Effect of Entamoeba Histolytica Toxins on Isolated Human Polymorphonuclear Leukocytes' Phagocytic Function
From the Department of Physiology, College of Medicine, King Saud University, Riyadh.
AS Al-Tuwaijri, Effect of Entamoeba Histolytica Toxins on Isolated Human Polymorphonuclear Leukocytes' Phagocytic Function. 1992; 12(4): 372-376
Abstract
This study was undertaken to investigate the effect of Entamoeba histolytica toxin (Ehp/t) obtained from HM1:IMSS strain on human polymorphonuclear leukocytes (PMNs) phagocytic function. A less virulent strain, i.e., NIH:200 was also used in this study for comparison. The results revealed that the toxin obtained from the strain HM1:IMSS inhibited the phagocytic activity of PMNs as measured by yeast uptake or chemiluminescence response while the toxin of strain NIH:200 did not show any significant effect. On the other hand, washing of PMNs that had been pre-exposed to the toxin of strain HM1:IMSS failed to reverse the effect of the toxin on PMNs. Heat inactivation of the toxin failed to alter its effect on PMNs. Addition of N-acetyl-D-galactosamine (GalNAc) to the PMNs did not alter the activity of Ehp/t toxin. These findings suggested that Ehp/t toxin might induce similar effect on PMNs of patients infected with ameba.
Entamoeba histolytica is a protozoan parasite of the large intestine of man which has a worldwide distribution. About 10% of the world population is estimated to be infected with the parasite [1]. This infection is mostly associated in communities where the standard of sanitation is poor [2]. A relatively high proportion of infected people usually develop amebiasis which is a type of disease resulting from the invasion of the parasite to the large intestine of the host.
Although several studies have been performed in an attempt to understand the host-parasite interaction in amebic infection, the exact mechanism of tissue damage is not as yet fully understood [3]. Both the toxin of the ameba parasite and host factors such as polymorphonuclear leukocytes (PMNs) have been implicated in the early events of the invasion by the ameba parasites [4,5]. The oxygen-free radicals released from PMNs upon stimulation may play a vital role in the severity of the tissue damage and in cecal tissue invasion in experimental amebic colitis [6].
Results obtained in this laboratory have shown that E. histolytica toxin (Ehp/t) depresses the luminol-dependent chemiluminescence responses of human PMNs in vitro [7]. Conversely, Salata and Ravdin [3] reported that the destruction of liver cell monolayers by E. histolytica was enhanced by lysed PMNs. These authors suggested that the tissue damage was not likely to be mediated by oxygen-free radicals released from stimulated PMNs. To further shed some light on the possible mechanisms of the pathogenesis of amebiasis, the present study was undertaken to examine the effect of a toxin obtained from a virulent strain of E. histolytica, i.e, HM1:IMSS, on the phagocytic activity of human PMNs. The luminol-dependent chemiluminescence (CL) responses and opsonized yeast uptake were utilized to assess the effect of Ehp/t toxins on phagocytosis. A less virulent strain NIH:200 was included in the study for comparison.
Methods
Entamoeba Histolytica Toxin
E. histolytica toxin (Ehp/t) was obtained from the virulent strain HM1:IMSS was kindly provided by Dr. Pittman (USA). Toxin of strain NIH:200 was prepared from axenic trophozoites as described by McGowan et al [12].
PMNs Isolation
Blood from apparently healthy donors was collected by venipuncture. Blood was collected in sterile heparinized containers (10 IU/ml), PMNs were isolated by sedimentation technique using Dextran T-500 and Nycodenz solution as previously described [7]. Ammonium chloride (0.87%) was used to lyse RBCs. PMN's viability was determined by trypan blue (0.2%).
Chemiluminescence (CL) Measurement
The PMNs oxidative burst was measured by CL as previously described [7–9]. The CL assay was carried out in a LKB (WALLAC) 1251 luminometer. The principle of oxidation of luminol, 5-amino-2,3-dihydro-1,4-phtalazinedione, by reactive oxygen species generated during PMNs phagocytosis. Luminol (0.1 mmol/L) was employed to enhance the amount of light emission [10] and Phorbol Myristate acetate (PMA, 0.002 g/L) was employed as a soluble agent to stimulate PMNs. The light output (CL response) in mV was continuously recorded on a LKB (WALLAC) chart recorder.
Measurement of Phagocytosis
Phagocytosis was measured by using opsonized dead yeast cells. The yeast cells were prepared by first washing 5 grams of pure bakers yeast cells twice with PBS. The yeast cells were then resuspended in PBS and heat-killed by boiling for 30 minutes. The final concentration was adjusted to 2 × 108 cells/ml. This stock suspension was kept frozen until used. Opsonization of yeast cells was performed by incubating dead yeast with normal human serum at 37°C for 30 minutes. The opsonized yeast was washed twice with PBS. The isolated human PMNs were suspended in an Eagle minimum essential medium with Earle salts supplemented with heat-inactivated fetal calf serum (5%) opsonized dead yeast and Ehp/t were added to the PMN's suspension and phagocytosis was measured. The cell suspension was immersed in ice to stop further phagocytosis. The number of yeast particles engulfed in a total of 200 PMNs was counted. The phagocytic index (K) was calculated as follows: K = a/b; where a is the total number of yeast cells within the PMNs and b is the number of PMNs counted.
Effect of Entamoeba Histolytica Toxin on PMNs
Various concentrations of Ehp/t toxin of both strains (0–1 g/L) were incubated for 30 minutes with PMNs (5 × 109/L) at 37°C. Opsonized yeast was added to PMNs and further incubated for 30 minutes. A selected concentration of Ehp/t toxin of strain HM1:IMSS (0.6 g/L) was incubated at different time intervals (0 to 30 minutes) with PMNs at 37°C and the phagocytic index of PMNs was calculated. In order to assess the effect of heat on the stability of the toxin, 0.6 g/L was boiled at 90°C for one hour. The toxin was incubated (30 minutes) at 37°C with PMNs, washed with PBS, and the phagocytosis and PMNs-CL response was assessed. Since N-acetyl galactosamine (GalNAc) is known to inhibit the adhesion of E. histolytica trophozoites to the host cells, the effect of the Ehp/t toxin on PMNs in the presence of GalNAc was examined. GalNAc (10 mmol/L) was pre-incubated for 30 minutes with PMNs, then the toxin (0.6 g/L) was added and incubated for 30 minutes. The phagocytic activity was determined as above.
Statistical Analysis
The results of CL maximum peak response were expressed as Mean ± S.E. Results were analyzed for significance between different groups using the two-tailed student's t-test. Results were considered significant when P < 0.05.
Results
Influence of E. Histolytica Toxin (Ehp/t) on Phagocytosis of PMNs
Phagocytosis of PMNs was markedly inhibited when the phagocytes were pre-incubated with various concentrated of Ehp/t toxin strain HM1:IMSS (0.1–1 g/L). This inhibition was found to be concentrated and time-dependent (Table 1). The percentage of inhibition of phagocytosis was 51, 78, 90, and 94 when PMNs were incubated with Ehp/t toxin at 37°C for 5, 10, 20, and 30 minutes, respectively. In contrast, Ehp/t toxin obtained from strain NIH:200 did not induce any significant effect on phagocytosis. Only Ehp/t obtained from strain HM1:IMSS inhibited PMNs-CL response (Table 2). To examine if the inhibit ory effect of Ehp/t toxin (HMI:1MSS) on PMNs activity was reversible, Ehp/t toxin (0.6 g/L) was pre-incubated with isolated PMNs for 30 minutes and then washed with PBS. The phagocytic function of PMNs was assessed by either determination of phagocytosis of opsonized yeast or CL response. Table 2 summarizes the influence of Ehp/t toxin (strain HM1:IMSS) on PMNs after washing the toxin from the incubation medium. There was no significant difference in the phagocytic index or PMA-induced CL between the washed and the unwashed samples which indicated that the effect was irreversible.
Table 1. Effect of various concentrations of E. histolytica toxin (Ehp/toxin) on the phagocytic activity of human polymorphonuclear leukocytes.
|
|
Phagocytic index M ± S.E. |
|
|
Concentrations of Ehp/t toxin (g/L) |
HM1:IMSS Ehp/t toxin |
NIH:200 Ehp/t toxin |
|
Control (PBS) |
7.48 ± 0.45 |
7.04 ± 0.20 |
|
0.1 |
6.88 ± 0.18 |
5.93 ± 0.20 |
|
0.4 |
2.79 ± 0.14 |
6.6 ± 0.30 |
|
0.6 |
0.56 ± 0.80 |
7.0 ± 0.20 |
|
1.0 |
0.44 ± 0.11 |
7.4 ± 0.20 |
Data are expressed as MEAN ± S.E. of 5 observations; PMN count = 5 × 109 cells/L; incubation temperature = 37°C; incubation time = 30 minutes; phagocytic index (k) was determined by:
Table 2. The reversibility effect of washing E. histolytica toxin (Ehp/t) obtained from strain HM1:IMSS with PBS from the incubation medium on the chemiluminescence and phagocytosis of opsonized yeast of isolated human polymorphonuclear leukocytes.
|
Group |
Ehp/t toxin |
Phagocytic index |
PMACL maximum peak (mV) |
|
Unwashed |
– |
7.50 ± 0.7 |
350.2 ± 0.3 |
|
Unwashed |
+ |
0.56 ± 0.1* |
20.2 ± 0.9* |
|
Washed |
– |
7.6 ± 0.8 |
281.1 ± 0.6 |
|
Washed |
+ |
0.73 ± 0.5* |
1.6 ± 0.3* |
Effect of Heating Ehp/t Toxin
In addition, the effect of heat on Ehp/t toxin HM1:IMSS activity was also examined by boiling the toxin for one hour. The cytotoxic effect of the Ehp/t toxin obtained from HM1:IMSS on the viability of PMNs was abolished when the toxin was boiled at 90°C for one hour (Table 3).
Effect of N-Acetyl-D-Galactosamine
In order to study whether the cytotoxic effect of the Ehp/t toxin HM1:IMSS can be modified by receptor inhibitors such as N-acetyl-D-galactosamine (GalNAc), PMNs were incubated with Ga1NAc for 30 minutes, and the toxin was added to the incubation media for an additional 20 minutes prior to the assessment of the PMNs activity (Table 4). GalNAc had no effect on the cytotoxic effect of Ehp/t toxin. The activity of the PMNs alone was also not significantly affected when various concentrations of GalNAc were added alone (data are not included).
Table 3. The effect of heat on E. histolytica toxin strain HM1:IMSS (EhpIt)-induced inhibition of isolated human polymorphonuclear leukocytes (PMNs) viability, phagocytosis of yeast uptake and chemiluminescence (CL) responses.
|
Group |
% cell viability |
Phagocytic index (K) M±S.E. |
PMA CL maximum peak (mV) M ± S.E. |
|
Control |
96 |
7.46 ± 0.86 |
430.6 ± 0.7. |
|
Ehp/t toxin |
55 |
2.7 ± 0.44 |
12.8 ± 0.6 |
|
Heat denatured Ehp/t toxin |
89 |
0.28 ± 0.08* |
2.4 ± 0.3* |
All data was calculated as Mean ± S.E.; PMN count = 5 × 109 cells/L; opsonized yeast = 2 × 1010/L; Ehp/t toxin (0.6 g/L) was denaturated by boiling the toxin at 90°C for one hour; phagocytic index (K) was calculated as under Table 1; luminol (0.1 mmol), PMA (0.002 g/L) was used to measure chemiluminescence response;P < 0.001.
Discussion
In an earlier study in this laboratory, we have shown that Ehp/t toxin (HM1:IMSS) depresses PMA-induced CL. Such depression was concentration-dependent [7]. The results have also suggested that PMNs may play a vital role in amebic tissue invasion mechanisms. The present study has shown that various concentrations of HM1:IMSS toxin markedly inhibits phagocytosis of PMNs. This inhibition is a dose-dependent phenomenon. When a fixed dose of Ehp/t toxin (0.6 g/L) was exposed to PMNs at different time intervals the rate of inhibition was directly proportional to the exposure time.
The inhibition induced by the amebic toxin could either be ascribed to a lytic effect of the toxin on the cell membrane of the PMNs which lysed 50% after 30 minutes of incubation, or that the toxin has a direct lytic action on the intracellular organelles of the PMNs. A study by Ravdin and Guerrant [11] have demonstrated that the killing of target cells by E. histolytica can only be achieved by direct contact and the cytolethal effect occurs at the amebic surface and before phagocytosis. This inhibition in phagocytosis could be due to interference of the toxin with the binding or attachment of the opsonized yeast particles on the receptors of the PMNs.
Table 4. Influence of N-acetyl galatosamine (GalNAc) on E. histolytica toxin strain HM1:IMSS (Ehp/t)-induced inhibition of isolated human polymorphonuclear leukocytes (PMNs) viability, phagocytosis of opsonized yeast and chemiluminescence responses.
|
Group |
% cell viability |
Phagocytic index (K) M ± S.E. |
PMA CL maximum peak (mV) M ± S.E. |
|
Control |
94 |
60.8 ± 3.0 |
442.6 ± 4 |
|
GalNAc |
91 |
47.0 ± 0.4 |
453.7 ± 10 |
|
Ehp/t toxin |
60 |
23.2 ± 0.4* |
80.5 ± 3 |
|
Ehp/t toxin ± GalNAc |
50 |
21.8 ± 0.3 |
83.6 ± 2 |
In contrast to the marked inhibitory effect on the phagocytosis induced by HM1:IMSS toxin, the toxin of the less virulent strain NIH:200 did not produce a significant inhibitory effect on the activity of PMNs. This is in agreement with the report by McGowan et al [12] who demonstrated that strain HM1:IMSS possessed marked cytotoxic activity on PMNs while strain NIH:200 exhibited a moderate activity.
N-acetyl-D-galactosamine (GalNAc) has been shown to inhibit the adherence of ameba trophozoite to the host cells [13]. Strain HMITMSS is more sensitive to such inhibition than strain 200:NIH. Additionally, exposure of the amebic trophozoite to GAlNAc can inhibit its killing ability and allow the neutrophils to kill the ameba. In the present study we attemped to demonstrate this observation on the HM1:IMSS toxin. The results, however, did not show any inhibitory effect on the interaction between PMNs and Ehp/t toxin.
Inactivation of the Ehp/t toxin by boiling for one hour at 95°C did not alter the cytotoxic activity on PMNs. This may suggest that the enzymes produced by the parasite have no role in the inhibitory process or that the boiling for one hour is not enough time to denature the toxin. Washing PMNs which were pretreated with HM1:IMSS toxin, did not change the inhibitory effect of the toxin indicating that the action of the toxin is irreversible. The present findings suggested that the inhibitory effect of Ehp/t toxin may occur in amebic infected humans which needs further investigation.
Acknowledgments
The author thanks Drs. R. Al-Rashed, A. Mahmoud, and I. Al-Mofleh for their suggestions; Mr. C. A. Victoria Jr. and Ms. O. S. Borbon for their technical assistance; Mr. R. M. Rebleza for maintenance and propagation of the parasites. This work was supported by Grant No. AR-10-10, from the King Abdul Aziz City for Science and Technology.
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