Conditioned Immunosuppression in Orally Immunized Mice

BRAIN, BEHAVIOR, AND IMMUNITY ARTICLE NO.

10, 44–54 (1996)

0004

Conditioned Immunosuppression in Orally Immunized Mice EDELTON FLAVIO MORATO,* MARIA GERBASE-DELIMA,† AND REGINALD M. GORCZYNSKI‡ Department of *Microbiologia, Imunologia and Parasitologia, Universidade Federal de Santa Catarina, Brazil; †Department of Pediatria, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Brazil; and ‡Department of Surgery and Immunology, University of Toronto, M5G 2C4, Toronto, Ontario, Canada Mice were given oral immunization after pretreatment with a regimen (cyclophosphamide and a novel taste in the drinking water, chocolate milk (CHM)) which leads to suppression of the antibody response to intravenously administered antigens given concurrently with CHM. Following this treatment mice were reexposed to CHM and IgM and IgA antibody forming cells (AFC) were measured in spleen and Peyer’s patch cells. Conditioned immunosuppression of AFC production was most marked (úfivefold) for IgA-AFC in Peyer’s patch, with effects of lesser magnitude for IgM-AFC in Peyer’s patch (twofold) and both IgM- and IgA-AFC in spleen. Analysis of cytokine production from stimulated Peyer’s patch and splenic T cells in vitro showed significant decreased production of both IL-2 and IL-4, with the latter being the predominant cytokine produced in Peyer’s patch cells of control animals. q 1996 Academic Press, Inc.

INTRODUCTION

There is a large body of evidence suggesting that both humoral and cell-mediated immune responses can be modified using Pavlovian conditioning procedures (for a review see Ader, Felten, & Cohen, 1991). One of the most used models for such analysis is represented by animals previously exposed to a flavored drinking solution (conditioned stimulus; CS) in the context of an intraperitoneal injection with cyclophosphamide (unconditioned stimulus). These conditioned animals subsequently show a diminished antibody response to sheep erythrocytes (SRBC) when reexposed to the CS concomitant with immunization (Ader & Cohen, 1975; Rogers, Reich, Strom, & Carpenter, 1976; Wayner, Flannery, & Singer, 1978; Gorczynski & Kennedy, 1984; Bovbjerg, Kim, Siskind, & Weksler, 1987; Gorczynski, 1987a). The gastrointestinal (G.I.) tract is extremely important in innate immune protection, since in many instances mucosal surfaces are the sites where pathogenic microorganisms first interact with the host. The gut-associated lymphoreticular tissue, including Peyer’s patches (PP), the appendix, and the solitary lymphoid nodules, is responsible for the selective uptake of antigens passing through the G.I. tract (Fujihashi, Kiyono, Beagley, Eldridge, & McGhee, 1988). PP contain regulatory T cells and other immunocompetent cells that may be important for determining both the quality and degree of immune response which occurs in response to orally administered antigens (Kiyono & McGhee, 1987). Current consensus suggests that there are at least two types of CD4/ Th cells, Th1 and Th2, defined by the patterns of cytokines they produce. Th1-type cells produce predominantly IL-2 and IFNg on stimulation, while Th2-type cells produce IL-4, IL-10, and IL-13 (Street & Mosmann, 1991; D’Andrea, Aste-Amegaza, Valiante, Ma, Kubin, & Trinchieri, 1993; de Waal Malefyt, Figdor, Huijbens, MohanPeterson, Bennet, Culpepper, Dang, Zurawski, & de Vries, 1993; Gorczynski, 1995a). Activation of different populations of Th cells may regulate development of different 0889-1591/96 $18.00

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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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types of immune responses (Fitch, McKisic, Lancki, & Gajewski, 1993). Thus studies in vitro showed that Th1-stimulated clones are involved preferentially in IgG2a synthesis, probably due to the action of IFNg and enhancement by IL-2 (Coffman, Seymour, Lebman, Hiraki, Christiansen, Shrader, Cherwinski, Savelkoul, Finkelman, Bond, & Mosmann, 1988). In contrast, IL-4 and IL-5/IL-6 (Th2 cytokines) are involved, respectively, in the production of IgE, IgG1, IgA, and other IgG subclasses (Leibson, Gefter, Zlotinic, Marrack, & Kappler, 1984; Coffman et al., 1988). TGFb, another cytokine produced locally in mucosal lymphoid tissue, may play a key role in regulation of mucosal immunity (Staats, Jackson, Marinaro, Takahashi, Kiyono, & McGhee, 1994). Despite numerous studies on conditioned immunosuppression (for a review see Ader et al., 1991) and on the role of the gastrointestinal tract in regulation of immune responses (for a review see Ogra, Mestecky, Lamm, Strober, & McGhee, 1994), there are few studies concerning the effects of conditioning in mice immunized with antigen by the oral route. The present study was undertaken to investigate the capacity of spleen and PP-T cells from orally immunized and conditioned mice to cooperate with B cells for Ig production. METHODS

Mice. Balb/c male mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were housed five per cage and allowed food and water ad libitum (except where on restricted water schedule; see text). Animals were on a 12 h on, 12 h off light–dark cycle (artificial lighting from 7:00–19:00 daily). Mice were entered into experiments at 10 weeks of age. Oral and systemic immunizations. SRBC from a single donor were obtained every 2 weeks from Dr. J. Hay (Sunnybrook Hospital, Toronto) and washed three times in phosphate-buffered saline (PBS) before use. Nonanesthetized mice were immunized at 9:00 with SRBC by a single intravenous injection of 5 1 108 SRBC (lateral tail vein) or by oral route, with a gavage needle, using 2 1 109 SRBC/mouse/day for 4 consecutive days according to Andre´, Bazin, and Heremans (1973). Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem (San Diego, CA). Mice were inoculated with KLH by gavage feeding with a dose of 1 mg/mouse/day for 4 consecutive days or by a single intravenous injection of 100 mg/mouse. Sodium dinitrobenzene sulfonate (DNBS; Eastman Kodak) was coupled to bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) as described elsewhere (Good, Wofsy, Henry, & Kimura, 1980). Mice were immunized with DNP–BSA using three subcutaneous immunizations at 21-day intervals with 100 mg DNP–BSA in incomplete Freund’s adjuvant. Animals were used as cell donors for the assays described below from 4 to 8 weeks after the last immunization. Conditioning of mice. Mice were maintained on a daily watering schedule in which water was available for a 30-min period (8:00 to 8:30 AM). After 10 days, experimental animals were exposed to chocolate milk (CHM; conditioned stimulus) in their drinking supply and immediately thereafter received an intraperitoneal (ip) injection of cyclophosphamide (CY; unconditioned stimulus) at a dose of 100 mg/kg in 0.5 ml of PBS. Reexposure to chocolate milk and cyclophosphamide was repeated on two further occasions at 21-day intervals (CHM:CY group). Four groups of mice were used as controls, as described elsewhere (Gorczynski, 1987b). In brief, these were: (a) mice receiving CHM followed by an ip injection of PBS (CHM:PBS group); (b) mice receiving water followed by an ip injection of CY (H2O:CY group); (c) mice receiving

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FIG. 1. Conditioning trials and oral immunization of mice. a CHM represents chocolate-flavored drinking milk. H2O is plain water in the drinking supply. CY indicates ip injection with cyclophosphamide (100 mg/kg). All groups consisted of a minimum of four/ group. b Oral immunization was daily (for 4 days) with 2 1 109 SRBC or 1 mg/mouse KLH, 28 days after the last trial. Mice in different groups were reexposed to CHM or H2O in the drinking supply as shown. * Unless shown otherwise, mice received H2O to drink.

water followed by an ip injection of PBS (H2O:PBS group); and (d) conditioned mice (CHM:CY) receiving no exposure to CHM after immunization (conditioned but nonreexposed to cues). In all groups CHM and injections were given on two further occasions at 21-day intervals. Mice in all groups were orally immunized 28 days after the last exposure to CY:CHM. Further reexposure to CHM on Days 0, 2, and 4, and where applicable on Day 6, after immunization was as described in Fig. 1 and shown in individual experiments (Tables 1–4). Cell preparation and culture. Spleen cells were removed aseptically in PBS and single-cell suspensions were prepared as described elsewhere (Gorczynski, MacRae, & Kennedy, 1982). PP were gently excised from the intestinal wall and dissociated with an appropriate enzyme mixture of Dispase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and Collagenase (Sigma Chemical Co., St. Louis, MO) to obtain single-cell preparations, according to methods described elsewhere (Kiyono, McGhee, Wannemuehler, Frangakis, Spalding, Michalek, & Koopman, 1982). Nylon-woolpurified T cells and anti-Thy 1.2-treated spleen (B) cells were obtained as described elsewhere (Gorczynski, 1987b). Monoclonal antibody and rabbit complement used to deplete T cells were obtained from Cedarlane Laboratories (Hornby, Ontario, Canada). B cells from mice immunized ip with DNP–BSA were incubated in vitro with T cells obtained from spleen or Peyer’s patch of mice immunized orally with SRBC or KLH. Irradiated normal spleen cells were used as feeder cells in culture, and 0.1% TNP– SRBC (30 ml/ml culture) or TNP–KLH (1 mg/ml culture) was used as antigen (Gorczynski & Cunningham, 1978). TNP (Sigma Chemical Co., St. Louis, MO) was coupled to KLH or to SRBC according to Henry (1980). Antibody-forming cells were enumerated on Day 5 of culture. Cultures designed to test proliferation and IL-2 and IL-4 production were set up with spleen and PP cells as previously described (Gorczynski & Kiziroglu, 1994). In all the experiments described below, cells were prepared from individual mice in all groups and tested separately in all assays. Assay for antibody-forming cells (AFC). The anti-SRBC Ig responses were assayed in modified Cunningham chambers, as described elsewhere (Gorczynski & Cunning-

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ham, 1978). Anti-TNP AFC responses were assayed in microwell plates (Kappler, 1974), using TNP lightly conjugated to SRBC (Rittenberg & Pratt, 1969). IgA-AFCs were determined using rabbit anti-mouse IgA antibodies (Serotec, Oxford, UK). The number of IgA-AFCs was calculated from differences in counts from slides assayed with/without anti-IgA antiserum. All assays were performed in triplicate. Cell proliferation and measurement of IL-2 and IL-4. Proliferation (as assessed by 3 HTdR uptake) was measured by harvesting culture contents onto glass fiber filter papers and counting in a b-counter (Gorczynski & Kiziroglu, 1994). Supernatants from stimulated cultures were assayed for IL-2 or IL-4 production, as described in detail elsewhere (Gorczynski & Wojcik, 1994). In brief, IL-2 activity was assayed using the IL-2-dependent cell line CTLL-2. Recombinant IL-2 for standardization of assays was purchased from Genzyme (Cambridge, MA). All assays were set up in triplicate in the presence of 11B11 mAb to block potential stimulation of CTLL-2 with IL-4. IL-4 activity was assayed with the IL-4-responsive line CT.4S, provided by Dr. G. Mills, Toronto General Hospital. Recombinant IL-4 for standardization of IL-4 assays was purchased from Pharmingen (San Diego, CA). All assays for IL-4 were set up in triplicate in the presence of the mAb S4B6 to block IL-2-mediated stimulation. Both the IL-2 and the IL-4 assays detected at least 0.2 U of recombinant lymphokine added to cultures. Statistical analysis. Differences in the responses between groups were assessed by one-way analysis of variance (ANOVA). RESULTS

T Cells from Conditioned Mice Challenged with Oral Antigen Have Decreased Helper Activity for B-Cell IgA-AFC Production Preliminary studies showed that in mice immunized by the oral route, using two different antigens for T-cell priming, one particulate, SRBC, and the other soluble, KLH, and sacrificed at Day 7 to provide a source of ‘‘helper T cells’’ for antibody responses in vitro (a time proven optimal by previously determined kinetics), PP rather than splenic T cells proved more effective in cooperation with spleen B cells for IgA-AFC production. Thus typical AFC responses for PP vs splenic T cells were 466 { 128 vs 42 { 19 and 395 { 84 vs 38 { 14, respectively, for SRBC or KLH stimulation. For IgM-AFC production, no significant difference between spleen and PP-T cells was detected. No significant IgA-AFC responses were observed with either of the two T-cell preparations following iv immunization with SRBC or KLH (data not shown). In order to investigate the role of conditioning upon T-cell cooperation for AFC production, we used T cells from conditioned SRBC-immunized mice instead of T cells from normal mice. As can be seen in Table 1 (typical data for one of four such studies), these results confirmed that PP-T cells are more efficient than spleen T cells in providing help for IgA production by B cells. There was an inhibition of this cooperation when T cells from conditioned mice reexposed to CHM were used. The number of IgM-AFC in cultures with PP-T cells, as well as the number of IgA-AFC with either spleen or PP-T cells, was significantly reduced in the conditioned -CHMexposed group, compared to nonconditioned CY-treated mice or conditioned mice not reexposed to CHM after immunization (Table 1, rows 13–15). Note that in separate studies where animals were exposed to CY at the time of immunization, no detectable

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TABLE 1 Effect of Conditioning on the Cooperation of Spleen (SP) and Peyer’s Patches (PP) T Cells from SRBC Orally Immunized Mice for IgM- and IgA-AFC Production by DNP–BSA-Primed B Cells, after in vitro Challenge with TNP–SRBC Anti-TNP AFC/culturea Pretreatment

Test trial

T cell

B cell

IgM

IgA

H2O:PBS

H2O:SRBC

0 SP PP

/ / /

8{ 5 64 { 31 96 { 36

0{ 0 19 { 10 166 { 44

H2O:CYb

CHMc:SRBC

0 SP PP

/ / /

9{ 4 31 { 11 107 { 33

7{ 4 11 { 8 187 { 48

CHM:PBS

H2O:SRBC

0 SP PP

/ / /

18 { 9 58 { 22 110 { 40

16 { 5 21 { 9 168 { 34

CHM:CY

CHM:SRBC

0 SP PP

/ / /

CHM:CY

H2O:SRBC

0 SP PP

/ / /

9{ 7 11 { 9 38 { 16* 8{ 5 27 { 12 112 { 36

6{ 3 0 { 0* 39 { 21* 8{4 9{ 4 216 { 39

a Arithmetic mean ({SD) of results obtained in cultures from individual cell suspensions of four mice per group. All cultures for each suspension were assayed in triplicate. b CY, cyclophosphamide. c CHM, chocolate milk. * p õ .05, compared with controls.

TNP-AFC above the background in nonimmunized control mice were detected (data not shown). To extend this example of conditioning in T-cell cooperation to a situation where a soluble antigen was the immunogen used, T cells from conditioned KLH-immunized mice were tested with DNP–BSA-primed B cells. As can be seen in Table 2 (data from one of four studies), conditioned mice reexposed to CHM (Table 2, rows 10– 12) again showed a significant reduction in the number of IgM-AFC when T cells were obtained from PP, but not from spleen T cells. For IgA-AFCs, significant reductions were observed with both spleen and PP-T cells obtained from conditioned mice. IgM- and IgA-AFC Production in Conditioned Mice Immunized by the Oral Route When we investigated the kinetics of IgM- and IgA-AFC production in spleen and PP following oral immunization, optimal anti-SRBC IgM- and IgA-AFC responses occurred simultaneously between Days 8 and 10. We thus next investigated antiSRBC IgM- and IgA-AFC production by spleen and PP cells from different groups of mice following conditioning and oral immunization 9 days following immunization, as shown in Table 3 (one of four studies). Conditioned animals exposed to CHM on Days 0, 2, 4, and 6 following immunization showed a significant reduction in IgA-

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TABLE 2 Effect of Conditioning on the Cooperation of Spleen (SP) and Peyer’s Patches (PP) T Cells from KLH Orally Immunized Mice for IgM- and IgA-AFC Production by DNP–BSA-Primed B Cells, after in Vitro Challenge with TNP–KLH Antigen Anti-TNP AFC/culturea Pretreatment

Test trial

T cell

B cell

IgM

IgA

H2O:PBS

H2O:KLH

0 SP PP

/ / /

7{ 4 36 { 19 152 { 43

3{ 2 66 { 29 355 { 104

H2O:CYb

CHMc:KLH

0 SP PP

/ / /

6{ 4 45 { 21 166 { 54

2{ 2 56 { 29 310 { 106

CHM:PBS

H2O:KLH

0 SP PP

/ / /

10 { 4 62 { 29 186 { 45

5{ 3 78 { 38 360 { 156

CHM:CY

CHM:KLH

0 SP PP

/ / /

CHM:CY

H2O:KLH

0 SP PP

/ / /

8{ 4 31 { 19 44 { 16* 6{ 5 38 { 19 135 { 39

0{ 0 14 { 6* 49 { 20* 3{ 2 51 { 24 249 { 88

Note. Superscripts a,b, and c are the same as for Table 1. * p õ .05, compared with controls.

AFC production by both spleen and PP cells compared with other groups, in particular with conditioned mice not reexposed to CHM (Table 3, rows 9 and 10). For IgMAFC production, a significant reduction was observed with spleen cells but not with PP cells from conditioned:CHM mice (Table 3, row 7). Cellular Proliferation and Cytokine Production by Spleen and PP-T Cells in Conditioned Mice Immunized with KLH by the Oral Route Based on the higher production of anti-TNP IgA-AFC using T cells from mice fed with KLH (Table 2) in comparison to those fed with SRBC (Table 1), in this experiment we utilized KLH for oral immunization. The results of the experiment designed to evaluate cellular proliferation and IL-2 and IL-4 cytokine secretion by spleen and PP cells are shown in Table 4 (one of three studies). Cells from all groups except those taken from conditioned mice reexposed to CHM proliferated when stimulated with KLH in vitro. IL-2 production from both spleen and PP populations was also similar in all groups except for the conditioned:CHM group, where significant suppression of IL-2 production was seen (Table 4, rows 8 and 9). No detectable IL-4 was observed in spleen cell suspensions from all groups. PP cells produced IL-4 at equivalent levels in the control groups, whereas the production of IL-4 was again essentially abolished in the conditioned:CHM group (Table 4, row 9).

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TABLE 3 IgM and IgA-AFC Production on Day 9 after Oral SRBC Immunization by Spleen (SP) and Peyer’s Patches (PP) Cells of Conditioned Mice and Controls Anti-SRBC AFC (106/cells)a Pretreatment

Test trial

Cells

IgM

IgA

H2O:PBS

H2O:SRBC

SP PP

154 { 61 51 { 23

612 { 206 159 { 64

H2O:CYb

CHMc:SRBC

SP PP

164 { 65 42 { 19

494 { 183 128 { 43

CHM:PBS

H2O:SRBC

SP PP

166 { 24 45 { 29

585 { 168 169 { 69

CHM:CY

CHM:SRBC

SP PP

CHM:CY

H2O:SRBC

SP PP

89 { 39* 29 { 14 178 { 48 52 { 19

109 { 68* 48 { 29* 525 { 174 149 { 38

Note. Superscripts a, b, and c are the same as for Table 1. * p õ .05, compared with controls.

DISCUSSION

It is well established that PP cells possess all of the immunocompetent cells, including regulatory T cells, that are necessary for induction and regulation of the IgA response (Suzuki, Kitamura, Kiyono, Kurita, Green, & McGhee, 1986). When a thymus-dependent antigen, SRBC, was used to prime mice by the oral route, PP cells became sensitized and showed a preferential IgA isotype response (Kiyono et al., 1982). The studies reported above verify this capacity of T cells from SRBC or KLHorally immunized mice to cooperate with B cells (obtained from mice immunized by intraperitoneal injection) for AFC production and show that PP-T cells preferentially potentiate IgA antibody formation. These data suggest that gavage feeding preferentially primes a Th2 response at the PP level for an IgA-AFC response. This in turn is in accordance with a recent report that showed a selective Th2 response in PP, followed by a migration of these cells to the spleen, in mice fed with SRBC (XuAmano, Aicher, Taguchi, Kiyono, & McGhee, 1992). Our results showing that the conditioning regime used alters splenic T-cell cooperation mainly for IgA-AFC production (Table 3) further supports this hypothesis of the migration of PP-primed T cells to the spleen. The possibility of neurohormonal/neuroendocrine control of the migration of T cells within the mucosal immune system, under the control of, for instance, vasointestinal peptide (VIP), has been extensively discussed by Ottaway (1984). Note, however, that we could not detect IL-4 production by splenic cells cultured with KLH on different days despite detectable IL-4 production by PP cells from Day 5 post oral immunization (data not shown). The modification of the immune response to systemically injected SRBC by conditioning has been well demonstrated (Ader & Cohen, 1975; Gorczynski & Kennedy, 1984; Gorczynski, MacRae, & Kennedy, 1984; Gorczynski, 1987a; Bovbjerg et al.,

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TABLE 4 Proliferation (IL-2 or IL-4) Production by Spleen (SP) and Peyer’s Patches (PP) Cell Suspensions from Conditioned Mice Fed with KLH and Challenged in Vitro with KLH

Pretreatment

Test trial

Cells

(cpm)a

0

0

SP

1060 { 316

IL-2 (U/ml)a

IL-4 (U/ml)a

0

0

H2O:PBS

H2O:KLH

SP PP

5168 { 486 6065 { 1211

5.6 { 1.1 3.1 { 1.2

0 6.6 { 1.7

H2O:CY

CHM:KLH

SP PP

4135 { 685 5120 { 904

6.1 { 1.5 2.0 { 0.6

0 5.1 { 2.1

CHM:PBS

H2O:KLH

SP PP

4560 { 1061 5560 { 1125

8.1 { 1.8 2.8 { 0.7

0 5.7 { 1.7

CHM:CY

CHM:KLH

SP PP

2195 { 503* 1390 { 319*

2.9 { 1.4* 0.4 { 0.3*

0 1.0 { 0.5*

CHM:CY

H2O:KLH

SP PP

5350 { 619 5815 { 1120

7.6 { 1.7 2.5 { 0.5

0 6.2 { 2.3

a See text, Materials and Methods, and footnotes to previous Tables for more details. All data represent arithmetic means ({SD) of triplicate cultures from four individuals/group.

1987). The present data extend these findings to orally immunized mice and show that conditioning can both locally and systemically modify the IgA immune response of mice fed with SRBC. One interesting observation of the conditioning effect on the AFC response was that the reduction of IgA-AFC (between 2.5- and 3.5-fold and 4.5and 5.5-fold for PP and spleen, respectively) was greater than the reduction of IgMAFC (between 1.4- and 1.7-fold and 1.7- and 1.8-fold reduction for PP and spleen, respectively). Whether this reflects a primary mechanism of conditioned immunosuppression at the ‘‘helper T cell’’ level, which is thus most manifest in AFC responses dependent upon T-cell cooperation (IgA, IgG, etc.) rather than in relatively T-independent AFC responses (IgM), remains to be seen. A further point of interest in this study is the negligible residual effect of CY on IgA responses by PP cells (Table 3), in contrast to the reported residual CY effect on IgG responses of cells seen after systemic immunization (Gorczynski, 1987a,b). Again, it is not clear to what extent this may reflect the different regulatory mechanisms operating for IgG and IgA responses and or systemic vs orally administered antigen. The analysis of cytokine production in conditioned mice given oral immunization demonstrated a decrease in Th activities, with a marked loss of Th2 function and IL4 production from PP cells (Table 4). This result is consistent with the data cited above, where a predominant activation of Th2 in Peyer’s patch was observed in mice fed with particular or soluble T-cell-dependent antigens. Note, however, that we recently reported that at least in mice challenged intracutaneously with Leishmania major, there was an apparent difference in the susceptibility of Th1 and Th2 cells to conditioned immunosuppression (Gorczynski, 1995b). Whether this reflects a difference related to the route of antigen administration, the nature of the antigen, or both of these (and other) variables is as yet unclear. Nevertheless, the possibility of

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conditioning immune responses to oral immunization has possible important clinical implications (e.g., in oral vaccines) for immunization of human populations (Staats et al., 1994). MacQueen et al. (MacQueen, Marshall, Perdue, Siegel, & Bienenstock, 1989) reported that release of mast cell protease II from the mucosal lamina propria of the intestine and lung could be conditioned using audiovisual cues. In addition to the studies of Ottaway on VIP, discussed above (Ottaway, 1984), there are independent reports that cholecystikinin can regulate IgA secretion in the rat intestine (Freier, Eran, & Faber, 1987). However, there have been no comparative studies, to our knowledge, of the ‘‘conditionability’’ of mucosal vs systemic immunity, though one might, a priori, anticipate that the system providing the interface between the organism and its external environment would indeed be subject to multiple levels of control, including neural regulation. Since the present study demonstrates that conditioning can affect the immune response of mice immunized orally with T-cell-dependent antigens, studies are now directed toward examining the possibility that the response to T-cell-independent antigens given by the oral route might also be amenable to conditioned immunoregulation. The effect of conditioning upon the response to systemically injected T-cell-independent antigens is currently unresolved (Cohen, Ader, Green, & Bovbjerg, 1979; Schulze, Benson, Paule, & Roberts, 1988; Ader & Cohen, 1991). ACKNOWLEDGMENT This work was supported by the Canadian Ileitis and Colitis Foundation.

REFERENCES Ader, R., & Cohen, N. (1975). Behaviorally conditioned immunosuppression. Psychosom. Med. 37, 333– 340. Ader, R., & Cohen, N. (1991). Conditioning of the immune response. Neth. J. Med. 39, 263–273. Ader, R., Felten, D. L., & Cohen, N., Eds. (1991). Psychoneuroimmunology, 2nd ed. Academic Press: New York. Andre´, C., Bazin, H., & Heremans, J. F. (1973). Influence of repeated administration of antigen by the oral route on specific antibody-producing cells in the mouse spleen. Digestion 9, 166–175. Bovbjerg, D., Kim, Y. T., Siskind, G. W., & Weksler, M. E. (1987). Conditioned suppression of plaqueforming cell response with cyclophosphamide. Ann. N. Y. Acad. Sci. 496, 588–594. Coffman, R. L., Seymour, B. W., Lebman, D. A., Hiraki, D. D., Christiansen, J. A., Shrader, B., Cherwinski, H. M., Savelkoul, H. F. J., Finkelman, F. D., Bond, M. W., & Mosmann, T. R. (1988). The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol. Rev. 102, 5– 28. Cohen, N., Ader, R., Green, N., & Bovbjerg, D. (1979). Conditioned suppression of a thymus independent antibody response. Psychosom. Med. 41, 487–492. D’Andrea, A., Aste-Amegaza, M., Valiante, N. M., Ma, X., Kubin, M., & Trinchieri, G. (1993). Interleukin 10 (IL-10) inhibits human lymphocyte interferon g-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178, 1041–1048. de Waal Malefyt, R., Figdor, C. G., Huijbens, R., Mohan-Peterson, S., Bennet, B., Culpepper, J., Dang, W., Zurawski, G., & de Vries, J. E. (1993). Effects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes: Comparison with IL-4 and modulation by IFNg or IL-10 J. Immunol. 151, 6370–6381. Fitch, F. W., McKisic, M. D., Lancki, D. W., & Gajewski, T. F. (1993). Differential regulation of murine T lymphocyte subsets. Annu. Rev. Immunol. 11, 29–48. Freier, S., Eran, M., & Faber, J. (1987). Effect of cholecystikinin and of its antagonist, of atropine, and of food on the release of immunoglobulin A and immunoglobulin G specific antibodies in the rat intestine. Gastroenterology 93, 1242–1246.

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