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Model system for study of virus-specific immune responses in gut-associated lymphoid tissue (GALT)
A904
AGA ABSTRACTS
Intestinal coccidiosis: using targeted mutagenesis to examine the roles of c¢1~and ?8 T-cells in the primary and secondary host response to Einseria. S.L Roberts, A. Smith, L. Wen, J.L. Viney, R.C. Findiy, M.J. Owen, A.B. West, and A.C. Hayday. Departments of Biology and Pathology, & Section of Immunobiology, Yale University School of Medicine, New Haven, CT; Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London, U.K.; Animal Health Division, Pfizer Inc., Groton, CT.
GASTROENTEROLOGY, VoI. IO8, No. 4
• EFFECT OF PLA2 INHIBITORS UPON THE INFLAMMATORY RESPONSE IN AN IN VIVO MODEL OF NECROTIZING ENTEROCOLITIS. JK Roche. L VanHoru, RL Guerrant. Department of Internal Medicine, University o f Virginia Health Sciences Center, Charlottesville, VA. ,o Neerotizing enterocolitis (NEe) is the most serious gastroenterological disorder affecting premature infants. It is characterized by segmental bowel inflammation and sometimes perforation, biliary pneumatosis, and failure to gain weight. We chose to study how local mucosal substances might modify pathophysiological mechanisms responsible for the intestinal lesions, through ,m,,i.l..,,. pharmacological blocking of requisite pro-inflamFigure1. Effectof quintmatory mediators. The model system, adapted trine (Q)on inte,tin~ seeretot7 response to from that of Clark et a/. (Ped. Res. 19:919), used acidified casein (1) at 3 ligated rat intestinal segments exposed to acidified hours. casein injected intraluminslly (small bowel and ,.0 colon), where inflammatory lesions are quantitated as an enhanced volume/length (V/L) ratio (indicating a secretory response) as Well as by macroscopic appearance and histology of the intestinal mucosa. Quinacrine was found to reduce the inflammatory secretion by > 50%, when injected intraluminally with the inciting agent (acidified Figure 2. Effect of casein) (Fig. 1). This result was found in both arietoloehi¢ acid on colon and small bowel, at several points after intestinal secret.or/ relesion induction (3 and 6 hours), and did not rely sponse to aeidlfiedcasein on systemic absorption of the quinecrine, e.g., lesions developed in intestinal loops adjacent to other quinicrine-treated ones. We next confirmed this finding using aristolochic ;acid, a more specific PLA2 inhibitor. Similar to quinacrine, this inhibitor reduced markedly both the V/L ratio as well as the inflammatory appearance of the luminal contents (Fig. 2). We conclude that PLA2 inhibitors are useful in reducing or preventing intestinal inflammatory lesions in vivo, and may be of benefit in human disorders such as necrotizing enterocolitis, where derivitives of arachadonic acid may be the principal pro-inflammatory elements active in mucosa. .
The crypt epithelial cells of the routine small intestine are targets for infection by Emeria vermiformis. Other species of this genus of protozoan parasites target many vertebrates including birds and reptiles. In fact, Eimeria infection of poultry is of considerable commercial importance. Eimeria infection induces crypt hyperplasia, munosal inflammation, and villous atrophy. However, the mechanism by which animals become immune to Eimeria is poorly understood. To approach this issue, we have studied the immune response to Eimeria in immunocompetent mice, and in mice rendered deficient in cO o r ~ T-cells or B cells by targeted gene disruption. The duration and severity of infection was monitored by counting the number of infectious oocysts shed in the feces. We can conclude from our studies that infection elicits increases in both the cO and "~ T-cell repertoires in the local lymph nodes,
and within the epithelium itself. However,attenuation of primary infection and the development of parasite-specific immunity are dependent entirely on cO T cells, und not on either ~ T-cells or B-cells. We are currently studying the contribution of ~6 T-cells to the regulation of epithelial celi turnover and the control of inflammation during the infection. Recent data will be presented.
• MODEL SYSTEM FOR STUDY OF VIRUS-SPECIFIC IMMUNE RESPONSES IN GUT-ASSOCIATED LYMPHOID TISSUE (GALT). JK Roche, V Braciele. Beirne Carter Center for Immunology Research, University of Virginia Health Sciences center, Charlottesville, VA. Although viruses are common antigens for stimulation of GALT in vDo, few model systems exist with which to study the virus-specific function of GALTderived T lymphocytes and their epitope specificity. We chose to investigate this, using the in-bred mouse species Balb/c challenged once orally with a virus which is trophic for mucosal epithelium (influenza), with subsequent characterization of meseateric lymph node ceils taken two to six weeks later. SlCr cytotoxicity was quantitated with infected (I) and non-infected (NI) P815 cells (6-hour assay), and specific proliferation measured using I and NI irradiated syogeneic splenocytes O-day assay). By the ~H-thymidine uptake/proliferatlon g" assay, GALT-derived lymphocytes demonstrated i ' ~ ~ J ~ _ specific stimulation for virus-infected cells in the presence of IL-2 (Fig. 1). A 60% loss of prolif'-eration without IL-2 suggested that lymphocytes were predominandy of CD8 ÷ lineage. A strong cytotoxic response of GALT-derived lymphocytes ~ "~ ......... /,~+,/...... against syngeneic virus-infected splenocytes was Figure l. ~H-Tuptake. demonstrated, even at E/T of 1/I, which was comparable t° the killing capacity °f a spleen'de- ! i ~ ~ d ~ l rived clone, tested concomitantly (Fig. 2). Lyric ~ .. units (calculated at 50% of maximum cytotoxicity) were 3 (GALl') and 6 per l04 target cells respec- ~ + tively. Flow cytometric analysis confirmed that CD8 cells predominated in most preps isolated " " . . .,/, . . .,:~ . . . . .+:~ . .,:,,,, from G A L T . E p R• o p e specificity of splenocytes of E ~.~To _ .~/, animals orally challenged with virus, showed that ~'" ~..... "" "~ ........"°~ "~"'" the predominant response was to the epitope 2 I0Figure2. StCreytotoxleity. 219, as determined using nonamers with binding for class I MHC. We conclude that this model system is useful in the study of virus-initiated GALT responses and may be used in the future to investigate the influence of the epithelium on the fine specificity and regulation of immune cells to viruses trophic for the intestinal epithelial cull
.
.
.
• MODULATION OF INTESTINAL INFLAMMATION: NEURO-PEPTIDE VIP DOWN-REGULATES T LYMPHOCYTE EFFECTS ON EPITHELIUM. JK Roche. L VanHorn, J Klein, S Strong, S Planchon. Dept of Interual Meal., Univ. of Va. Health Sciences Center, Charlottesville, VA; and the Research Dept., Cleveland Clinic Fdn., Cleveland, OH. la vDo, we have found that VIP reduces the secretory response which follows rat intestinal macosal exposure to C. d~clle toxin A (Fig. 1). However, the mechanism of this effect is not clear, and could involve either direct effects on the intestinal epithelium, inhibition of a mononuclear cell cytokine, or direct effects upon T lympbocytes in mucosa. To study this, we investigated human epithelial cell monolayers (T84), having a Figure 1 Effectof rip o~ quantifiable barrier function, which we have shown toxin A-inducedaccretionin t~vo. previously is reduced in the presence of intestinederived human lamina propria mononudear cells (LPMC) (Gastro. I04:A771). Effects of VIF directly upon epithelial barrier function were minimal, when monolayers were co-incubated with 106-10"~° M VIP and the transmonolayer electrical potential difference was used to quantitate epithelial resistance (Fig. 2). Likewise, VIP seemed to have little effect upon the lFN-v-induced decrement in epithelial barrier function. Howevel, VIP at 10+ M did reduce by > 50% the epithelial barrier function-disrupting effect of human colon-derived LPMC (Fig. 3). This effect may be partially explained by the ability of VIP to reduce release of lymphokines (IL-2, for example) from human colon-derived LPMC under Coo A stimulation (Fig. 4). We conclude that VIP as well as perhaps other neuropeptides may be potent down-regulators of local mucosal inflammatin~, primarily due to a direct effect on T lymphocytes and on the release of their secreted products. ! ]
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Figu~ 2. O, mediaonly; a, IFlq~/poaaiv©control.PGE2 ¢oneemlm~nl:A. IO~M;v, IO~M;II, 10-UM.
oo , + ~ Figure3. 0, mediaonly; A, LPMC only; •, LPMC + IOtMVIP; A. LPMC+ In+ M VIP; e. IFN-q,positive control.
.,~
-,o '-~e w'--
Figul~ 4. Inhibitionof LPMC lymphokin© re.lease.
AGA ABSTRACTS
Intestinal coccidiosis: using targeted mutagenesis to examine the roles of c¢1~and ?8 T-cells in the primary and secondary host response to Einseria. S.L Roberts, A. Smith, L. Wen, J.L. Viney, R.C. Findiy, M.J. Owen, A.B. West, and A.C. Hayday. Departments of Biology and Pathology, & Section of Immunobiology, Yale University School of Medicine, New Haven, CT; Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London, U.K.; Animal Health Division, Pfizer Inc., Groton, CT.
GASTROENTEROLOGY, VoI. IO8, No. 4
• EFFECT OF PLA2 INHIBITORS UPON THE INFLAMMATORY RESPONSE IN AN IN VIVO MODEL OF NECROTIZING ENTEROCOLITIS. JK Roche. L VanHoru, RL Guerrant. Department of Internal Medicine, University o f Virginia Health Sciences Center, Charlottesville, VA. ,o Neerotizing enterocolitis (NEe) is the most serious gastroenterological disorder affecting premature infants. It is characterized by segmental bowel inflammation and sometimes perforation, biliary pneumatosis, and failure to gain weight. We chose to study how local mucosal substances might modify pathophysiological mechanisms responsible for the intestinal lesions, through ,m,,i.l..,,. pharmacological blocking of requisite pro-inflamFigure1. Effectof quintmatory mediators. The model system, adapted trine (Q)on inte,tin~ seeretot7 response to from that of Clark et a/. (Ped. Res. 19:919), used acidified casein (1) at 3 ligated rat intestinal segments exposed to acidified hours. casein injected intraluminslly (small bowel and ,.0 colon), where inflammatory lesions are quantitated as an enhanced volume/length (V/L) ratio (indicating a secretory response) as Well as by macroscopic appearance and histology of the intestinal mucosa. Quinacrine was found to reduce the inflammatory secretion by > 50%, when injected intraluminally with the inciting agent (acidified Figure 2. Effect of casein) (Fig. 1). This result was found in both arietoloehi¢ acid on colon and small bowel, at several points after intestinal secret.or/ relesion induction (3 and 6 hours), and did not rely sponse to aeidlfiedcasein on systemic absorption of the quinecrine, e.g., lesions developed in intestinal loops adjacent to other quinicrine-treated ones. We next confirmed this finding using aristolochic ;acid, a more specific PLA2 inhibitor. Similar to quinacrine, this inhibitor reduced markedly both the V/L ratio as well as the inflammatory appearance of the luminal contents (Fig. 2). We conclude that PLA2 inhibitors are useful in reducing or preventing intestinal inflammatory lesions in vivo, and may be of benefit in human disorders such as necrotizing enterocolitis, where derivitives of arachadonic acid may be the principal pro-inflammatory elements active in mucosa. .
The crypt epithelial cells of the routine small intestine are targets for infection by Emeria vermiformis. Other species of this genus of protozoan parasites target many vertebrates including birds and reptiles. In fact, Eimeria infection of poultry is of considerable commercial importance. Eimeria infection induces crypt hyperplasia, munosal inflammation, and villous atrophy. However, the mechanism by which animals become immune to Eimeria is poorly understood. To approach this issue, we have studied the immune response to Eimeria in immunocompetent mice, and in mice rendered deficient in cO o r ~ T-cells or B cells by targeted gene disruption. The duration and severity of infection was monitored by counting the number of infectious oocysts shed in the feces. We can conclude from our studies that infection elicits increases in both the cO and "~ T-cell repertoires in the local lymph nodes,
and within the epithelium itself. However,attenuation of primary infection and the development of parasite-specific immunity are dependent entirely on cO T cells, und not on either ~ T-cells or B-cells. We are currently studying the contribution of ~6 T-cells to the regulation of epithelial celi turnover and the control of inflammation during the infection. Recent data will be presented.
• MODEL SYSTEM FOR STUDY OF VIRUS-SPECIFIC IMMUNE RESPONSES IN GUT-ASSOCIATED LYMPHOID TISSUE (GALT). JK Roche, V Braciele. Beirne Carter Center for Immunology Research, University of Virginia Health Sciences center, Charlottesville, VA. Although viruses are common antigens for stimulation of GALT in vDo, few model systems exist with which to study the virus-specific function of GALTderived T lymphocytes and their epitope specificity. We chose to investigate this, using the in-bred mouse species Balb/c challenged once orally with a virus which is trophic for mucosal epithelium (influenza), with subsequent characterization of meseateric lymph node ceils taken two to six weeks later. SlCr cytotoxicity was quantitated with infected (I) and non-infected (NI) P815 cells (6-hour assay), and specific proliferation measured using I and NI irradiated syogeneic splenocytes O-day assay). By the ~H-thymidine uptake/proliferatlon g" assay, GALT-derived lymphocytes demonstrated i ' ~ ~ J ~ _ specific stimulation for virus-infected cells in the presence of IL-2 (Fig. 1). A 60% loss of prolif'-eration without IL-2 suggested that lymphocytes were predominandy of CD8 ÷ lineage. A strong cytotoxic response of GALT-derived lymphocytes ~ "~ ......... /,~+,/...... against syngeneic virus-infected splenocytes was Figure l. ~H-Tuptake. demonstrated, even at E/T of 1/I, which was comparable t° the killing capacity °f a spleen'de- ! i ~ ~ d ~ l rived clone, tested concomitantly (Fig. 2). Lyric ~ .. units (calculated at 50% of maximum cytotoxicity) were 3 (GALl') and 6 per l04 target cells respec- ~ + tively. Flow cytometric analysis confirmed that CD8 cells predominated in most preps isolated " " . . .,/, . . .,:~ . . . . .+:~ . .,:,,,, from G A L T . E p R• o p e specificity of splenocytes of E ~.~To _ .~/, animals orally challenged with virus, showed that ~'" ~..... "" "~ ........"°~ "~"'" the predominant response was to the epitope 2 I0Figure2. StCreytotoxleity. 219, as determined using nonamers with binding for class I MHC. We conclude that this model system is useful in the study of virus-initiated GALT responses and may be used in the future to investigate the influence of the epithelium on the fine specificity and regulation of immune cells to viruses trophic for the intestinal epithelial cull
.
.
.
• MODULATION OF INTESTINAL INFLAMMATION: NEURO-PEPTIDE VIP DOWN-REGULATES T LYMPHOCYTE EFFECTS ON EPITHELIUM. JK Roche. L VanHorn, J Klein, S Strong, S Planchon. Dept of Interual Meal., Univ. of Va. Health Sciences Center, Charlottesville, VA; and the Research Dept., Cleveland Clinic Fdn., Cleveland, OH. la vDo, we have found that VIP reduces the secretory response which follows rat intestinal macosal exposure to C. d~clle toxin A (Fig. 1). However, the mechanism of this effect is not clear, and could involve either direct effects on the intestinal epithelium, inhibition of a mononuclear cell cytokine, or direct effects upon T lympbocytes in mucosa. To study this, we investigated human epithelial cell monolayers (T84), having a Figure 1 Effectof rip o~ quantifiable barrier function, which we have shown toxin A-inducedaccretionin t~vo. previously is reduced in the presence of intestinederived human lamina propria mononudear cells (LPMC) (Gastro. I04:A771). Effects of VIF directly upon epithelial barrier function were minimal, when monolayers were co-incubated with 106-10"~° M VIP and the transmonolayer electrical potential difference was used to quantitate epithelial resistance (Fig. 2). Likewise, VIP seemed to have little effect upon the lFN-v-induced decrement in epithelial barrier function. Howevel, VIP at 10+ M did reduce by > 50% the epithelial barrier function-disrupting effect of human colon-derived LPMC (Fig. 3). This effect may be partially explained by the ability of VIP to reduce release of lymphokines (IL-2, for example) from human colon-derived LPMC under Coo A stimulation (Fig. 4). We conclude that VIP as well as perhaps other neuropeptides may be potent down-regulators of local mucosal inflammatin~, primarily due to a direct effect on T lymphocytes and on the release of their secreted products. ! ]
b, +
i"t
"o
i
,
=
+
Figu~ 2. O, mediaonly; a, IFlq~/poaaiv©control.PGE2 ¢oneemlm~nl:A. IO~M;v, IO~M;II, 10-UM.
oo , + ~ Figure3. 0, mediaonly; A, LPMC only; •, LPMC + IOtMVIP; A. LPMC+ In+ M VIP; e. IFN-q,positive control.
.,~
-,o '-~e w'--
Figul~ 4. Inhibitionof LPMC lymphokin© re.lease.