximum and minimum levels were similar in all sample groups (see figure). Samples taken from patients with kala-azar or malaria had geometric mean TNF levels nearly five times higher than any of the other sample groups (table n). In this respect TNF serum levels in samples from patients with parasitic diseases were significantly different from all others tested. Discussion Of the diseases studied, kala-azar and malaria alone were associated with substantially raised serum TNF levels. This
1. Matthews N. Anti-tumour
cytotoxin produced by human anti-tumour cytotoxin produced by human monocytes: Studies on its mode of action. Br J Cancer 1983;
48: 405-10. 2. Helson L, Helson C, Green S. Effects of murine tumour necrosis factor on heterotransplanted human tumors. Expl Cell Biol 1979, 47: 53-60 3. Sugarman BJ, Aggarwal BB, Hass PE, Palladino MA, Shepard HM. Recombinant human tumour necrosis factor-alpha: Effects on proliferation of normal and transformed cells in vitro. Science 1985; 230: 943-45. 4. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR. Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factors. Nature 1986; 319: 516-18. 5. Taveme J, Depledge P, Playfair JHL. Differential sensitivity in vivo of lethal and nonlethal malarial parasites to endotoxin-induced serum factor. Infect Immun 1982, 37: 927-34.
6. Taveme J, Matthews N, Depledge P, Playfair HL. Malarial parasites and tumour cells are killed by the same component of tumour necrosis serum. Clin Exp Immunol 1984; 57: 293-300. 7. Beutler B, Mahoney J, Trang NL, Pekala P, Cerami A. Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264 7 cells. J Exp Med 1985; 161: 984-95. 8. Penica D, Nedwin GE, Hayflick JS, et al. Human tumour necrosis factor: Precursor structure, expression and homology to lymphotoxin. Nature 1984; 312: 724-29. 9. Blackwell JM. Receptors and recognition mechanisms of leishmania species. Roy Soc Trop Med Hyg 1985; 79: 606-12. 10. Beutler B, Greenwold D, Hulmes JD, et al. Identity of tumour necrosis factor and macrophage-secreted factor cachectin. Nature 1985; 316: 552-54. 11. Torti FM, Dieckmann B, Beutler B, Cerami A, Ringold GM. A macrophage factor inhibits adipocyte gene expression: An in vitro model of cachexia. Science 1985; 229: 867-71. 12. Chang KP, Dwyer DM. Multiplication of human parasite (Leishmama donovani) in phagolysosomes of hampster macrophages in vitro. Science 1976; 193: 678-80. 13. Pearson RD, Steigbigel RT. Mechanism of lethal effect of human serum upon Leishmania donovani. J Immunol 1980; 125: 2195-201. 14. El-On J, Schnur LF, Greenblatt CL. Leishmania donovani: Physiochemical, immunological, and biological characterisation of excreted factor from promastigotes. Exp Parasitol 1979; 47: 254-69. 15. Hoover DL, Berger M, Oppenheim MH, Hockmeyer WT, Meltzer MS Cytotoxicity of human serum for Leishmania donovani amastigotes: Antibody facilitation of alternate complement pathway-mediated killing. Infect Immun 1985; 47: 247-52.