Peyer's patches are required for oral tolerance to proteins

Abstract

To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treated in utero with lymphotoxin beta-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Peyer's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4(+) T-cell-derived IFN-gamma, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.

Figure 1

OVA-specific IgG Ab responses (A) in serum and IgG AFC (B) in spleen of mice from dams treated with LTβR-Ig, LFA-3-Ig, or untreated BALB/c mice orally immunized with 25 mg of OVA (□) or PBS (■). Both groups of mice were challenged by the s.c. route with 100 μg of OVA in 100 μl of complete Freund's adjuvant 7 days after feeding. Fourteen days after s.c. OVA/CFA challenge, serum was assessed for OVA-specific IgG Ab responses. Numbers of splenic OVA-specific IgG AFCs were determined by enzyme-linked immunospot assay. The results represent the mean values ± SEM for 12 mice in each experimental group and from three separate experiments.

Figure 2

Effects of oral administration of OVA on DTH (A) and T-cell-proliferative responses (B) in mice from dams treated with LTβR-Ig or LFA-3-Ig, or from untreated BALB/c mice. (A) Thirteen days after i.p. immunization, PBS (20 μl) containing 10 μg of OVA was injected into the left ear pinna, and the control right ear pinna was injected with PBS. The DTH response is expressed as the increase in ear swelling after OVA injection over the level of swelling in the control ear pinna receiving PBS. (B) Fourteen days after s.c. immunization, splenic CD4⁺ T cells were cultured with or without OVA for 5 days. The stimulation index was determined as cpm of wells with Ag/cpm of wells without Ag (controls). The levels of thymidine incorporated in each control well were between 500 and 1,000 cpm. The results represent the mean values ± SEM from three separate experiments (triplicate wells/experiment).

Figure 3

TNP-specific IgG Ab responses in serum (A) and IgG AFC in spleen (B) of mice from dams treated with LTβR-Ig, LFA-3-Ig, or untreated BALB/c mice. Mice were fed 0.25 ml of PBS or PBS containing 10 mg of TNBS on days 0 and 7. On day 14, mice were sensitized with picryl chloride (TNCB) and 14 days later, TNP-specific serum IgG Ab responses were determined. The numbers of splenic IgG AFCs were determined by enzyme-linked immunospot. The results represent the mean values ± SEM for 12 mice in each experimental group and were representative of three separate experiments.

Figure 4

The effects of oral administration of TNBS on DTH (A) and T-cell-proliferative (B) responses 7 and 14 days after TNCB sensitization. The details of the assays are described in Materials and Methods. The stimulation index was determined as described in the legend of Fig. 2. The level of thymidine incorporation for each control well was between 500 and 1,000 cpm. The results represent the mean values ± SEM from three separate experiments (triplicate wells/experiment).

Figure 5

The effects of oral administration of TNP-OVA on TNP-specific DTH responses. Mice from dams treated with LTβR-Ig and untreated BALB/c mice were fed OVA (45 mg), TNP-OVA (containing 7 mg of TNP and 45 mg of OVA), or TNBS (7 mg) on days 0, 3, and 7. The results represent the mean values ± SEM from two separate experiments.

Figure 6

Serum OVA levels after oral administration of OVA. Mice were fed 25 mg of OVA and serum samples were collected at 30, 60, and 120 min after feeding. Serum OVA levels were determined by ELISA as described in Materials and Methods. The results represent the mean values ± SEM for 10 mice in each experimental group and were representative of two separate experiments.