Oral administration of bovine whey proteins to mice elicits opposing immunoregulatory responses and is adjuvant dependent

Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (beta-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-beta was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory outcome and this is also reflected by the systemic cytokine profile.     

The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration.

We have investigated the immunological consequences of feeding a protein antigen to previously immunized animals. BALB/c mice were systemically primed with ovalbumin (OVA) in complete Freund's adjuvant (CFA) and fed with high (10 mg/g body weight), medium (1 mg/g body weight) or low (1 microgram/g body weight) doses of OVA once (Day 1, 7 or 14) or sequentially for 5 days (Days 1-5, 7-11, 14-18). The specific IgG antibody response was suppressed only by early feeds of high-dose OVA (Days 1-5). Medium-dose OVA fed on Day 14 or low-dose OVA fed at any stage after immunization enhanced the IgG antibody response. In contradistinction, systemic delayed-type hypersensitivity responses (DTH) were usually suppressed by early feeds of high or medium doses of OVA but never after feeding low-dose OVA. The results suggest that systemic DTH and IgG antibody responses to oral antigen are subject to different control mechanisms in previously primed animals. Such responses depend on the immune status of the animal and are controlled by antigen dose, time and frequency of feeding. The immunological effects observed are also demonstrable following adoptive transfer of spleen cells collected 14 days after multiple feeds of high-dose OVA to immunized mice. Our findings suggest that oral hyposensitization after systemic immunization is regulated by (suppressor) spleen cells which are activated by gut-processed antigen.     

The role of antigen recognition and suppressor cells in mice with oral tolerance to ovalbumin.

The induction of tolerance by feeding proteins may prevent potentially harmful delayed-type hypersensitivity (DTH) reactions to food antigens. Suppressor T cells (Ts) are present in mice with tolerance of systemic DTH after feeding ovalbumin (OVA) but, as other immunoregulatory mechanisms have also been described, the exact role of Ts in maintaining tolerance is not known. In this study, we have used the ability of native and denaturated OVA to cross-react at the level of helper/effector T cells, but not Ts, to re-examine the role of Ts in oral tolerance to OVA. Mice immunized with native OVA (nOVA) or denatured OVA (dOVA) in adjuvant had fully cross-reacting DTH to either nOVA or dOVA, but intravenous administration of antigen induced Ts which were specific for the appropriate form. Mice fed nOVA or dOVA had identical tolerance of systemic DTH to both forms of OVA, and feeding nOVA induced splenic Ts which suppressed the DTH response to both nOVA and dOVA. Splenic Ts could not be detected in mice fed dOVA. The results support the hypothesis that tolerance of systemic DTH in mice fed native proteins is due to Ts. Although, for the moment, there is no complementary evidence for a role for Ts in oral tolerance to denatured proteins, this study is consistent with the idea that Ts are the mechanism which normally prevent enteropathy due to DTH against dietary proteins. In addition, our study underlines the differences between orally and parenterally induced Ts and reinforces the view that fed proteins induce Ts after processing by the gut or its lymphoid accessory cells.     

Priming of systemic and local delayed-type hypersensitivity responses by feeding low doses of ovalbumin to mice.

We have examined the effects on both systemic and intestinal immunity of feeding different doses of ovalbumin (OVA) to mice. A single feed of doses of more than 1 mg OVA produced significant suppression of subsequent delayed-type hypersensitivity (DTH) and IgG antibody responses. Feeding 100 micrograms-1 mg OVA had no net effect on systemic immunity, but mice fed 10-50 micrograms OVA had consistently enhanced systemic DTH responses when immunized subsequently with OVA in adjuvant. Oral challenge of these mice with OVA produced alterations in mucosal architecture and in intra-epithelial lymphocyte counts, consistent with the presence of an intestinal DTH response. Similar changes were not found in mice fed tolerogenic doses of OVA. Although feeding low doses of OVA primed both systemic and intestinal DTH responses, this had no effect on serum IgG responses and very little systemic DTH could be revealed in OVA-fed mice without systemic challenge with OVA in adjuvant. We conclude that feeding certain low doses of protein antigens can induce priming of local and systemic DTH responses rather than the immune tolerance which is normally found. The development of clinical food hypersensitivities may be highly dependent on the dose of dietary antigen at the time of first encounter.     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.     

Aberrant Tolerance Induction with Cationic Antigens

We studied the induction of tolerance in female C57B1/6 mice by oral and intravenous (i.v.) administration of protein antigens before immunization. Native proteins [chicken egg albumin (OVA), bovine serum albumin (BSA)] and their cationic derivatives [amidated (a)OVA, aBSA, methylated (m)BSA] were compared in their capacity to suppress cell-mediated immunity (CMI), as measured by a delayed type hypersensitivity test (DTH). Oral feeding of 0.5 mg negative proteins gave a clear suppression. By contrast, cationic derivatives (50 μ g to 50 mg) did not suppress CMI. Data from cross experiments, where aOVA was fed in OVA-immune mice, showed no suppression at all. When OVA was fed in aOVA-immune mice, only a partial suppression was achieved.
The potency to induce tolerance differed also when the antigens were administered i.v.: 25 μg OVA was sufficient to induce a clear suppression, whereas a much higher amount ofaOVA (500 μ g ) caused marginal suppression in OVA- and aOVA-immune mice, respectively. Nevertheless, when concentrations of aOVA up to 2.5 mg were tested in a chronic model of arthritis, a significant suppression was achieved.
The differences in inducing a CMI suppression may have implications for the feasibility of immunointervention. Successful modulation of human arthritis may be highly dependent on the nature of the antigen involved.     

Depletion of suppressor T cells by 2'-deoxyguanosine abrogates tolerance in mice fed ovalbumin and permits the induction of intestinal delayed-type hypersensitivity.

We have re-examined the role of suppressor T cells (Ts) in regulating immune responses to fed proteins by investigating the effect of 2'-deoxyguanosine (dGuo) on systemic and intestinal immunity in mice fed ovalbumin (OVA). Administration of dGuo for 10 days abrogated the suppression of systemic delayed-type hypersensitivity (DTH) and antibody responses normally found after feeding OVA, and also prevented the generation of OVA-specific Ts. In parallel, mice given dGuo and fed OVA developed sensitization to OVA in the gut-associated lymphoid tissues (GALT) after oral challenge with OVA and had increased intraepithelial lymphocyte (IEL) counts and crypt cell production rates (CCPR) in the jejunal mucosa, indicating the presence of a local DTH response. These findings confirm the importance of Ts in preventing hypersensitivity to dietary protein antigens and suggest that enteropathies associated with food hypersensitivity are due to a defect in Ts activity.     

The immunological consequences of feeding cholera toxin. II. Mechanisms responsible for the induction of oral tolerance for DTH.

The mechanisms behind the induction of oral tolerance after feeding cholera toxin (CT) were examined using cell and serum transfer protocols. The feeding of CT or cholera toxoid (TD) induced a splenic cell capable of inhibiting the induction of systemic delayed-type hypersensitivity (DTH) but not humoral immunity. Depletion studies showed that this cell was Thy-1.2 positive. Transfer experiments suggested that suppressor cell activity was present in the mesenteric lymph nodes (MLN) and spleens of donor mice 1 week but not 3 days after feeding CT. When spleen cells were transferred to syngeneic recipients at various times after immunization, they were more effective at inhibiting systemic DTH when transferred within a short time of immunization. If the cells were transferred 6 days after immunization they no longer suppressed the development of DTH, which suggested that they inhibit the afferent limb of this immune response. This has been confirmed by the failure of a tolerogenic dose of CT, administered by gavage, to suppress the activity of mature effector TDTH cells. Serum collected 1 hr after feeding CT also suppressed the induction of systemic DTH. However, the tolerogenic activity of CT-fed serum was abrogated by the pretreatment of recipients with cyclophosphamide (Cy) (100 mg/kg), suggesting that this activity is mediated through the induction of suppressor cells. Transfer of fed serum, however, did not induce the splenic suppressor cell described above and we would suggest that several mechanisms may operate in the mucosal regulation of systemic DTH.     

Immunological responses to fed protein antigens in mice. I. Reversal of oral tolerance to ovalbumin by cyclophosphamide

Feeding ovalbumin over a wide range of doses is known to reduce subsequent systemic immune responses to parenteral immunization. In the present study, we have fed mice 2 mg and 25 mg ovalbumin (OVA) 2 weeks before systemic immunization and followed the resulting humoral antibody and cell-mediated immune (CMI) responses. The results indicate that while 25 mg OVA will reduce subsequent IgM, IgG and CMI responses to OVA, feeding 2 mg OVA will only suppress CMI responses and to a lesser extent the IgM response. Furthermore, the tolerant state induced by feeding 25 mg OVA was only partially prevented by 100 mg/kg cyclophosphamide (CY) while the suppressed CMI after feeding 2 mg OVA was completely blocked by CY pretreatment. These findings suggest that the humoral and cell-mediated limbs of the immune response may be controlled by different regulatory systems after feeding antigen, and that activation of these systems is dependent on the dose of oral antigen use. In addition, the results are in agreement with our previous finding that CY pretreatment will allow the development of CMI in the gut and gut-associated lymphoid tissue (GALT) after oral OVA and suggest that this phenomenon is related to breakdown of oral tolerance induction.     

Orally induced bystander suppression in experimental autoimmune uveoretinitis occurs only in the periphery and not in the eye

Oral administration of retinal soluble antigen (S-Ag) suppresses the induction of S-Ag-mediated experimental autoimmune uveitis (EAU) in Lewis rats. EAU induced with interphotoreceptor retinoid-binding protein (IRBP), another retinal autoantigen, can also be suppressed by oral administration of IRBP. It has been speculated that feeding with one retinal autoantigen could suppress induction of uveitis with the other retinal protein by means of bystander suppression. Both uveitogenic effector and suppressor cells should find their antigens within the retina, where the suppressor cells would be expected to act on the effector cells. However, reciprocal combinations of antigens used for induction and suppression of uveitis failed to prevent onset of disease, demonstrating that bystander suppression obviously does not occur in the eye. To investigate further the localization of suppressor mechanisms, we fed Lewis rats either with retinal S-Ag or with ovalbumin (OVA) and then immunized the animals either with a mixture of S-Ag and OVA or with each antigen separately, injected into contralateral hind legs. Feeding of S-Ag prior to immunization led to suppression of uveitis, whereas feeding of OVA had no tolerizing effect when S-Ag and OVA were injected into different legs. Nevertheless, immunizing rats with a mixture of S-Ag and OVA after OVA feeding suppressed uveitis to a high degree. These findings suggest that orally induced bystander suppression might not occur in the target organ, but rather peripherally at the site of induction of the autoimmune T cells.     

Bystander suppression of the immune response to human serum albumin in rats fed ovalbumin.

Bystander suppression of delayed-type hypersensitivity (DTH) and the antibody response to human serum albumin (HSA) were studied in young normal rats and in young rats made partially tolerant to ovalbumin (OVA) by feeding an OVA-containing diet for 4 weeks from weaning. At 2 months of age, the animals were intracutaneously immunized with a mixture of OVA and HSA in Freund's complete adjuvant (FCA) at one site of the back, or separately at two different sites on the back. All rats made orally tolerant to OVA showed a significantly reduced IgE and IgG anti-OVA antibody production and DTH response to OVA, compared to the controls. OVA-fed rats subsequently immunized with a mixture of OVA + HSA had significantly lower IgE and DTH responses to HSA than the controls. When rats were immunized with OVA and HSA at two different sites, however, there was no difference in the response to HSA between the OVA-fed rats and the control rats, which rules out the possibility of shared epitopes between the antigens. Ear-challenge with the mixture of OVA + HSA gave a significantly lower DTH reaction in the tolerant rats immunized with a mixture of the antigens, compared to the control rats. However, suppression of the DTH reaction was not seen when tolerant and control rats were immunized with HSA alone and challenged with the mixture of OVA + HSA in one ear. These results present evidence that young rats orally tolerant to one antigen show a suppressed T-cell and antibody response to an unrelated antigen, provided that the two antigens are given in a mixture during the inductive phase. There was no evidence for bystander suppression of the T-cell response at the effector site.     

Kinetics of oral tolerance: study of variables affecting tolerance induced by oral administration of antigen

The induction of antigen-specific tolerance by oral administration of hen egg albumin (OVA) was shown to be time- and dose-dependent. The IgE response was the most effectively suppressed by antigen feeding prior to or after parenteral immunization. The effect on IgG response paralleled that observed on the IgE response, but the magnitude of suppression was less pronounced. The IgA response was enhanced rather than suppressed if the interval between feeding and parenteral immunization was short or if the animals were antigen-fed after the intraperitoneal priming.     

Helper CD4+ T cells for IgE response to a dietary antigen develop in the liver

BACKGROUND: Although T-cell responses to food antigens are normally inhibited either by deletion, active suppression, or both of antigen-specific T cells, T helper cells for IgE response to a food antigen still develop by unknown mechanisms in a genetically susceptible host. OBJECTIVE: We determined the site at which those IgE helper T cells develop. METHODS: We administered ovalbumin (OVA) orally to DO11.10 mice and studied CD4+ T cells in Peyer's patches, the spleen, and the liver. Helper activity for IgE response was assessed by adoptively transferring those CD4+ T cells to naive BALB/c mice, followed by systemic immunization with OVA. RESULTS: OVA-specific CD4+ T cells were deleted by cell death in the liver and Peyer's patches of DO11.10 mice fed OVA. OVA-specific CD4+ T cells that survived apoptosis in the liver expressed Fas ligand and secreted IL-4, IL-10, and transforming growth factor beta(1). CD4+ T cells producing IFN-gamma were deleted in the liver by repeated feeding of OVA. On transfer of CD4+ T cells to naive mice and systemic immunization with OVA, a marked increase in OVA-specific IgE response developed only in the mice that received hepatic CD4+ T cells from OVA-fed mice, the effect of which was not observed in the recipients of hepatic CD4+ T cells deficient in IL-4. In addition, significant suppression of delayed-type hypersensitivity and IgG(1)/IgG(2a) responses to OVA was observed in the recipients of hepatic CD4+ T cells, and this suppression required Fas/Fas ligand interaction. CONCLUSION: Together, these results suggested that a food antigen might negatively select helper T cells for IgE response to the antigen by preferential deletion of T(H)1 cells in the liver.     

Interleukin 6 inhibits delayed-type hypersensitivity and the development of adjuvant arthritis

We studied the effect of interleukin 6 (IL 6) on the delayed-type hypersensitivity (DTH). In mice immunized with sheep red blood cells (SRBC), a DTH response was evoked by antigen challenge into the hind paw 5 days after immunization. The magnitude of the response was assessed by footpad swelling measured 24 h after antigen challenge. IL 6 significantly suppressed the DTH in its induction phase in a dose-dependent manner when administered s.c. into the back at a dose of greater than 2.5 micrograms twice a day (5 micrograms/day) for 5 consecutive days from the day of immunization (day 0) to 1 day before antigen challenge (day 4). Heat-inactivated IL 6 did not suppress the DTH response. Furthermore, the suppressive activity of IL 6 was completely abolished by affinity chromatography on an anti-IL 6 antibody. This suppression was also obtained when IL 6 was administered only on day 0 and day 1, but not on days 3 and 4. This indicates that IL 6 acts on the early part of the induction phase of DTH development. Furthermore, footpad swelling was suppressed even by the administration of IL 6 after antigen challenge. These results show that IL 6 suppresses both the induction and effector phases of DTH. To confirm further this inhibitory effect of IL 6, we examined its effect on the development of adjuvant arthritis in rats. Administration of IL 6 also significantly suppressed the development of adjuvant arthritis.     

A genetically determined lack of oral tolerance to ovalbumin is due to failure of the immune system to respond to intestinally derived tolerogen

In this study we have examined whether differences between mouse strains in the induction of tolerance after feeding ovalbumin (OVA) are due to differences in intestinal processing of OVA or are determined by the systemic immune system. Compared with major histocompatibility complex (MHC)-congenic BALB/c mice, BALB/B mice develop much less tolerance of systemic delayed-type hypersensitivity (DTH) and humoral immunity after feeding OVA and this defect is also expressed partially in (BALB/B x BALB/c)F1 animals. Serum taken from either BALB/c or BALB/B mice fed OVA 1 h before produced significant suppression of systemic DTH responses in BALB/c, but not in BALB/B mice. Although OVA-fed BALB/B serum was slightly less tolerogenic than BALB/c serum, we conclude that the defective induction of oral tolerance in BALB/B mice is due primarily to a MHC-influenced defect with the immune system. These findings support the idea that clinical food-sensitive enteropathy reflects an immune response gene-controlled defect in tolerance to dietary proteins.     

Oral tolerance to ovalbumin in mice: studies of chemically modified and 'biologically filtered' antigen.

Suppression of systemic immunity after the feeding of antigen was investigated in mice by means of serum transfer experiments. Serum collected from mice 1 hr after a single intragastric dose of 25 mg OVA induced suppression of systemic DTH when injected intraperitoneally into recipient mice. This suppression was found to be restricted to the cell-mediated limb of immunity and was antigen-specific. A postulated function of the intestine, conversion of antigen into tolerogenic form by means of intestinal antigen processing, was studied by attempting to mimic intestinal alteration of OVA by chemical modification of the antigen. Parenteral injection of mice with either deaggregated or denatured OVA did not produce the typical pattern of unresponsiveness seen in animals given intestinally processed OVA. Intestinal processing was also shown to be distinct from systemic antigen processing. Mice injected with serum containing systemically 'filtered' OVA did not become tolerant to OVA in the manner of recipients of serum from OVA-fed mice.     

Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and are immunogenic by the oral route

Induction of all forms of protective immunity by oral immunization with subunit vaccines is an ideal goal for the development of novel vaccines, but creates several theoretical problems from the point of view of antigen processing mechanisms. We show here that incorporation of the protein antigen ovalbumin (OVA) in lipophilic immune-stimulating complexes (ISCOMS) induces very strong primary immune responses in mice and requires very small amounts of antigen. OVA ISCOMS were particularly efficient at stimulating T-cell-mediated immunity in vivo, including delayed-type hypersensitivity (DTH) and potent class I major histocompatibility complex (MHC)-restricted cytotoxic T-cell responses. Furthermore, unlike native protein, OVA in ISCOMS was immunogenic when given orally. Thus, ISCOMS seem to allow protein to enter both the endogenous and exogenous pathways of antigen processing and overcome the usual induction of tolerance after feeding antigen. ISCOMS could provide potentially useful adjuvants for the development of oral subunit vaccines.     

Specific Suppression of Humoral and Delayed Hypersensitivity Responses by Cyclophosphamide in an Experimental Model of Autoimmunity

ABSTRACT: The aim of this report is to investigate the effect of cyclophosphamide (CY) in an experimental model of autoimmunity to rat male accessory glands. The results indicated that 100 mg/kg of this drug suppressed humoral immune response that persisted for at least 45 days when administered 3 days after the first immunization of rats with modified rat male accessory glands (MRAG) in complete Freund's adjuvant (CFA). Administration of the drug 3 days before ID injection of antigen caused a shorter suppression of antibody formation. Delayed type hypersensitivity (DTH) studied 13 days after the first immunization was suppressed only in the animals that were administered CY after the antigen. The specificity of the immunosuppression was studied in rats treated with CY after the first immunization with MRAG using aggregated human γ-globulin (AHGG) as an unrelated antigen. The studies demonstrated significant suppression of DTH (p < 0.005) and humoral immunity only against MRAG. On the contrary, the response to AHGG was not significantly modified.     

Independent genetic control of B- and T-cell tolerance in strains of mouse selected for extreme phenotypes of oral tolerance

Two strains of mice were genetically selected for susceptibility (TS-Ab/HetS strain) or resistance (TR-Ab/HetS strain) to oral tolerance of the humoral response by using ovalbumin (OVA). The progressive interstrain divergence produced by bi-directional selective breeding during 15 generations demonstrated the polygenic nature of oral tolerance. This paper shows the humoral and delayed-type hypersensitivity (DTH) responses, after intragastric administration of OVA and subsequent immunization with that immunogen in complete Freund adjuvant (CFA). Only the TS-Ab/HetS mice were tolerant for immunoglobulin (Ig)G production with its tolerance degree being the same as that obtained when Al ((OH)(3)) was employed. The DTH reactivity was not correlated to the antibody responsiveness, because both the TS-Ab/HetS and TR-Ab/HetS strains had their DTH reactions suppressed. The cyclophosphamide (Cy) pretreatment prevented DTH suppression on TR-Ab/HetS but do not in TS-Ab/HetS mice. Interstrain difference was also observed for the splenic index in the Cy-treated groups, although the number of splenocytes was the same. Flux cytometry cell analysis showed the recovery of CD3(+) cell numbers in both strains, but only the TR-Ab/HetS mice had their CD4/CD8 pattern restored. These results suggest: firstly, the independent control of DTH and humoral tolerance responsiveness; secondly no support for the clonal anergy concept; and thirdly the matrix proteins neo-synthesis after Cy treatment may facilitate the tolerance abrogation.     

Influences of anti-mouse interleukin-6 receptor antibody on immune responses in mice

In the present study, we examined the effect of anti-IL-6 receptor antibody (MR16-1) on humoral and cellular immune responses in mice. MR16-1 did not affect antigen-specific antibody production in either the primary or secondary response in mice immunized with dinitro-phenyl (DNP)-keyhole limpet haemocyanin (KLH) in saline. DNP-KLH immunization with complete Freund's adjuvant (CFA) markedly augmented anti-DNP antibody production and induced interleukin 6 (IL-6) production in serum. In this case, MR16-1 significantly suppressed antibody production and further increased serum IL-6 levels. Regarding the cellular response, we studied the effect on the delayed-type hypersensitivity (DTH) response. DTH response was induced in mice by the immunization with Mycobacterium butyricum with incomplete Freund's adjuvant and following antigen challenge into the footpad 14 days after immunization. When MR16-1 was injected immediately after immunization, the DTH response was significantly suppressed and enlargement of the spleen was also suppressed. This suppressive effect was observed, when MR16-1 was administered on day 0, but not on days 5 and 10. Again, serum IL-6 levels were much higher in MR16-1-treated mice compared with controls. Furthermore, spleen cells from control mice released IL-2 and INFgamma by the stimulation of antigen in vitro. In contrast, spleen cells from MR16-1-treated mice produced these cytokines at a marginal level. In contrast, MR16-1 did not suppress the DTH response, when it was injected immediately after antigen challenge. Our results suggest that IL-6 does not always involve antibody production, although IL-6 augments antibody production, and that IL-6 is essential for the induction of Th1 cells.