Cell-mediated immune response to unrelated proteins and unspecific inflammation blocked by orally tolerated proteins

Abstract

Oral tolerance promotes a generalized decrease in specific immune responsiveness to proteins previously encountered via the oral route. In addition, parenteral immunization with a tolerated protein also triggers a significant reduction in the primary responsiveness to a second unrelated antigen. This is generally explained by 'innocent bystander suppression', suggesting that the transient and episodic effects of inhibitory cytokines released by contact with the tolerated antigen would block responses to the second antigen. In disagreement with this view, we have previously shown that: (i) these inhibitory effects do not require concomitance or contiguity of the injections of the two proteins; (ii) that intravenous or intragastric exposures to the tolerated antigen are not inhibitory; and (iii) that the inhibitory effect, once triggered, persists in the absence of further contact with the tolerated protein, possibly by inhibition of secondary responsiveness (immunological memory). The present work confirms that immunological memory of the second unrelated antigen is hindered by exposure to the tolerated antigen and, in addition, shows that this exposure: (i) inhibits the inflammation triggered by an unrelated antigen through the double effect of inhibiting production of leucocytes in the bone marrow and blocking their migration to inflammed sites; and (ii) significantly blocks footpaw swelling triggered by carrageenan. Taken together, these results conclusively demonstrate that inhibitory effects of parenteral injection of tolerated antigens are much more general than suggested by the 'innocent bystander suppression' hypothesis.

Figure 1

Injection of ovalbumin (OVA) into OVA orally tolerant mice inhibits delayed-type hypersensitivity (DTH) triggered by dinitrophenylated OVA (DNP-OVA) or dinitrophenylated keyhole lympet haemocyanin (DNP-KLH). The footpad increased in sensitized mice 24 hr after challenge with DNP-OVA in the right footpad and DNP-KLH in the left footpad, as indicated in the figure. Open bars: control, non-tolerant mice, subcutaneously (s.c.) immunized with DNP-OVA plus DNP-KLH in complete Freund’s adjuvant (CFA). Solid bars: OVA orally tolerant mice s.c. immunized with DNP-OVA plus DNP-KLH in CFA. Values are mean ± standard error of the mean (for five or six animals). *P<0·05 compared with non-tolerant mice (given water).

Figure 2

Injection of ovalbumin (OVA) into OVA orally tolerant mice inhibits anti-haemoglobin (Hb) antibody production. Total levels of serum anti-OVA (a) and anti-Hb (b) antibodies from sensitized mice killed 24 hr after intraperitoneal (i.p.) challenge with the antigens indicated in the figure were determined. Open bars: control, non-tolerant mice, subcutaneously (s.c.) immunized with OVA plus Hb. Solid bars: OVA orally tolerant mice s.c. immunized with OVA plus Hb. Values are mean ± standard error of the mean (for five or six animals). *P<0·05 compared with non-tolerant mice (given water).

Figure 3

Injection of ovalbumin (OVA) into OVA orally tolerant mice inhibits peritonitis triggered by snail haemoglobin (Hb). Total and differential leucocyte counts (× 10⁵ cells/ml) in the peritoneal lavage fluid 24 hr after intraperitoneal (i.p.) challenge with the antigens indicated in the figure were determined. Open bars: control, non-tolerant mice, subcutaneously (s.c.) immunized with OVA plus Hb. Solid bars: OVA orally tolerant mice s.c. immunized with OVA plus Hb. Values are mean ± standard error of the mean (for five or six animals). *P<0·05 compared with non-tolerant mice (given water).

Figure 4

Injection of ovalbumin (OVA) into OVA orally tolerant mice inhibits the increase in peripheral blood and bone marrow eosinophils triggered by snail haemoglobin (Hb). Total eosinophil counts in the peripheral blood (a) and bone marrow (b) 24 hr after intraperitoneal (i.p.) challenge with the antigens indicated in the figure were determined. Open bars: control, non-tolerant mice, subcutaneously (s.c.) immunized with OVA plus Hb. Solid bars: OVA orally tolerant mice s.c. immunized with OVA plus Hb. Values are mean ± standard error of the mean (for five or six animals). *P<0·05 compared with non-tolerant mice (given water).

Figure 6

Injection of ovalbumin (OVA) into OVA orally tolerant mice, at different time intervals, inhibits carrageenan-induced paw oedema. Mice submitted to oral OVA treatment were immunized with OVA plus Al(OH)₃ at 24 hr before, 4 hr before, or 4 hr after paw oedema initiation with carrageenan. Values are mean ± standard error of the mean (six to eight animals), indicating the oedema inhibition percentage at 24 hr compared with the control carrageenan group.

Figure 5

Injection of ovalbumin (OVA) into OVA orally tolerant mice inhibits development of the delayed phase of carrageenan-induced paw oedema. Animals that had previously been treated with OVA orally (closed triangles) or that had been drinking only water (open circles) were injected with OVA plus Al(OH)₃ intraperitoneally (i.p.) 24 hr before the intraplantar injection of carrageenan. The control group (open squares) consisted of animals that received only carrageenan. Each point represents the mean of six to eight animals and vertical lines are the standard error of the mean (SEM). *P<0·05 compared with the control group.

Figure 7

Injection of ovalbumin (OVA) into OVA orally tolerant mice inhibits polymorphonuclear (PMN) and mononuclear (MN) leucocyte influx into inflamed paws injected with carrageenan. Myeloperoxydase (MPO) and N-acetyl-glucosaminidase (NAG) activities were quantified as indirect indicators of PMN and MN cell influx, respectively. Animals that had been drinking only water (white bars) or water plus OVA (black bars) received an intraperitoneal (i.p.) injection of OVA plus Al(OH)₃ 24 hr before paw oedema initiation with carrageenan. Enzyme activity was measured 24 hr after carrageenan injection. Control groups were naïve animals that received only a carrageenan intraplantar injection (positive control; dark grey bars) or a saline intraplantar injection (negative control; light grey bars). Values are mean ± standard error of the mean (six to eight animals). *P<0·05 compared with the negative control group; ^#P<0·05 compared with the positive control group.