The IL-6R alpha chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo - PubMed (original) (raw)

. 2005 Feb;115(2):313-25.

doi: 10.1172/JCI22433.

Tatjana Eigenbrod, Norbert Krug, George T De Sanctis, Michael Hausding, Veit J Erpenbeck, El-Bdaoui Haddad, Hans A Lehr, Edgar Schmitt, Tobias Bopp, Karl-J Kallen, Udo Herz, Steffen Schmitt, Cornelia Luft, Olaf Hecht, Jens M Hohlfeld, Hiroaki Ito, Norihiro Nishimoto, Kazuyuki Yoshizaki, Tadamitsu Kishimoto, Stefan Rose-John, Harald Renz, Markus F Neurath, Peter R Galle, Susetta Finotto

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The IL-6R alpha chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo

Aysefa Doganci et al. J Clin Invest. 2005 Feb.

Erratum in

Abstract

The cytokine IL-6 acts via a specific receptor complex that consists of the membrane-bound IL-6 receptor (mIL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). In this study, we investigated the role of IL-6R components in asthma. We observed increased levels of sIL-6R in the airways of patients with allergic asthma as compared to those in controls. In addition, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130-fraction constant led to suppression of Th2 cells in the lung. By contrast, blockade of mIL-6R induced local expansion of Foxp3-positive CD4+CD25+ Tregs with increased immunosuppressive capacities. CD4+CD25+ but not CD4+CD25- lung T cells selectively expressed the IL-6R alpha chain and showed IL-6-dependent STAT-3 phosphorylation. Finally, in an in vivo transfer model of asthma in immunodeficient Rag1 mice, CD4+CD25+ T cells isolated from anti-IL-6R antibody-treated mice exhibited marked immunosuppressive and antiinflammatory functions. IL-6 signaling therefore controls the balance between effector cells and Tregs in the lung by means of different receptor components. Furthermore, inhibition of IL-6 signaling emerges as a novel molecular approach for the treatment of allergic asthma.

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Figures

Figure 1

Figure 1

sIL-6R is increased in BALF of asthmatic patients as compared to control subjects, and its levels correlate with the number of IL-5–producing CD4+ T cells in BALF after allergen challenge. (A) sIL-6R was measured before (untreated) and 24 hours after allergen or saline challenge in control subjects (gray bars) or in subjects with asthma (black bars). In the asthmatic patients, sIL-6R levels were increased at baseline and were further increased after allergen challenge. *P < 0.05; ***P < 0.001. (B–D) In these patients, a positive correlation was found between the levels of sIL-6R and the number of CD4+ T cells (r = 0.9027; P < 0.0001) after allergen challenge (C). Furthermore, the value of sIL-6R positively correlated with the number of CD4+ T cells producing IL-5 in BALF (r = 0.9171; P = 0.0001) (D), while a lower correlation was found between sIL-6R and the number of eosinophils (Eos.) in BALF (r = 0.6059; P = 0.0282) (B). Furthermore, the value of sIL-6R in the airways of asthmatic subjects after allergen challenge correlates positively with the respective sIL-6R of IL-5 (E) and IL-13 (F) in BALF.

Figure 2

Figure 2

Local blockade of sIL-6R by gp130-Fc downregulates IL-4, IL-5, and IL-13 levels and reduces GATA-3 expression in experimental asthma. BALB/c mice were sensitized and challenged with OVA whereas control mice were given saline. Some OVA-sensitized mice received additional treatment with gp130-Fc to block sIL-6R function in vivo, as indicated. gp130-Fc treatment was associated with a significant decrease in IL-4 (A), IL-5 (B), and IL-13 (C) levels in BALF of OVA-sensitized mice. *P < 0.05; **P < 0.01; ***P < 0.001. Data represent mean values ± SEM from 5 mice per group. (D) Total lung proteins were isolated from gp130-Fc–treated mice and untreated control mice at day 28 and analyzed by Western blot analysis after immunoblotting with a monoclonal antibody directed against GATA-3. Furthermore, ERK2 expression was determined on the same blot after membrane stripping and incubation with an anti–ERK-2 antibody. Each lane in the Western blot was loaded with 50 μg proteins isolated from different mice (saline, n = 4; OVA, n = 5; OVA + gp130-Fc, n = 5). Quantification of Western blots by densitometry is reported in E and shows decreased GATA-3 expression after i.n. delivery of gp130-Fc in OVA-sensitized and -challenged mice (*P < 0.05; ***P = 0.00056). (F) RT-PCR for GATA-3 and T-bet in 105 lung CD4+ T cells per group after total RNA extraction. Both anti–IL-6R antibody and gp130-Fc treatment led to upregulation of T-bet, while GATA-3 remained unchanged in lung OVA-specific CD4+ T cells.

Figure 3

Figure 3

Antiinflammatory mechanism of local treatment with anti–IL-6R antibodies. Blockade of IL-6R led to a downregulation of IL-4 (P = 0.05) (A), and, at higher doses (100 μg/day), of IL-5 (P = 0.029) (B) in the lungs of treated mice. These findings were accompanied by downregulation of the total number of eosinophils (P = 0.0002) in BALF (C) and CD4+ cells (P = 0.05) in the airways of treated mice (D and E). (D) CD4+ lung cells were stained by using monoclonal anti-CD4 antibodies (BD), and immunohistochemistry was performed as previously described (34). Pictures were taken with an Olympus inverted microscope connected to a digital camera. Original magnification, ×400. (E) The number of CD4+ lung cells obtained from 1 lung after CD4 isolation is reported for different groups. *P < 0.05; ***P < 0.001.

Figure 4

Figure 4

Blockade of mIL-6R through i.n. application of anti–IL-6R antibodies ameliorates AHR and induces IFN-γ and IL-10 levels in BALF in a mouse model of asthma after OVA sensitization. (A) Eight to 10 BALB/c mice per group were sensitized to OVA-alum (OVA-sensitized mice) followed by local treatment with OVA alone or treatment with OVA plus either gp130-Fc, IgG, or anti–IL-6R antibody. Control mice were sensitized with saline-alum and exposed to saline aerosol (saline). Transpulmonary resistance was performed 24 hours after the last local treatment in all mice, as specified in Methods. Dose-response curves to MCh were obtained after administering indicated doses of intravenous MCh. OVA-sensitized mice reacted with an increase of airway resistance at low doses of MCh as compared to that of mice given saline. Anti–IL-6R–treated, OVA-sensitized mice were more protected from the development of AHR compared to untreated (P = 0.049) or IgG-treated, OVA-sensitized mice. Moreover, blockade of sIL-6R by gp130-Fc was less effective compared to anti–IL-6R antibody treatment. (B and C) Local anti–IL-6R antibody treatment induced significant release of IFN-γ (P = 0.048) (B) and IL-10 (P = 0.020) (C) in BALF of OVA-senstitized mice as compared to untreated, OVA-sensitized mice (5 < n < 15). Mean values ± SEM are shown. *P < 0.05.

Figure 5

Figure 5

IL-10–producing CD4+ T cells in the lungs of anti–IL-6R antibody–treated mice. (A and B) CD4+ T cells were isolated from the lung of treated or untreated mice and cultured overnight in the presence of anti-CD3 antibodies. CBA was performed on the CD4+ T cell supernatants. CD4+ T cells isolated from the lung of anti–IL-6R antibody–treated mice secreted increased amounts of IL-10 and IFN-γ as compared to those of OVA-sensitized and -challenged, untreated or IgG-treated mice (P = 0.023 and P = 0.013 for IL-10 and IFN-γ, respectively). Levels of the Th2-type chemokine MCP-1 were not upregulated upon anti–IL-6R antibody treatment, however. (C and D) By contrast, lung CD4+ cells isolated from mice treated i.n. with gp130-Fc did not show changes either in IL-10 (C) or IFN-γ production (D).

Figure 6

Figure 6

Increased release of IL-10 from Foxp3+ CD4+CD25+ Tregs isolated from the lungs of anti–IL-6R–treated mice. CD4+CD25+ T cells and CD4+CD25– T cells were isolated from lung cells in the different experimental groups, after which cytokine production was analyzed. The purity of the CD4+CD25+ cell population was 95–98% as determined by FACS analysis during cell sorting. (A–C) Lung CD4+CD25+ T cells isolated from OVA-sensitized, anti–IL-6R antibody–treated mice released increased amounts of IL-10 (B) per cell as compared to CD4+CD25+ T cells from OVA-sensitized and -challenged, untreated or OVA-sensitized, IgG-treated mice. In contrast, lung CD4+CD25– T cells produced little IL-10 (B) but more TGF-β (A) and some IFN-γ (C). n = 6. By contrast, CD4+CD25+ isolated from gp130-Fc–treated mice released less IL-10 (B), IFN-γ (C), and TGF-β (A), while the CD4+CD25– isolated from the same mice released the same amount of IL-10 (B) and less TGF-β (A). (D) Expression of Foxp3 on CD4+CD25+ lung T cells upon anti–IL-6R antibody treatment. CD4+CD25+ and CD4+CD25– lung T cells from untreated or anti–IL-6R antibody–treated, OVA-sensitized mice were separated as described above. This was followed by RNA extraction and analysis of Foxp3 or β-actin expression by RT-PCR. (E) Real-time PCR for Foxp3 in CD4+CD25+ and CD4+CD25– cells is reported as the ratio of Foxp3 to HGPRT. Anti–IL-6R antibody treatment led to a significant upregulation of Foxp3 as compared to OVA treatment. This experiment was performed 3 times in duplicate. *P < 0.05; **P < 0.01.

Figure 7

Figure 7

Increased number and augmented immunosuppressive function of CD4+CD25+ T cells in the lungs of anti–IL-6R–treated, OVA-sensitized mice. (A) i.n. but not i.p. anti–IL-6R antibody treatment after OVA sensitization and challenge led to an induction of CD4+CD25+ T cell number in the lung (*P = 0.057). (B) IL-6 levels were increased in BALF of OVA-sensitized mice as compared to those of saline-treated mice. i.p. but not i.n. injection of anti–IL-6R antibodies led to a further increase of IL-6 in the airways. (C) CD4+CD25+ T cells isolated from the lungs of anti–IL-6R antibody–treated (i.n.) mice inhibited proliferation of CFSE-labeled target CD4+ spleen T cells more efficiently compared to CD4+CD25+ T cells isolated from the lungs of OVA-sensitized and -challenged, untreated mice. Mean values ± SEM; n = 5 mice per group; *P < 0.05. (D) Histograms of a representative cell population of CD4+ spleen cells labeled with CFSE and coincubated for 4 days with either CD4+CD25+ or CD4+CD25– cells isolated from the lungs of different groups. Percentages indicate the number of spleen CD4+/CFSE-labeled cells at day 4 (M1, 20 hours). (E) RT-PCR for the IL-6R α chain shows selective expression on Foxp3+ CD4+CD25+ lung T cells but not CD4+CD25– lung T cells. One representative experiment out of 3 is shown. (F) phospho–STAT-3 (pSTAT3) immunostaining in CD4+CD25+ cells. Spleen CD4+CD25+ T cells were incubated either with medium alone (left panel), with 20 ng/ml of IL-6 (middle panel), or with IL-6 (20 ng/ml) and anti–IL-6R antibodies (10 μg/ml) (right panel). Magnification, ×200.

Figure 8

Figure 8

CD4+CD25+ T cells from OVA-sensitized mice can inhibit CD4+CD25– T cell–induced experimental asthma in Rag1–/– mice. (A–F) Cotransfer of CD4+CD25+ and CD4+CD25– spleen T cells into Rag1–/– mice. CD4+CD25+ and CD4+CD25– T cells were isolated from the spleens of OVA-sensitized and -challenged mice given IgG-control antibodies or anti–IL-6R antibodies. CFSE-labeled CD4+CD25– spleen T cells from OVA-sensitized mice (5 × 105; CSFE-labeled indicated with asterisks) were cotransferred i.p. with either 5 × 105 unlabeled CD4+CD25– T cells (A and D) or CD4+CD25+ T cells (B, C, E, and F) into immunocompromised Rag1 knockout mice. Cotransfer of CD4+CD25+ T cells from anti–IL-6R–treated (C and F) or IgG-treated (B and E) mice suppressed allergic airway inflammation induced by transfer of CD4+CD25– T cells from OVA-sensitized mice. Magnification, ×100 (A–C), ×400 (D–F). Mice receiving only CD4+CD25– T cells showed CFSE-positive cells in the lung (H), whereas mice receiving CD4+CD25+ T cells as well showed a marked decrease in the number of CFSE-positive cells (G and I), suggesting a CD4+CD25+ T cell–mediated suppression of effector CD4+CD25– T cell proliferation. Results in G were obtained by calculating the average number of CFSE-positive cells per high power field (HPF) (n = 30). ***P = 0.001.

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