An interleukin 4 (IL-4)-independent pathway for CD4+ T cell IL-4 production is revealed in IL-4 receptor-deficient mice

Abstract

IL-4 receptor alpha chain (IL-4Ralpha)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Ralpha resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis. CD4+, CD62L-high, and CD62L-low T cell populations from uninfected IL-4Ralpha-/- mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4Ralpha-/-, beta2-microglobulin-/- double homozygous mice produced less IL-4 than did either IL-4Ralpha-/- or wild-type mice. These results indicate that an IL-4-independent, beta2-microglobulin-dependent pathway exists through which the CD62L-low CD4+ population has acquired IL-4-producing capacity in vivo, strongly suggesting that these cells are NK T cells.

Figure 1

Targeting of IL-4Rα. The IL-4Rα locus was disrupted in BALB/c ES cells. (A) The IL-4Rα gene, targeting construct, and locus after homologous recombination are shown. Exons 7–9 (4.5 kb) were replaced by a PGKneo gene orientated antiparallel to the IL-4Rα gene. The targeting vector contained 6.3-kb homology to the genomic sequence. (B) Southern blot analysis of EcoRI-digested DNA obtained from offspring of heterozygous matings. The 12.7-kb band indicates the presence of the wild-type allele. The 4.9-kb band reveals the disrupted IL-4Rα locus.

Figure 2

IL-4Rα is not expressed in targeted mice. (A) IL-4Rα expression and induction of IL-4Rα and class II MHC were measured by flow cytometry. Spleen cells from IL-4Rα^−/− mice and wild-type littermates were cultured at 1 × 10⁶/ml in cRPMI medium for 40 hr in medium alone or with IL-4 (1000 units/ml). For detection of IL-4Rα, splenocytes were stained with 20 μg/ml rat anti-mouse IL-4Rα (M1) and detected with biotinylated mouse anti-rat IgG2a in the presence of 10 μl normal mouse serum, followed by streptavidin-PE. Class II MHC Ia^b,d expression was gated on B220⁺ cells. Dotted line, unstained cells; thin line, medium alone; bold line, with IL-4. (B) STAT6 EMSA. Spleen cells were cultured with IL-4 (10,000 units) for 10 min. Cell lysates were incubated with a double-stranded STAT6-element probe and run on a acrylamide gel. Oct1 sequences were used to control for loading. A polyclonal anti-STAT6 antibody supershifted the complex (Right).

Figure 3

TH2 cytokines are reduced after N. brasiliensis infection. (A) Mesenteric lymph nodes were removed from noninfected controls (n.i.) and mice infected 7 days with N. brasiliensis. Purified CD4⁺ cells (10⁶/ml) were stimulated for 24 hr with immobilized anti-CD3 and supernatant cytokine content measured by ELISA. (Inset) The graph compares IL-4 production in noninfected (Left) and infected (Right) IL-4Rα^−/− mice. (B) Serum IgE, IgG1, and IgG2a were measured at days 0 and 13 after infection. Error bars represent standard deviations (n = 3).

Figure 4

IL-4 is produced by CD62L-low CD4⁺ cells independent of IL-4 priming. (A) Purified CD4⁺ T cells were sorted for CD4⁺, CD62L-high and CD4⁺, CD62L-low populations. Cells were stimulated (10⁶/ml) for 24 hr with immobilized anti-CD3 and supernatants measured by ELISA. (B) CD4⁺ T cells were purified and sorted as described and cultured with soluble anti-CD3 with T- depleted splenocytes from BALB/c IL-4^−/− mice along with IL-2, anti-IFN-γ, anti-IL-12, and either IL-4 or anti-IL-4 (11B11). After 4 days, cells were washed and rechallenged (10⁶/ml) for 24 hr with immobilized anti-CD3, and supernatants were assayed for IL-4 production.

Figure 5

CD4⁺, NK1.1+ T cells produce IL-4 in IL-4Rα^−/− mice. (A) For measurement of IL-4 from CD4⁺, NK1.1+ T cells, mice were injected i.v. with anti-CD3. After 90 min spleens were removed and cells cultured for 1 hr. Supernatants were measured for IL-4 by bioassay. Error bars represent standard deviations from three individual mice. (B) CD4 T cells were purified from mesenteric lymph node cells from offspring of IL-4Rα^+/− × β₂m^+/− matings. Cells were sorted for CD4⁺, CD62L-low populations and stimulated (10⁶/ml) for 24 hr with immobilized anti-CD3 and supernatants measured for IL-4 and IL-2 by ELISA.