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Giardia lamblia infection of human intestinal epithelial cell cultures inhibits epithelial nitric oxide production
GASTROENTEROLOGY Vol. 114, No. 4
A970 AGAABSTRACTS • G3975 GIARDIA INDUCES PROLIFERATION AND INTERFERON-T PRODUCTION BY INTESTINAL LYMPHOCYTES BUT NOT CYTOTOXIC ACTIVITY OR MIGRATION. EC Ebert*, RE Brolint, AI Roberts*. Depts. of Medicine* and Surgeryt, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. In intestinal infection with the parasite Giardia intestinalis, the organisms attach to epithelial cells of the proximal jejunum, which may lead to their interaction with the intestinal immune system. Intraepithelial lymphocytes (IELs) may directly encounter these organisms if they invade the intraepithelial space or when IELs emigrate to the unstirred water layer along the epithelium. In addition, antigens shed by the trophozoites may stimulate both IELs and lamina propria lymphocytes (LPLs). The reactivity of IELs and LPLs from normal human jejunum toward giardia organisms in vitro was examined and compared to control peripheral blood lymphocytes (PBLs). The proliferative response of 2 x 105 lymphocytes in a 3d coculture with lxl04 live giardia, measured by 3H-thymidine incorporation, and the amount of interferon-'/ produced in a parallel culture, as assayed by ELISA, are indicated in the Table. Proliferation (cpm) IEL LPL PBL
2,133± 233 15,045± 2381 13,235:t: 3220
Interferon-'/(pg/ml) (pg/ml) + Giardia control not done not done 8300± 2200 2750± 2 1 0 0 132 ± 92 60 ± 40
Responder Freque0¢y not done 1/3,950 1/7,500
Only CD4÷ T cells proliferated, as anti-CD4 immunomagnetic depletion eliminated the response. The giardia-associated stimulus has mitogenic rather than antigenic characteristics, since 1. proliferation of LPLs peaked on day 3, not day 6, 2. the frequency of responding cells, determined by limiting dilution analysis, was in the range of mitogen (Table), and 3. no anemnestic response occurred when LPLs were reexposed to giardia after the initial 5 day coculture. The ability of human IELs and LPLs to lyse 51Cr-labelled giardia was tested since murine IELs are capable of doing so. Whether used fresh (spontaneous cytotoxicity), after stimulation for 3 days with IL-2 or IL-15 (lymphokineactivated killing), or after a 5 day coculture with giardia (cytotoxic T lymphocyte activity), there was no lysis. Serine esterase release by IELs under the same conditions was undetectable. The migratory response of IELs and LPLs toward giardia organisms or medium conditioned by their culture was measured in the transwell system. Lymphocyte movement was no higher than background levels. In conclusion, a giardia-associated mitogen stimulates CD4 ÷ T lymphocytes from the intestine and the circulation to proliferate and produce interferon-y, but does not induce cytotoxic activity or migration. (Supported by NIH grant #DK42166) • G3976 TRIGGERING OF ACTIVATED CD2 MAKES LAMINA PROPRIA LYMPHOCYTES (LPLS) REFRACTORY TO CD3/TCR STIMULATION. EC Ebert and AI Roberts. Dept. of Medicine, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Human intestinal LPLs demonstrate greater proliferation and cytokine secretion in response to the ligation of CD2 rather than CD3. These cells are already activated upon isolation and proliferate without exogenous stimulation. This pre-activated state may be related to their low CD3-responsiveness. The mechanisms of these events was determined. The spontaneous proliferation of freshly isolated LPLs (lxl05) from normal jejunal mucosa averaged 1825 -+ 844 cpm compared to a response by PBL of 201 + 53 cpm after a 3d culture, as assessed by 3H-thymidine incorporation. The LPL response was inhibited up to 56% by blocking MAbs against CD2, CD58, CD80; neutralizing MAbs against IL-1, IL-2, and TNFet; and by ILlra added at the start of the culture. The same antibodies, except CD58, abrogated LPL proliferation in response to CD2 stimulation with the mitogenic MAbs, TI 12 and T113, suggesting that the spontaneous response depends on the CD2 pathway. When LPLs were cocultured with irradiated peripheral blood monocytes, but no additional stimulus, proliferation (4400 ± 843 cpm) was greater than spontaneous and was blocked by MAbs against CD2 and CD58 (955 -+ 226, 1818 + 572 cpm, respectively, p<0.05). PBLs responded minimally. Thus, supplementary antigen-presenting ceils bearing CD58 (a CD2 ligand) boosted spontaneous proliferation by LPLs, implicating the CD2-CD58 interaction. Immunofluorescence revealed the activated form of CD2 (Tll 2 and Tll 3 epitopes) on LPLs, but only T112 on PBLs. The activation events occurring in LPLs in response to CD3 ligation or mitogen were examined. Calcium influx was measured fluorometrically in Fura-2 loaded cells. 1L2 receptors were detected by immunofluorescence and flow cytometry, and quantitated by radioligand binding. Upon CD3 ligation, few IL2 receptors were detected. Calcium influx was transient, but occurred only after a 3d culture. Phytohemagglutinin resulted in IL2 receptor expression similar to that of PBLs, but no calcium inflows. Therefore, the low responsiveness of LPLs is due to incomplete activation, the defect varying with the stimulus. LPLs were stimulated mitogenically for 5d with anti-CD2 MAbs (or control MAb) to mimic activation in vivo. Subsequent proliferation to CD3 ligation
was low (500 -+ 100 v. 7100 _+2600, p<0.05). Suppressor cells were involved since the CD3-response of fresh LPLs was reduced by 30 _+3% when antiCD2-prestimulated LPLs were added. Partly implicated was apoptosis that was greater after CD2 rather than CD3 ligation, as revealed by apo-2.7 expression. In conclusion, the spontaneous proliferation of LPLs is likely a consequence of their bearing activated CD2 which is triggerable by CD58. This leads to suppressor activity and apoptosis that attenuate CD3-responsiveness by inhibiting activation events. A refractory response of LPLs to CD3FFCR stimulation may maintain tolerance in an antigen-filled environment. Supported by NIH grant #DK42166. • G3977 GIARDIA LAMBLIA INFECTION OF HUMAN INTESTINAL EPITHELIAL CELL CULTURES INHIBITS EPITHELIAL NITRIC OXIDE PRODUCTION. L. Eckmann. F. Laurent, F.D. Gillin, M.F. Kagnnff; Depts. of Medicine and Pathology, Univ.of Calif., San Diego, La Jolla, CA. Giardia lamblia is a strictly lumen-dwelling human pathogen that resides in the small intestine in close proximity to intestinal epithelial cells. Epithelial cells can release nitric oxide (NO) after cytokine stimulation, and NO was reported to kill G. lamblia trophozoites in vitro. In the present studies, we asked whether G. lamblia has evolved the ability to modulate epithelial cell NO production. Methods: Confluent cultures of HT-29 human intestinal epithelial cells and polarized Caco-2 epithelial cells were infected with G. lamblia trophozoites, and stimulated with a combination of IFN-~/, TNF~, and IL-I~. NO production was assessed by assaying the stable NO end products, nitrite and nitrate, in the culture supematants. Results: Infection of HT-29 cultures with G. lamblia trophozoites dose-dependently inhibited by >95% epithelial NO production induced by cytokine stimulation, whereas numbers of viable epithelial cells were not affected. Half-maximal inhibition was observed at a MOI of 1 Giardia:20 epithelial cells. Similarly, apical infection of polarized Caco-2 monolayers with G. lamblia inhibited epithelial NO production by >80%. Inhibition of epithelial NO production required viable Giardia trophozoites but not cell:cell contact, suggesting release of an inhibitory activity by G. lamblia or a lack of crucial substrates for epithelial NO production as possible mechanisms underlying the inhibition of epithelial NO production. In support of the latter, addition of arginine, which is the substrate for NO synthase to produce NO and an important metabolic fuel for Giardia, to Giardia-infected HT-29 cultures almost completely reversed the inhibition of epithelial NO production. Conclusion: The strictly lumendwelling human pathogen G. lamblia can inhibit the production of the Giardia-toxic effector molecule NO by intestinal epithelial cells through limiting epithelial access to arginine. Supported by NIH grant DK35108 and a CCFA Award. • G3978 A RANDOMIZED,
SINGLE-BLIND, PHARMACOKINETIC AND DOSERESPONSE STUDY OF SUBCUTANEOUS METHOTREXATE, 15 AND 25 MG/WEEK, FOR REFRACTORY ULCERATIVE COLITIS AND CROHN'S DISEASE. LJ Egan 1, WJ Sandborn I, WJ Tremaine 1, JA Leighton 2, DC Mays 1, MG PikO, AR Zinsmeister 1, JJ Lipsky 1. Mayo Clinic, Rochester, MN1; Scottsdale, AZ2. Background: Methotrexate (MTX), 25 rag/week by i.m. injection, has been
shown to induce remission in 39% of refractory Crohn's disease (CD) patients, but toxicity limited therapy in 17% (Feagan NEJM 1995;332:292). MTX 12.5 rag/week by mouth was ineffective in ulcerative colitis (UC), but toxicity was rare (Oren Gastroenterology 1996;110:1416). Erythrocyte and plasma MTX concentrations may be useful predictors of response and toxicity. Aims: 1) To determine if circulating concentrations of MTX predict response or toxicity. 2) To compare the efficacy of MTX, 15 or 25 mg/week by s.c. injection for refractory UC and CD. Methods: 31 patients with refractory UC, n=14 and CD, n=17, defined as inability to discontinue prednisone without disease flare, were enrolled and prednisone was adjusted to 20 mg/d for 2 weeks. Patients were stratified for balancing into 4 groups: by disease, UC or CD, and by activity, inflammatory bowel disease questionnaire (IBDQ) <170 (steroid refractory), or _> 170 (steroid dependant), and then randomized to MTX 15 vs 25 mg/week s.c. (patients blinded). A prednisone taper was begun at 2 weeks, but if disease flared, prednisone could be increased. Every 2 weeks the IBDQ was administered and trough concentrations of erythrocyte MTX (competitive protein binding assay), plasma MTX and plasma 7OHMTX (HPLC) were determined. Toxicity was defined as any event leading to MTX dose reduction or discontinuation. After 16 weeks, clinical outcomes were assessed for remission (IBDQ > 170 and off prednisone) and response (remission, or increase in IBDQ _> 16, or IBDQ > baseline and off prednisone). Results: 1 patient was eliminated after screening and 2 patients were censored from analysis for non-compliance. Median overall trough erythrocyte MTX, plasma MTX and plasma 7OHMTX concentrations were not different between and 15 and 25 mg/week groups. Mean -+ SE MTX concentrations: *Erythrocyte MTX *PlasmaMTX *Plasma7OHMTX Yes No Yes No Yes No UC response 181±25 169±11 0.7±0.3 0.5±0.2 0.5±0.1 2.0±0.5 CD response 205±21 214+31 0.7±0.1 0.7±0.3 2.0±0.8 3.1±2.0 UC+CD toxicity 143±3 197±12 0.5±0.4 0.7+0.1 1.0±0.7 2.0±0.5 *No associationsbetweenresponseor toxicity and concentrations(logisticregression).
A970 AGAABSTRACTS • G3975 GIARDIA INDUCES PROLIFERATION AND INTERFERON-T PRODUCTION BY INTESTINAL LYMPHOCYTES BUT NOT CYTOTOXIC ACTIVITY OR MIGRATION. EC Ebert*, RE Brolint, AI Roberts*. Depts. of Medicine* and Surgeryt, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. In intestinal infection with the parasite Giardia intestinalis, the organisms attach to epithelial cells of the proximal jejunum, which may lead to their interaction with the intestinal immune system. Intraepithelial lymphocytes (IELs) may directly encounter these organisms if they invade the intraepithelial space or when IELs emigrate to the unstirred water layer along the epithelium. In addition, antigens shed by the trophozoites may stimulate both IELs and lamina propria lymphocytes (LPLs). The reactivity of IELs and LPLs from normal human jejunum toward giardia organisms in vitro was examined and compared to control peripheral blood lymphocytes (PBLs). The proliferative response of 2 x 105 lymphocytes in a 3d coculture with lxl04 live giardia, measured by 3H-thymidine incorporation, and the amount of interferon-'/ produced in a parallel culture, as assayed by ELISA, are indicated in the Table. Proliferation (cpm) IEL LPL PBL
2,133± 233 15,045± 2381 13,235:t: 3220
Interferon-'/(pg/ml) (pg/ml) + Giardia control not done not done 8300± 2200 2750± 2 1 0 0 132 ± 92 60 ± 40
Responder Freque0¢y not done 1/3,950 1/7,500
Only CD4÷ T cells proliferated, as anti-CD4 immunomagnetic depletion eliminated the response. The giardia-associated stimulus has mitogenic rather than antigenic characteristics, since 1. proliferation of LPLs peaked on day 3, not day 6, 2. the frequency of responding cells, determined by limiting dilution analysis, was in the range of mitogen (Table), and 3. no anemnestic response occurred when LPLs were reexposed to giardia after the initial 5 day coculture. The ability of human IELs and LPLs to lyse 51Cr-labelled giardia was tested since murine IELs are capable of doing so. Whether used fresh (spontaneous cytotoxicity), after stimulation for 3 days with IL-2 or IL-15 (lymphokineactivated killing), or after a 5 day coculture with giardia (cytotoxic T lymphocyte activity), there was no lysis. Serine esterase release by IELs under the same conditions was undetectable. The migratory response of IELs and LPLs toward giardia organisms or medium conditioned by their culture was measured in the transwell system. Lymphocyte movement was no higher than background levels. In conclusion, a giardia-associated mitogen stimulates CD4 ÷ T lymphocytes from the intestine and the circulation to proliferate and produce interferon-y, but does not induce cytotoxic activity or migration. (Supported by NIH grant #DK42166) • G3976 TRIGGERING OF ACTIVATED CD2 MAKES LAMINA PROPRIA LYMPHOCYTES (LPLS) REFRACTORY TO CD3/TCR STIMULATION. EC Ebert and AI Roberts. Dept. of Medicine, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. Human intestinal LPLs demonstrate greater proliferation and cytokine secretion in response to the ligation of CD2 rather than CD3. These cells are already activated upon isolation and proliferate without exogenous stimulation. This pre-activated state may be related to their low CD3-responsiveness. The mechanisms of these events was determined. The spontaneous proliferation of freshly isolated LPLs (lxl05) from normal jejunal mucosa averaged 1825 -+ 844 cpm compared to a response by PBL of 201 + 53 cpm after a 3d culture, as assessed by 3H-thymidine incorporation. The LPL response was inhibited up to 56% by blocking MAbs against CD2, CD58, CD80; neutralizing MAbs against IL-1, IL-2, and TNFet; and by ILlra added at the start of the culture. The same antibodies, except CD58, abrogated LPL proliferation in response to CD2 stimulation with the mitogenic MAbs, TI 12 and T113, suggesting that the spontaneous response depends on the CD2 pathway. When LPLs were cocultured with irradiated peripheral blood monocytes, but no additional stimulus, proliferation (4400 ± 843 cpm) was greater than spontaneous and was blocked by MAbs against CD2 and CD58 (955 -+ 226, 1818 + 572 cpm, respectively, p<0.05). PBLs responded minimally. Thus, supplementary antigen-presenting ceils bearing CD58 (a CD2 ligand) boosted spontaneous proliferation by LPLs, implicating the CD2-CD58 interaction. Immunofluorescence revealed the activated form of CD2 (Tll 2 and Tll 3 epitopes) on LPLs, but only T112 on PBLs. The activation events occurring in LPLs in response to CD3 ligation or mitogen were examined. Calcium influx was measured fluorometrically in Fura-2 loaded cells. 1L2 receptors were detected by immunofluorescence and flow cytometry, and quantitated by radioligand binding. Upon CD3 ligation, few IL2 receptors were detected. Calcium influx was transient, but occurred only after a 3d culture. Phytohemagglutinin resulted in IL2 receptor expression similar to that of PBLs, but no calcium inflows. Therefore, the low responsiveness of LPLs is due to incomplete activation, the defect varying with the stimulus. LPLs were stimulated mitogenically for 5d with anti-CD2 MAbs (or control MAb) to mimic activation in vivo. Subsequent proliferation to CD3 ligation
was low (500 -+ 100 v. 7100 _+2600, p<0.05). Suppressor cells were involved since the CD3-response of fresh LPLs was reduced by 30 _+3% when antiCD2-prestimulated LPLs were added. Partly implicated was apoptosis that was greater after CD2 rather than CD3 ligation, as revealed by apo-2.7 expression. In conclusion, the spontaneous proliferation of LPLs is likely a consequence of their bearing activated CD2 which is triggerable by CD58. This leads to suppressor activity and apoptosis that attenuate CD3-responsiveness by inhibiting activation events. A refractory response of LPLs to CD3FFCR stimulation may maintain tolerance in an antigen-filled environment. Supported by NIH grant #DK42166. • G3977 GIARDIA LAMBLIA INFECTION OF HUMAN INTESTINAL EPITHELIAL CELL CULTURES INHIBITS EPITHELIAL NITRIC OXIDE PRODUCTION. L. Eckmann. F. Laurent, F.D. Gillin, M.F. Kagnnff; Depts. of Medicine and Pathology, Univ.of Calif., San Diego, La Jolla, CA. Giardia lamblia is a strictly lumen-dwelling human pathogen that resides in the small intestine in close proximity to intestinal epithelial cells. Epithelial cells can release nitric oxide (NO) after cytokine stimulation, and NO was reported to kill G. lamblia trophozoites in vitro. In the present studies, we asked whether G. lamblia has evolved the ability to modulate epithelial cell NO production. Methods: Confluent cultures of HT-29 human intestinal epithelial cells and polarized Caco-2 epithelial cells were infected with G. lamblia trophozoites, and stimulated with a combination of IFN-~/, TNF~, and IL-I~. NO production was assessed by assaying the stable NO end products, nitrite and nitrate, in the culture supematants. Results: Infection of HT-29 cultures with G. lamblia trophozoites dose-dependently inhibited by >95% epithelial NO production induced by cytokine stimulation, whereas numbers of viable epithelial cells were not affected. Half-maximal inhibition was observed at a MOI of 1 Giardia:20 epithelial cells. Similarly, apical infection of polarized Caco-2 monolayers with G. lamblia inhibited epithelial NO production by >80%. Inhibition of epithelial NO production required viable Giardia trophozoites but not cell:cell contact, suggesting release of an inhibitory activity by G. lamblia or a lack of crucial substrates for epithelial NO production as possible mechanisms underlying the inhibition of epithelial NO production. In support of the latter, addition of arginine, which is the substrate for NO synthase to produce NO and an important metabolic fuel for Giardia, to Giardia-infected HT-29 cultures almost completely reversed the inhibition of epithelial NO production. Conclusion: The strictly lumendwelling human pathogen G. lamblia can inhibit the production of the Giardia-toxic effector molecule NO by intestinal epithelial cells through limiting epithelial access to arginine. Supported by NIH grant DK35108 and a CCFA Award. • G3978 A RANDOMIZED,
SINGLE-BLIND, PHARMACOKINETIC AND DOSERESPONSE STUDY OF SUBCUTANEOUS METHOTREXATE, 15 AND 25 MG/WEEK, FOR REFRACTORY ULCERATIVE COLITIS AND CROHN'S DISEASE. LJ Egan 1, WJ Sandborn I, WJ Tremaine 1, JA Leighton 2, DC Mays 1, MG PikO, AR Zinsmeister 1, JJ Lipsky 1. Mayo Clinic, Rochester, MN1; Scottsdale, AZ2. Background: Methotrexate (MTX), 25 rag/week by i.m. injection, has been
shown to induce remission in 39% of refractory Crohn's disease (CD) patients, but toxicity limited therapy in 17% (Feagan NEJM 1995;332:292). MTX 12.5 rag/week by mouth was ineffective in ulcerative colitis (UC), but toxicity was rare (Oren Gastroenterology 1996;110:1416). Erythrocyte and plasma MTX concentrations may be useful predictors of response and toxicity. Aims: 1) To determine if circulating concentrations of MTX predict response or toxicity. 2) To compare the efficacy of MTX, 15 or 25 mg/week by s.c. injection for refractory UC and CD. Methods: 31 patients with refractory UC, n=14 and CD, n=17, defined as inability to discontinue prednisone without disease flare, were enrolled and prednisone was adjusted to 20 mg/d for 2 weeks. Patients were stratified for balancing into 4 groups: by disease, UC or CD, and by activity, inflammatory bowel disease questionnaire (IBDQ) <170 (steroid refractory), or _> 170 (steroid dependant), and then randomized to MTX 15 vs 25 mg/week s.c. (patients blinded). A prednisone taper was begun at 2 weeks, but if disease flared, prednisone could be increased. Every 2 weeks the IBDQ was administered and trough concentrations of erythrocyte MTX (competitive protein binding assay), plasma MTX and plasma 7OHMTX (HPLC) were determined. Toxicity was defined as any event leading to MTX dose reduction or discontinuation. After 16 weeks, clinical outcomes were assessed for remission (IBDQ > 170 and off prednisone) and response (remission, or increase in IBDQ _> 16, or IBDQ > baseline and off prednisone). Results: 1 patient was eliminated after screening and 2 patients were censored from analysis for non-compliance. Median overall trough erythrocyte MTX, plasma MTX and plasma 7OHMTX concentrations were not different between and 15 and 25 mg/week groups. Mean -+ SE MTX concentrations: *Erythrocyte MTX *PlasmaMTX *Plasma7OHMTX Yes No Yes No Yes No UC response 181±25 169±11 0.7±0.3 0.5±0.2 0.5±0.1 2.0±0.5 CD response 205±21 214+31 0.7±0.1 0.7±0.3 2.0±0.8 3.1±2.0 UC+CD toxicity 143±3 197±12 0.5±0.4 0.7+0.1 1.0±0.7 2.0±0.5 *No associationsbetweenresponseor toxicity and concentrations(logisticregression).