Conceived and designed the experiments: PSF MY. Performed the experiments: WW MY. Analyzed the data: WW PMH PSF MY. Contributed reagents/materials/analysis tools: PMH PSF MY. Wrote the paper: PMH PSF MY.
The authors have declared that no competing interests exist.
Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4+ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues.
We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4+ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4+ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4+ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation.
These results suggest that different points of eosinophilic inflammatory processes in allergic airway disease may be differentially regulated by the activation of STAT6-dependent and -independent pathways.
Eosinophilic inflammation is a hallmark feature of allergic diseases of the lung (asthma), gastrointestinal tract (allergic eosinophilic gastroenteritis), skin (eczema), other systemic diseases (idiopathic hypereosinophilic syndrome and eosinophilic pneumonia) and parasitic helminth infection
IL-5 is the most important factor that regulates the expansion, growth and survival of eosinophils although it is dispensable for eosinophil development under homeostatic conditions
Once eosinophils are produced specific chemotactic factors, namely the chemokines eotaxin-1, -2 and -3, cooperate with IL-5 to critically regulate their migration and activation during allergic inflammation
Many immune cells, in particular CD4+ T-helper type 2 lymphocytes (Th2 cells), CD8+ T cells, and NK cells but also mast cells and eosinophils produce IL-5. Of these cells, Th2 cells are the predominant source of IL-5 during allergic responses
Clinical and experimental investigations have demonstrated the obligatory role of Th2 cells in the pathogenesis of eosinophilic inflammation and allergic disorders
In this study we assessed the role of STAT6 in the development of eosinophilic inflammation in a mouse model of allergic airways inflammation using wild type (WT) and STAT6-deficient mice. We also determined the roles of IL-5, eotaxins, CD4+ and CD8+ T cells and NK cells in the development of STAT6-independent eosinophilic inflammation.
Specific pathogen free WT and STAT6-deficient BALB/c mice (male and female, 6–8 weeks) were obtained from the University of Newcastle and Australian National University. STAT6-deficient mice were backcrossed for 12 generations onto the BALB/c background. All experiments were performed with approval from the animal ethics committees of The University of Newcastle (ID 899 and 974) and the Australian National University (The early part of experiments was conducted at ANU and ANU ID was expired and not archived).
Mice were sensitized at 6–8 wk of age by i.p. injection (day 0) with 50 µg of ovalbumin (OVA) (fraction V, Sigma, St Louis, MO, USA) admixed with 1 mg Alhydrogel (Reheis Inc., Berkeley Heights, NJ, USA) in 200 µl of 0.9% sterile saline on day 0 and 12. Non-sensitized mice were injected with 1 mg Alhydrogel in 200 µl of 0.9% sterile saline. On days 24, 26, 28 and 30, all groups of mice were challenged with aerosolized OVA (10 mg/ml in 0.9% saline) for 3×30 minutes with 30 minutes break using an ultrasonic nebuliser. On day 23, 25, and 27, 29 and 31, eosinophils as a percentage of leukocytes in peripheral blood were assessed. On day 31 inflammatory responses in bronchoalveolar lavage, airway sections and lung tissue, and eosinophil numbers in the bone marrow were assessed as previously described
Bronchoalveaolar lavage was collected, cells isolated and stained and differential inflammatory cell counts performed as previously described
Blood was collected by heart bleed and cell-free serum prepared. IL-5, eotaxin-1 and -2 were determined by ELISA cytokine according to the instructions of the manufacturer (BD Pharmingen, San Diego, CA, USA)
The method for quantitative PCR has been described in detail previously
Bone marrow was isolated as previously described
Mice were injected i.p. with anti-IL-5 (200 ug, TFK5, rat anti-mouse monoclonal IgG1, ATCC, Manassas, VA, USA) or isotype control (rat IgG1) monoclonal antibody (mAb) on days 22, 26 and 30 during the OVA challenge as previously described
CD4+ or CD8+ cells were depleted by i.p. injection with 500 µg anti-CD4 (Clone GK1.5, rat anti-mouse monoclonal IgG2b)
NK cells were depleted by i.v. injection with 50 µl anti-ASIALO GM1 polyclonal antibody (Wako Chemicals, Osaka, Japan) or rabbit serum on day 22, 26 and 30, according to manufacturer's instructions as previously described
An initial one-way analysis of variance (or a Kruskal-Wallis test for non-parametric data) was followed by appropriate comparisons to test for differences between means of groups. Values are reported as the mean ± SEM for each experimental group. The number of mice in each group ranged from 8–12. Differences in means were considered significant if P<0.05.
We first investigated the role of STAT6 in eosinophil accumulation in the lung. WT and STAT6-deficient mice were sensitized and challenged with OVA/OVA or SAL/OVA and leukocyte numbers were determined in the BALF. Pronounced infiltrations of inflammatory cells were detected in the BALF of OVA/OVA treated WT mice, compared to SAL/OVA treated controls (
OVA/OVA treated WT mice had significantly increased numbers of eosinophils in (A) BALF and (B) peribronchial and perivascular regions of the lung, compared to SAL/OVA treated controls. The levels of BALF neutrophil in OVA/OVA treated STAT6-deficient mice were significantly higher than that in SAL/OVA WT control but less than that in OVA/OVA treated WT mice. No significant infiltration of eosinophils was observed in OVA/OVA treated STAT6-deficient mice, which were at the same levels as SAL/OVA treated WT mice. #P<0.05 compared to SAL/OVA treated WT, *P<0.05 compared to OVA/OVA treated WT mice.
Histological examination of lung tissue showed that a significant infiltration of eosinophils occurred into the peribronchial and perivascular regions of OVA/OVA treated WT mice compared to SAL/OVA treated controls (
To determine the point of the inflammatory pathways where eosinophil influx in the lung was disrupted in STAT6-deficient mice, eosinophil levels in the blood and bone marrow were assessed. The percentages of eosinophils in peripheral blood of OVA/OVA treated WT and STAT6-deficient mice were significantly greater than in respective SAL/OVA treated controls (
OVA/OVA treatment of WT and STAT6-deficient mice significantly increased (A) levels of eosinophils as percentages of leukocytes in peripheral blood and (B) numbers of eosinophils in bone marrow compared to SAL/OVA treated controls. OVA/OVA treated STAT6-deficient mice had a greater percentage of eosinophils in peripheral blood but significantly fewer eosinophils in the bone marrow than OVA/OVA treated WT controls. #P<0.05 compared to other groups. *P<0.05 compared to SAL/OVA treated controls.
OVA/OVA treatment of WT and STAT6-deficient mice resulted in increased numbers of eosinophil in bone marrow compared to the respective SAL/OVA controls (
IL-5 and eotaxins are critical regulators of the expansion and chemotaxis of eosinophils
OVA/OVA treated WT mice had significantly increased levels of (A) serum IL-5 and mRNA encoding (B) IL-5, (C) eotaxin-1 and (D) eotaxin-2 in lung tissue compared to SAL/OVA treated controls. OVA/OVA treated STAT6-deficient mice also had significantly increased levels of IL-5 in serum and lung but these levels were lower than OVA/OVA treated WT mice. There was no increase the expression of eotaxin-1 and -2 in OVA/OVA treated STAT-6 deficient or SAL/OVA treated controls. #P<0.05 compared to other groups. *P<0.05 compared to respective SAL/OVA treated control.
We then assessed whether the accumulation of eosinophils in blood and development in bone marrow in the absence of STAT6 was associated with the STAT6-independent production of IL-5. OVA/OVA treated STAT6-deficient mice were administered IL-5 neutralizing mAb or isotype control every four days from day 22 d of the OVA/OVA treatment regime. Anti-IL-5 mAb treatment completely abolished the increases in the percentage of eosinophils in the peripheral blood of OVA/OVA treated STAT6-deficient mice (
The increased (A) percentages of eosinophils in peripheral blood and (B) numbers of eosinophils in bone marrow in response to OVA/OVA treatment were completely abolished by neutralization of IL-5 with anti-IL-5 antibody in STAT6-deficient mice. #P<0.05 compared to other groups.
CD4+- and CD8+-T cells and NK cells produce IL-5 and may regulate the expansion of eosinophils in bone marrow. Therefore, we then assessed the contribution of these cells on the STAT6-independent development of eosinophils. OVA/OVA treated STAT6-deficient mice were administered CD4+, NK, CD4+/NK or CD8+ cell neutralizing antibodies or isotype control every four days from day 22 d of the OVA/OVA treatment regime. Some studies have suggested that anti-ASIALO GM1 antibody may target other cells (e.g. CD8+ T cells or NK cells)
(A) Anti-CD4 and (B) anti-NK cell (anti-ASIALO GM1) antibody significantly suppressed the increased development of eosinophils in the bone marrow of OVA/OVA treated STAT6-deficient mice. Combined treatment with (C) anti-CD4 and anti-NK antibodies completely abolished the increased development of eosinophils. By contrast, (D) anti-CD8 antibody had no effect. #P<0.05 compared to other groups. *P<0.05 compared to SAL/OVA treated mice.
Here we demonstrate that STAT6 is a critical mediator of eosinophil influx into the lung. However, we also demonstrate that an alternative pathway that is independent of STAT6 controls the movement of eosinophils into the blood and partially mediates their development in the bone marrow during allergic airway inflammation. These effects were associated with reduced but still elevated levels of IL-5 in serum and the inhibition of the expression of eotaxins in the lung. We also show that IL-5 production, most likely from CD4+ and NK cells, may mediate STAT6-independent eosinophil accumulation in the blood and development in the bone marrow.
Other studies by Kuperman
In our study, STAT6 deficiency led to reduced levels of eosinophils in bone marrow (
Characterization of mRNA levels of chemokines in the lung revealed that the expression of eotaxin-1 and -2 were exclusively dependent on STAT6. Other chemokines, including MDC, TECK, TARC, RANTES, MCPs and MIPs were expressed independently of STAT6 in response to OVA/OVA treatment (unpublished data). The absence of eotaxin-1 and -2 in the lung, however, may have prevented the trafficking of eosinophils from the blood into lung tissue, and resulted in greater numbers of eosinophils in the circulation. Interestingly, although IL-5 was still produced in the lung (although markedly decreased) of OVA/OVA treated STAT6-deficient mice, the levels of eosinophils in the lung were not increased. These results confirm the importance of the STAT6-dependent production of these eotaxins and IL-5, by contrast to other chemokine subfamilies, in the recruitment of eosinophils to the airways during allergic inflammation.
CD4+ Th2 cells are the predominant cellular sources of IL-5
The depletion of CD4+ cells and NK cells in the absence of STAT6 inhibited eosinophilic responses in the bone marrow and the production of IL-5. These results suggest that although STAT6 pathways play a central role in the induction of eosinophilic responses, other pathways involving CD4+ T cells and NK cells may also contribute by independently producing IL-5 and inducing the development of eosinophilic responses in blood and bone marrow. Indeed increased numbers of NK cells (CD3−CD49b+FcεRII−) were detected in the bone marrow of OVA/OVA treated STAT6-deficient mice (unpublished data). These results indicate that a cooperative mechanism of STAT6-independent production of IL-5 may exist in CD4+ cells and NK cells and highlights the potential differential regulation of eosinophilic responses that is mediated by the surrounding inflammatory environment.
In summary, eosinophilic responses in allergic airway inflammation are differentially regulated by the activation of STAT6-dependent and -independent pathways at different parts of the inflammatory process. STAT6-dependent pathways critically regulate eosinophil migration into the lung, that involves the induction of eotaxin-1 and -2 expression. However, STAT6-independent IL-5-regulated eosinophilic pathways operate in blood and bone marrow compartments. These alternative pathways may be regulated by CD4+ and NK cells which produce IL-5 and contribute to the development of eosinophils in the bone marrow and their subsequent release into the blood. Therefore, therapeutic strategies that inhibit eosinophilia in eosinophil-mediated diseases should consider both pathways, even though both pathways are critically dependent on IL-5.