Chronic helminth infection induces alternatively activated macrophages expressing high levels of CCR5 with low interleukin-12 production and Th2-biasing ability

Abstract

Helminth infections induce Th2-type biased immune responses. Although the mechanisms involved in this phenomenon are not yet clearly defined, antigen-presenting cells (APC) could play an important role in this process. Here, we have used peritoneal macrophages (F4/80+) recruited at different times after challenge with Taenia crassiceps as APC and tested their ability to regulate Th1/Th2 differentiation. Macrophages from acute infections produced high levels of interleukin-12 (IL-12) and nitric oxide (NO), paralleled with low levels of IL-6 and prostaglandin E(2) (PGE(2)) and with the ability to induce strong antigen-specific CD4+ T-cell proliferation in response to nonrelated antigens. In contrast, macrophages from chronic infections produced higher levels of IL-6 and PGE(2) and had suppressed production of IL-12 and NO, associated with a poor ability to induce antigen-specific proliferation in CD4+ T cells. Failure to induce proliferation was not due to a deficient expression of accessory molecules, since major histocompatibility complex class II, CD40, and B7-2 were up-regulated, together with CD23 and CCR5 as infection progressed. These macrophages from chronic infections were able to bias CD4+ T cells to produce IL-4 but not gamma interferon (IFN-gamma), contrary to macrophages from acute infections. Blockade of B7-2 and IL-6 and inhibition of PGE(2) failed to restore the proliferative response in CD4+ T cells. Furthermore, studies using STAT6(-/-) mice revealed that STAT6-mediated signaling was essential for the expansion of these alternatively activated macrophages. These data demonstrate that helminth infections can induce different macrophage populations that have Th2-biasing properties.

FIG. 1.

Course of T. crassiceps infection in BALB/c mice and Th1/Th2 markers. (a) Female BALB/c mice were inoculated i.p. with 20 cysticerci of T. crassiceps, and parasite loads and total IgE levels were monitored at times indicated. (b) Endpoint titers for antigen-specific antibody production of IgG1 and IgG2a. (c) Antigen-specific production of IFN-γ, IL-4, and IL-13 by splenocytes from T. crassiceps-infected mice as markers of Th1/Th2 environment. Data are representative of three independent experiments. Error bars represent ± standard deviations.

FIG. 2.

Cytokine production by macrophages in response to Th1-polarizing antigen (LPS). IL-12 (a), IL-6 (b), and IL-18 (c) production by macrophages from different weeks after T. crassiceps infection was determined by ELISA. Adherent PECs were obtained from infected mice at the times indicated, as well as from age-matched uninfected mice. Macrophages (10⁶) were stimulated in vitro with 5 μg of LPS/ml for 48 h. Data are the mean ± standard deviation for six animals at each time point. Asterisk, P < 0.05 with respect to uninfected mice and mice at 2 weeks postinfection; ND, not determined.

FIG. 3.

Other products of macrophages' activity are altered by T. crassiceps infection. Production of PGE₂ (a) and NO (b) as measured by ELISA or Griess reaction were assayed from the same culture supernatants from Fig. 2. Asterisk, P < 0.05 with respect to uninfected mice and mice at 2 weeks postinfection.

FIG. 4.

T. crassiceps infection alters the capacity of macrophages as antigen-presenting cells. Macrophages from different weeks after infection were loaded with KLH (a) or OVA (b) and cocultured with CD4⁺ cells (previously sensitized) for 5 days; proliferation was assayed by uptake of [³H]thymidine (3H-TdR). Data are representative of three different experiments, ± standard errors. Asterisk, P < 0.05; double asterisk, P < 0.01 (compared to uninfected mice or mice infected for 2 weeks).

FIG. 5.

T-cell-polarizing ability of alternatively activated macrophages in vivo during T. crassiceps infection. Adherent peritoneal macrophages were obtained from infected mice at the times indicated, as well as from age-matched uninfected mice. Cells (10⁶) were loaded in vitro with 50 μg of OVA for 2 h. CD4⁺ T cells (2 × 10⁶) from healthy mice previously immunized with OVA were cocultured, and 72 h later supernatants were analyzed for IFN-γ (a), IL-12 (b), IL-4 (c), and IL-6 (d). Data are representative of three independent experiments with at least four animals individually assayed. Error bars indicate ± standard deviations. Asterisk, P < 0.05 comparing with macrophages from healthy mice or early (2 weeks)-infected mice.

FIG. 6.

Phenotypes of peritoneal macrophages isolated from T. crassiceps infection. Cell surface expression of F4/80, MHC-II, CD40, B7-1, and B7-2 at 0, 2, 4, and 8 weeks after T. crassiceps infection are shown. Data are representative histograms of three separate experiments.

FIG. 7.

T. crassiceps infection up-regulates the expression of CD23/CCR5 in macrophages. Cell surface expression of F4/80-CD23 and F4/80-CCR5 on peritoneal macrophages obtained at different weeks after infection. Bottom, macrophages obtained from STAT6-KO mice 8 weeks after T. crassiceps infection.

FIG. 8.

Effect of fixation, blockade of IL-6/B7-2, inhibition of PGE₂, and absence of STAT6 signaling of macrophages in CD4⁺ T-cell proliferation and cytokine production. (a) Macrophages from mice at 8 weeks postinfection were used as APC and cocultured as for Fig. 4. In addition, blocking antibodies for IL-6 and B7-2, indomethacin, fixed macrophages, and STAT6-KO macrophages were used in these cocultures. (a) Proliferation was assayed by [³H]thymidine (3H-TDR) incorporation. (b to d) Cytokine levels detected in the presence and absence of blocking antibodies, indomethacin, fixed macrophages, or STAT6-KO macrophages. Asterisk, P < 0.05 compared to isotype control. ND, not determined.