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Direct and Antibody Dependent Cell Mediated Cytotoxicity Against Giardia Lamblia by Splenic And- Intestinal Lymphoid Cells in Mice

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Direct and Antibody Dependent Cell Mediated Cytotoxicity Against Giardia Lamblia by Splenic And- Intestinal Lymphoid Cells in Mice Gut: first published as 10.1136/gut.27.1.73 on 1 January 1986. Downloaded from Gut, 1986, 27, 73-77 Direct and antibody dependent cell mediated cytotoxicity against Giardia lamblia by splenic and- intestinal lymphoid cells in mice S S KANWAR, N K GANGULY, B N S WALIA, AND R C MAHAJAN From the Departments ofParasitology and Paediatrics, Postgraduate Institute ofMedical Education and Research, Chandigarh, India SUMMARY Direct cytotoxicity and antibody dependent cell mediated cytotoxicity against Giardia lamblia trophozoites exhibited by splenic, intraepithelial and lamina propria lymphocyte populations isolated from G lamblia infected mice were studied. Different patterns of cytotoxicity were found. Intraepithelial lymphocytes showed a direct cytotoxic activity of 20*6±5-6% before infection. It was significantly higher on the 20th (p<0.01) and 30th (p<005) day postinfection. Lamina propria lymphocytes showed a significantly augmented level of both direct cytotoxicity and antibody dependent cell mediated cytotoxicity on the 20th and 30th postinfection days. Direct cytotoxicity by splenic lymphocytes remained unchanged during infection but antibody dependent cell mediated cytotoxicity was significantly increased. The host response to G lamblia involves the immune weighing 10-12 g were used in this study. G lamblia system. Previous exposure to this infection is known cysts were obtained from the stool of a patient and a http://gut.bmj.com/ to increase resistance to a second challenge in both fixed inoculum of 10 000 cysts/0-2 ml was prepared man and animals.1 2 Smith et al3 reported that on a sucrose gradient9 and fed to the animals.1" Five human peripheral blood monocytes/macrophages animals were killed on each of the days 0, 10, 20, are spontaneously cytotoxic for G lamblia tropho- and 30 postinfection. zoites and this cytotoxicity is reduced in chronic giardiasis.4 In another study they reported defective QUANTITATION OF CYST AND TROPHOZOITE spontaneous cytotoxicity but normal antibody de- (a) Cyst count on September 28, 2021 by guest. Protected copyright. pendent cytotoxicity by resident macrophages iso- Two hour stool samples from each animal were lated from C3H/Hej mice.5 collected, homogenised in normal saline and layered Giardia lamblia has been shown to infect mice. on sucrose gradient.9 The results were expressed as Using a mouse model, we have examined the role of number of cysts/g of stool. cytotoxic effector cells in resistance of mice to this infection. Direct cytotoxicity of lymphocytes and (b) Trophozoite count antibody dependent cell mediated cytotoxicity The small intestine was flushed with a fixed amount against G lamblia are likely to be most relevant in of normal saline, and the trophozoites in the mucosae. We have therefore investigated the direct intestinal perfusate counted in a haemocytometer.6 and antibody mediated cytotoxicity of mucosal lymphocytes (intraepithelial lymphocytes and PREPARATION OF EFFECTOR CELLS lamina propria lymphocytes) against G lamblia. (a) Splenic lymphocytes These were removed and harvested according to the Methods method described earlier.6 They were purified by Ficoll-isopaque density gradient. ANIMALS AND MODE OF INFECTION Parasite free, 2-3 weeks old Swiss albino mice, (b) Intestinal lymphocytes Address for correspondence: Professor R C Mahajan, Head, Department of were isolated a modified Parasitology, Postgraduate Institute of Medical Education and Research, Gut lymphocytes by Chandigarh-160012, India. method of Davies and Parrott" and Taglibue et al.12 Received for publication 9 April 1985 Briefly, the small intestine was washed with calcium 73 Gut: first published as 10.1136/gut.27.1.73 on 1 January 1986. Downloaded from 74 Kanwar, Ganguly, Walia, and Mahajan and magnesium free Hank's balanced salt solution. hours at 37°C in a shaking water bath. 0*1 ml of The mesentery, adherent connective tissue and fat labelled trophozoites (5 x 10),5 suspended in incuba- were removed from the intestine and Peyers patches tion medium, were placed in sterile small test tubes. were dissected. The intestine was opened longitu- 0*1 ml of effector cells were also added (effector cell dinally and cut into 1-2 cm long pieces and washed density was adjusted to achieve 25:1, 50:1 and 100:1 thoroughly in calcium and magnesium free Hank's E:T ratios). To assay antibody dependent cell balanced salt solution. To isolate intraepithelial mediated cytoxicity, 0.1 ml of immune serum lymphocytes, gut pieces were incubated at 37°C for (1:3000 diluted) was added. The effector cells, 15 minutes in calcium and magnesium free Hank's labelled trophozoites and antiserum were mixed and balanced salt solution containing 5 mmol EDTA tubes were centrifuged at 100 g for five minutes. with constant stirring. This process was repeated These were then incubated at 37°C in a 5% CO2 three times. The supernatants were pooled, centri- humidified atmosphere for two hours. After incuba- fuged, resuspended in RPMI-1640, filtered through tion, the tubes were centrifuged and the amount of glass wool column and washed. Most of the lympho- radio-activity in the supernatant was measured in a cytes obtained by this method were from the gamma counter. The maximum release was ob- epithelial layer. " tained by treating the labelled trohozoites with For isolation of lymphocytes from the lamina 10% Triton-xlOO followed by processing as outlined propria, gut pieces were washed with calcium and above. The spontaneous release varied from 10- magnesium free Hank's balanced salt solution and 15% of the maximal release. The percentage specific incubated for 20 minutes in 25 ml RPMI-1640 cytotoxicity was calculated by the following formula: containing 5% heat inactivated fetal calf serum % specific cytotoxicity=experimental release (RPMI-FCS) and 2 mmol L-glutamine with stirring. (cpm)-spontaneous release (cpm) x 100/maximal re- These pieces were then incubated in 15 ml RPMI- lease (cpm)-spontaneous release(cpm). FCS containing 25 U/ml collagenase (Boehringer, W. Germany) for 15 minutes. The whole procedure STATISTICAL ANALYSIS was repeated three times. The supernatants were Student's t test was used to analyse all the data. pooled, filtered through a glass wool column and washed in RPMI-FCS. Results The lymphocyte rich preparations of intra- http://gut.bmj.com/ pethelial lymphocytes and lamina propria lympho- Figure 1 shows the kinetics of cyst excretion and cytes were loaded on a percoll density gradient trophozoite counts in the intestine during the 30 day (Pharmacia, Sweden) and the lymphocytes reco- period of study. The peak cyst excretion and peak vered from the 1*050/1 085 g/ml density interface. trophozoite count occurred on the 10th postinfec- Their viability was checked by the 0.2% trypan blue tion day. By the 30th day, cyst excretion ceased in dye exclusion test. The yield of intraepithelial all the animals, although a few trophozoites could lymphocytes obtained was 0-6-2Slx 10.7 still be detected in 30-40% of infected animals in the on September 28, 2021 by guest. Protected copyright. intestinal washings. ANTISERUM AGAINST G lamblia Different effector:target cell (E:T) ratios were Antiserum to G lamblia trophozoites was raised in used (25:1, 50:1 and 100:1) to test for direct rabbits13 and titrated in a cytotoxicity assay. The cytotoxicity and antibody dependent cell mediated serum was used at a dilution of 1:3000 in all cytoxicity. There was a slight but consistent increase experiments. At this concentration, the antiserum in direct cytotoxicity at E:T ratios of 50:1 and 100:1. itself had no giardiacidal activity. Normal rabbit A very low level of direct cytotoxicity (6.34±2.15%) serum was used at the same dilution. Both normal was observed at E:T ratios of 25:1 with splenic and immune sera were heated at 56°C for 30 minutes lymphocytes, while with intraepithelial lymphocytes before use. and lamina propria lymphocytes, the respective values were 19 03±2 08% and 18-30±3-92%. With CYTOTOXICITY ASSAY lamina propria lymphocytes, the antibody depen- Giardi lamblia trohozoites were purified by the dent cell mediated cytoxicity was much higher Feely and Erlandsen'4 method of sticking them to (44.98%), in comparison to the values obtained with polystyrene petri dishes, and washed with RPMI- intraepithelial lymphocytes and splenic lyphocytes 1640 containing 25 mM HEPES, 10% heat inacti- at E:T ratios of 100:1 (Fig. 2). vated fetal calf serum and 50 ,g/ml gentamicin. The Intraepithelial lymphocytes showed a cytotoxic cytotoxicity assay was a modified technique of Smith activity of 20-6±5*63% before infection. On the et al.S Trophozoites were labelled with sodium5' 20th postinfection day, the values were increased to chromate using 70 gCi/5 x 106 parasite/ml for 12 39*82±5*87% (p<OO1), whereas antibody depen- Gut: first published as 10.1136/gut.27.1.73 on 1 January 1986. Downloaded from Mediated cytotoxicity against Giardia lamblia in mice 75 50. p 0C -0 / / 0 / U U ._ 30 N 0, 0. 0. O- CLi in 20 10 Days after inocculation Fig. 1 Giardia lamblia cyst and tropozoite counts (log,o) on differentpostinfection days. Each point represents the mean ±SD offive animals. * @ 25:1 50:1 100:1 Effector: dent cell mediated cytoxicity for intraepithelial target ratio http://gut.bmj.com/ lymphocytes showed no significant change from the Fig. 2 Direct (continuous line) and antibody dependent preinfection value. In contrast, lamina propria (dotted line) cell mediated cytotoxic activity exhibited by lymphocytes showed a significantly higher direct splenic (L), IEL (@) and LPL(O) at different E:T ratios cytotoxicity and antibody dependent cell mediated against G lamblia trophozoites. Values represent the mean cytoxicity on day 20 and 30 (Table 1). ofthree experiments before infection. The level of direct cytotoxicity exhibited by splenic lymphocytes at E:T ratios of 50:1 before infection was 12.9±5-4%; this remained unchanged Discussion on September 28, 2021 by guest.
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