Maternal tolerance is not critically dependent on interleukin-4

Abstract

Pregnant animals can generate and maintain immune responses to fetal antigens. This however, does not usually lead to fetal loss. At least two types of immune response are recognized. T helper type 1 (Th1) responses support the generation of cellular cytotoxicity. In contrast, Th2-type responses support the production of non-cytotoxic antibody and suppress the Th1-type. One attempt to explain why the fetus is not generally rejected has been to suggest that during pregnancy Th2-type responses are dominant. These responses rely heavily on interleukin-4 (IL-4) for both functions. This work focuses on maternal immunity to the male antigen H-Y, which is expressed in male fetuses. When injected with male spleen cells, female mice of certain strains mount a cytotoxic immune response to H-Y. However, pregnant females immunized in this way do not deliver litters with fewer males. To help delineate the possible role of IL-4 in such maternal tolerance, female mice genetically deficient in IL-4 were studied. The results show that: (1) deficiency in maternal IL-4 does not affect fertility, (2) deficiency in IL-4 is not associated with selective loss of male offspring in unimmunized mice, (3) pregnancy does not obliterate anti-H-Y reactivity in immunized mice and (4) maternal immunity to H-Y in the absence of IL-4 does not result in loss of male offspring. The results suggest that IL-4-dependent Th2-type responses are not critical to maternal tolerance. Other cytokines must be examined for their role in this phenomenon.

Figure 1

The absence of IL-4 does not lead to a loss of male offspring in B6 mice. (a) Schema of experiment: 6- to 8-week-old B6 or B6-4KO females were mated twice with B6-4KO males. The numbers of male and female pups born in each litter were recorded. Three months after the first mating, the mice were killed and their spleen cells were assayed in vitro for anti-H-Y and anti-alloantigen reactivity. (b) The experimental data: y-axis, fraction of males born per litter; x-axis, mother's strain. Nine of 11 B6 and 11 of 12 B6-4KO mothers are represented because the litters of the other mice were eaten at birth. Each symbol represents one litter born to a B6 (circle) or B6-4KO (triangle) mother. Grey symbols, first litters; black symbols, second litters. Arrows point to litters born to mothers H-Y-sensitized by pregnancy.

Figure 2

Pregnancy does not permanently diminish the ability to respond to H-Y in B6 or B6-4KO mice. (a) Schema of experiment: B6 or B6-4KO females were injected with a priming dose of male B6-4KO male spleen cells. Two weeks later, females of both strains were mated with B6-4KO males. Within 3 days of mating, the female mice were given another dose of male spleen cells. They were then mated twice with B6-4KO males. The numbers of male and female pups born in each litter was recorded. Three months after the first mating, the mice were killed and their spleen cells were assayed in vitro for anti-H-Y and anti-alloantigen reactivity. (b) The experimental data: each symbol represents one mouse. Open symbols, mice never pregnant; grey symbols, mice pregnant with one litter; black symbols, mice pregnant with two litters. Y-axis, specific killing of male (circles, killing by B6 females; triangles, killing by B6-4KO females) or allogeneic (squares) targets; x-axis, groups of mice tested. Dotted lines link the data for each mouse. Crosses are the mean values of male-specific killing in each group.

Figure 3

Pregnancy with male pups in B6 or B6-4KO females does not impair the ability to reject a male skin graft, and the ability to reject a male skin graft does not lead to a decrease in normal or IL4-deficient male offspring. (a) Schema of experiment: B6 and B6-4KO females were treated as in Fig. 2, except that instead of being boosted with male spleen cells at the time of mating, they received a male B6-4KO (test) and female (control) tail skin graft. All mice rejected their grafts and within 2 months the mice were re-mated and the resulting litters were examined. Y-axis, days from placement of graft until if completely fell off; the cross refers to the average time for complete rejection in B6 females. X-axis, % males per litter born. Open symbols represent mice which either were never mated or never got pregnant. Solid symbols represent animals which became pregnant. Circles, B6 females (n = 8); triangles, B6-4KO females (n = 4). Grey, first litters; black, second litters. Individual mothers are depicted by number. Thus, immunized mouse no. 2 was mated and grafted, had a litter with 42% males, and completely rejected a male skin graft within 35 days. Her second litter contained approximately 28% males. Mice numbers 3, 5 and 6 ate their first litters at birth.

Figure 4

Survival of male offspring is not altered by sustained systemic anti-H-Y cytotoxicity in B6 or B6-4KO mothers. Test mice in Fig. 3 were examined in vitro for anti-H-Y and anti-alloantigen reactivity. Data from this experiment, the CTL experiments using the mice in Fig. 2 and the mice which were immunized by pregnancy alone were pooled and analysed for comparison of male pup survival to maternal anti-H-Y reactivity in the spleen of B6 and B6-4KO mothers 2 months after the last litter was born. Y-axis, fraction of males in a litter; x-axis, maternal spleen specific killing of male targets, divided into low or no (< 20%), medium (20–60%), or high (> 60%). Each symbol represents a litter. Circles, litters born to B6 mothers; triangles, litters born to B6-4KO females; grey, first litters; black, second litters. Arrows point to litters born to mothers sensitized by pregnancy alone. Numbers beside each litter are the exact values of maternal anti-H-Y and anti-alloantigen reactivity. Crosses represent the average fraction of males per litter in each group.