IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33–LacZ gene trap reporter strain (Il-33Gt/Gt) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti–IL-33 Abs, using Il-33Gt/Gt (Il-33–deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33–LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

Interleukin-33 (previously known as NF from high endothelial venules) (1, 2) is an IL-1–like cytokine that signals through the ST2 receptor (3) and drives production of cytokines and chemokines in target cells (4, 5), including mast cells (68), basophils and eosinophils (9), endothelial cells (10), Th2 lymphocytes (3), and invariant NKT and NK cells (11, 12). IL-33 is also a potent activator of type 2 innate immune cell populations that have been described recently, including natural helper cells from adipose tissues and lung (13, 14), nuocytes from mesenteric lymph nodes and spleen (15), and innate helper 2 cells from various tissues (16). These innate immune cell populations have been shown to respond to IL-33 by producing extremely high amounts of the Th2 cytokines IL-5 and IL-13 (1316), as well as to play important roles in innate immune responses after helminth infection in the intestine (13, 15, 16) or influenza virus infection in the lungs (14, 17). An important role for IL-33 in innate, rather than acquired, immunity is also supported by observations in IL-33–deficient mice (18).

We initially described IL-33 as an NF abundantly expressed in high endothelial venules (HEVs), specialized blood vessels mediating lymphocyte recruitment into lymph nodes (1). We later discovered its association with chromatin in the nucleus of HEV endothelial cells and concluded that IL-33 is a chromatin-associated cytokine in vivo (2). Further analyses revealed that IL-33 mimics Kaposi sarcoma herpesvirus for attachment to chromatin and docks through a short chromatin-binding peptide into an acidic pocket formed by histones H2A-H2B (19). Constitutive expression and nuclear localization of IL-33 has been observed in all endothelial cells along the vascular tree, indicating that the endothelium constitutes a major cellular source of IL-33 in normal human tissues (20, 21). Abundant expression of IL-33 was also detected in the nuclei of epithelial cells in human barrier tissues, such as the skin, gastrointestinal tract, and lungs (20, 22). The high levels of constitutive expression of IL-33 in blood vessels and epithelial barrier tissues, as well as its nuclear localization in vivo, suggested that it may function as a novel alarmin (intracellular alarm signal released after cell injury) to alert the immune system after endothelial or epithelial cell damage during trauma or infection (20). In agreement with this model, we and other investigators recently reported that full-length IL-331–270 is biologically active (2325) and that it can be released in the extracellular space after cellular damage (23, 24). We also showed that processing of full length IL-331–270 by caspases results in its inactivation (23, 24), rather than in its activation, as initially proposed (3).

Although the expression profile of endogenous IL-33 has been relatively well characterized in normal human tissues (20, 21), comparatively little is known about the expression of endogenous IL-33 protein in mouse tissues during homeostasis. Immunohistochemical analysis of lung tissue from wild-type and Il-33–deficient mice revealed that IL-33 exhibits constitutive nuclear expression in the alveolar epithelium (26). High levels of IL-33 mRNA have been reported in other mouse epithelial barrier tissues (i.e., stomach, skin) and lymphoid organs (i.e., lymph nodes, spleen) (3), but the cellular sources of IL-33 protein in these tissues have not been characterized. Similarly, nothing is known about the expression of IL-33 during embryogenesis. In the current study, we generated a novel Il-33–LacZ gene trap (Gt) reporter strain (Il-33Gt/Gt) and used this mouse model to analyze Il-33 promoter activity in mouse adult tissues and embryos. In addition, we investigated expression of the IL-33 protein in mouse tissues by immunohistofluorescence, using Il-33Gt/Gt mice (Il-33–deficient mice) as control. These analyses indicated that endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues. The results also revealed the existence of important species-specific differences between humans and mice, with a striking lack of IL-33 expression in mouse blood vessels during homeostasis.

C57BL/6J mice were obtained from Charles River Laboratories. Il-33Gt mice were generated by microinjecting an embryonic stem (ES) cell clone provided by Texas A&M Institute for Genomic Medicine (IST10946B6-Tigm) into host mouse blastocysts. The ES clone contains a unique Gt insertion in the first intron of the Il-33 locus. The locus has been disrupted by insertion of the Omnibank Gt Vector 76 where two viral long terminal repeats frame a splice acceptor upstream of the β-galactosidase and neomycin resistance fusion gene. Sequencing of the Il-33 locus in the clone IST10946B6 localized precisely the insertion site in intron 1 of the Il-33 gene, 3076 bp upstream of the ATG initiation codon. IST10946B6 ES cells were injected into C57BL/6J host mouse blastocysts, and the derived chimeric mice were outcrossed to C57BL/6 to follow germline transmission. The derived F1 mice were screened by PCR. We got five founders and used one of them to establish the colony (Il-33Gt(IST10946B6-Tigm)Girard). C57BL/6:Il-33−/− homozygous mice (Il-33Gt/Gt) were generated by crossing heterozygous C57BL/6:Il-33-LacZ mice (Il-33+/Gt).

Genotyping of the pups was performed by PCR on tail DNA using a DNA extraction kit (Sigma) and the following primers: P1: 5′-CATTCAAGACCAGCTATTTCCTG-3′, , P2: 5′-CTCAATTCCTCCTGGTACAGGCAG-3′, P3: 5′-CTTGCAAAATGGCGTTACTTAAGC-3′, P4: 5′-GCACAAAGCTAAGAAGCTGCAAGC-3′.

PCR made with P1/P4 and P2/P4 gave a band of 1051 and 402 bp, respectively, and revealed the wild-type allele; P3/P4 were used to detect the Gt allele and gave a band of 337 bp. All set of primers were annealed at 60°C for 30 cycles.

Colonic inflammation was induced by administering 3% dextran sulfate sodium (DSS; Sigma) in the drinking water for 1 wk. LPS (Escherichia coli serotype 0111:B4; Sigma)-induced endotoxin shock (20 mg/kg LPS injected i.p.) and papain-induced airway inflammation (5 mg/ml papain [Sigma] in 20 μl saline once a day for 3 d) were evaluated as described (18). All of the mice were bred under specific pathogen-free conditions and handled according to institutional guidelines under protocols approved by the Institut de Pharmacologie et de Biologie Structurale and Région Midi Pyrénées animal care committees.

Lung homogenates were lysed in RIPA lysis buffer solution (400 mM NaCl, 20 mM Tris [pH 7,4], 10 mM EDTA, 0.1% SDS, 1% Triton, 0.05 Tween 20) supplemented with a protease inhibitor mixture tablet (Roche). The supernatants were collected after centrifugation (16,000 × g) at 4°C for 10 min. Lung lysates were analyzed by SDS-PAGE and blotted. Membranes were blocked and incubated with goat anti-mouse IL-33 (1/1000; R&D Systems) and donkey anti-goat, HRP-conjugated (1/10,000; Promega) polyclonal Abs. The immunoreactive proteins were visualized with ECL plus reagents (ECL Western blotting Detection Reagents; Amersham).

β-Galactosidase detection with the chromogenic substrate X-Gal (X-Gal staining) was done overnight on 10-μm cryosections of wild-type and mutant tissues using a LacZ Tissue staining kit (InvivoGen), and sections were counterstained by nuclear Fast Red (Sigma) and mounted with Coversafe medium (Microm). Fifty-micrometer vibratome brain sections and embryo, liver, or lung whole-mount staining were realized on freshly dissected tissues, fixed for 2 or 1 h, respectively, with 4% paraformaldehyde; washed in PBS; permeabilized for 3 h in PBS, 2 mM MgCl2, 0.02% Nonidet P-40, and 0.01% sodium deoxycholate at 4°C; and stained from 30 min to overnight using a LacZ Tissue staining kit (InvivoGen). Clearing of embryos was done as described previously (27).

Immunohistofluorescence was performed on formalin-fixed mouse tissues. Five-micrometer paraffin-embedded sections were deparaffinized in Histo-clear (National Diagnostics) and rehydrated in graded alcohol series. After rehydration, the paraffin sections were boiled in a microwave oven for epitope retrieval in Sodium Citrate Buffer (10 mM [pH 6]) for 20 min. Sections were equilibrated in PBS and incubated with blocking solution MAXblock (Active Motif) for 1 h at room temperature. Polyclonal goat Ab anti-mouse IL-33 (1/200, AF326; R&D Systems), polyclonal rabbit Ab anti-Sox2 (1/100, AB5603; Millipore), rat mAb anti-mouse CD31 (1/20, clone SZ31; Dianova), rat mAb MECA-79 (1/100, clone MECA-79; Pharmingen), polyclonal rabbit Ab anti-von Willebrand Factor (vWF; 1/50, A0082; Dako), and mouse mAb anti–α-smooth muscle actin (α-SMA; 1/100, clone 1A4; Dako) diluted in PBS, MAXblock 20% were incubated overnight at 4°C. Sections were washed in PBS for 30 min and incubated with bovine anti-goat DyLight 549 (1/200; Jackson ImmunoResearch), donkey anti-rat IgG DyLight 488 (1/200; Jackson ImmunoResearch), donkey anti-rabbit IgG DyLight 488 (1/200; Jackson ImmunoResearch), or goat anti-mouse Cy2 (1/200; Amersham) secondary Abs, respectively, for 1 h at room temperature. Sections were counterstained with DAPI and mounted in Mowiol.

Immunohistofluorescence on normal human tissues was performed using two sources of formalin-fixed paraffin-embedded human tissue microarrays, as previously described (20).

Fluorescent images were visualized using an inverted microscope Eclipse TE300 Nikon with 40×/0.75 and 100×/0.5–1.3 objectives at room temperature and captured with a DXM 1200 digital camera using Nikon ACT1 software. Bright-field images were visualized using an Eclipse 80i Nikon microscope with 4×/0.10 and 40×/0.75 objectives at room temperature and captured through a Digital Sight DS 5M L1 Nikon camera using DS 5M L1 Nikon software. Whole-mount expression in embryos, liver, and lung and vibratome sections were visualized using a Leica MZ8 stereomicroscope with 0.63–5× objectives and a 16×/14B ocular. Bright-field images were captured with a Nikon Coolpix 950 digital camera. All images were processed using Adobe Photoshop CS2 software.

To further characterize the in vivo expression and function of IL-33, we inactivated the Il-33 gene using a Gt-inactivation strategy (Fig. 1A; Gt insertion in intron 1 of the Il-33 gene). The βgeo insertion disrupts production of the IL-33 protein and is useful to visualize the activity of the endogenous Il-33 promoter, through X-Gal staining (β-galactosidase activity). Mice heterozygous for the mutation (Il-33+/Gt) have been intercrossed, and viable homozygous Il-33 null mice (Il-33Gt/Gt) have been obtained, indicating that IL-33 is not essential for embryonic development, health, and fertility, as recently reported for other lines of Il-33–deficient mice generated independently (18, 26). Loss of IL-33 protein in the Il-33Gt/Gt mice was validated by Western blot analysis of lung tissue extracts (Fig. 1B). X-Gal staining in IL-33Gt/Gt adult mice revealed constitutive activity of the Il-33 promoter in mouse lymphoid organs, with strong signals in the T cell areas of peripheral lymph nodes and spleen (Fig. 1C). Similar β-galactosidase activity was observed in Il-33+/Gt heterozygous mice (Fig. 1C). In contrast, no signals were observed in lymphoid organs from wild-type mice, indicating that the X-Gal staining in Il-33Gt/Gt mice was specific. We concluded that the Il-33–LacZ Gt mouse is a useful tool to analyze IL-33 expression in vivo.

FIGURE 1.

In vivo analysis of Il-33 promoter activity in mouse lymphoid organs using a novel Il-33–LacZ Gt reporter strain. (A) Schematic representation of the Gt cassette insertion into intron 1 of the Il-33 locus. The Gt cassette consists of a promoter-less βgeo reporter/selectable marker gene flanked by splice acceptor (SA) and polyadenylation (polyA) sites. (B) Genotyping and Western blot analysis of Il-33+/+, Il-33+/Gt, and Il-33Gt/Gt mice. Analysis of IL-33 protein expression by Western blot was performed using whole-lung homogenates. *Nonspecific band. (C) Il-33 promoter-driven β-galactosidase expression in mouse lymphoid tissues. Lymph nodes and spleen from Il-33+/+, Il-33+/Gt, and Il-33Gt/Gt mice were stained with X-Gal. Scale bars are as indicated.

FIGURE 1.

In vivo analysis of Il-33 promoter activity in mouse lymphoid organs using a novel Il-33–LacZ Gt reporter strain. (A) Schematic representation of the Gt cassette insertion into intron 1 of the Il-33 locus. The Gt cassette consists of a promoter-less βgeo reporter/selectable marker gene flanked by splice acceptor (SA) and polyadenylation (polyA) sites. (B) Genotyping and Western blot analysis of Il-33+/+, Il-33+/Gt, and Il-33Gt/Gt mice. Analysis of IL-33 protein expression by Western blot was performed using whole-lung homogenates. *Nonspecific band. (C) Il-33 promoter-driven β-galactosidase expression in mouse lymphoid tissues. Lymph nodes and spleen from Il-33+/+, Il-33+/Gt, and Il-33Gt/Gt mice were stained with X-Gal. Scale bars are as indicated.

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We next performed immunofluorescence staining of mouse peripheral lymph node sections, using human lymphoid tissues as controls and Il-33–deficient mice (Il-33Gt/Gt) to validate the specificity of the immunostaining (Fig. 2). As previously reported (20), IL-33 was expressed in the nuclei of both HEV endothelial cells and α-SMA+ fibroblastic reticular cells (FRCs) in human lymph node and tonsil (Fig. 2A, 2C). Surprisingly, IL-33 expression was not observed in HEVs from wild-type mouse peripheral lymph nodes (Fig. 2B). In contrast, IL-33 was strongly expressed in α-SMA+ FRCs from mouse lymph nodes (Fig. 2D), similarly to human tonsil (Fig. 2C). Staining of α-SMA+ FRCs with anti–IL-33 Abs was specific because it was not observed in Il-33–deficient mice (Fig. 2E). Double staining with the DNA-binding dye DAPI indicated that IL-33 accumulates in the nuclei of α-SMA+ FRCs, with no evidence for cytoplasmic localization (Fig. 2F–H). Like in lymph nodes, IL-33 was constitutively expressed in the nuclei of α-SMA+ FRCs from mouse spleen (Fig. 2I–K). We concluded that IL-33 is constitutively and highly expressed in FRCs from mouse lymph nodes and spleen, but, contrary to humans, it is not expressed in HEVs.

FIGURE 2.

IL-33 protein is highly expressed in the nuclei of α-SMA+ FRCs but is not detectable in HEVs from mouse peripheral lymph nodes. Tissue sections from human peripheral lymph node (A) and tonsil (C), as well as from peripheral lymph nodes (B, D, F, G, H) and spleen (I, J, K) from wild-type mice (Il-33+/+), were double stained with anti–IL-33 Abs (red, A–G, I, J) and anti-CD31 (green, A), HEV-specific mAb MECA79 (green, B), or anti–α-SMA (green, C–G, I, J) mAbs. DNA was counterstained with DAPI (blue). Lymph nodes from Il-33–deficient mice (Il-33Gt/Gt) were used as controls (E). Large arrows, HEV endothelial cells; small arrows, nuclei of FRCs. Scale bars, 10 μm.

FIGURE 2.

IL-33 protein is highly expressed in the nuclei of α-SMA+ FRCs but is not detectable in HEVs from mouse peripheral lymph nodes. Tissue sections from human peripheral lymph node (A) and tonsil (C), as well as from peripheral lymph nodes (B, D, F, G, H) and spleen (I, J, K) from wild-type mice (Il-33+/+), were double stained with anti–IL-33 Abs (red, A–G, I, J) and anti-CD31 (green, A), HEV-specific mAb MECA79 (green, B), or anti–α-SMA (green, C–G, I, J) mAbs. DNA was counterstained with DAPI (blue). Lymph nodes from Il-33–deficient mice (Il-33Gt/Gt) were used as controls (E). Large arrows, HEV endothelial cells; small arrows, nuclei of FRCs. Scale bars, 10 μm.

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We then extended our analyses to nonlymphoid tissues. Strikingly, although IL-33 is constitutively expressed at high levels in the endothelium from normal human tissues (20), constitutive expression of IL-33 in endothelial cells was not observed in mouse tissues (Fig. 3). For instance, strong nuclear staining of blood vessel endothelial cells with anti–IL-33 Abs was observed in human colon, lung, cervix, and breast but not in the corresponding mouse tissues (Fig. 3A). Although IL-33 was not constitutively expressed in endothelial cells along the vascular tree, some sporadic expression in a few blood vessels from adipose tissue was occasionally seen in some mice. This observation suggested that expression of IL-33 in mouse blood vessels could be induced under certain conditions. Accordingly, we observed strong nuclear staining of IL-33 in vWF+ endothelial cells in the inflamed colon during DSS-induced colitis (Fig. 3B), a model for inflammatory bowel diseases, which was shown to be dependent (in part) on endogenous IL-33 (18). Together, these results revealed the existence of important species-specific differences in the regulation of IL-33 in blood vessels between humans and mice.

FIGURE 3.

IL-33 protein is not constitutively expressed along the vascular tree in mouse tissues. (A) Species-specific endothelial cell expression of IL-33 along the vascular tree. Tissue sections from representative human and mouse (Il-33+/+) tissues were double stained with anti–IL-33 (red) and anti-CD31 (green) Abs. DNA was counterstained with DAPI (blue). Scale bars, 10 μm. (B) IL-33 protein expression is induced in blood vessels from the inflamed colon in mouse during DSS-induced colitis. Sections were double-stained with anti–IL-33 (red) and anti-vWF Abs (green). DNA was counterstained with DAPI (blue). Scale bars, 20 μm.

FIGURE 3.

IL-33 protein is not constitutively expressed along the vascular tree in mouse tissues. (A) Species-specific endothelial cell expression of IL-33 along the vascular tree. Tissue sections from representative human and mouse (Il-33+/+) tissues were double stained with anti–IL-33 (red) and anti-CD31 (green) Abs. DNA was counterstained with DAPI (blue). Scale bars, 10 μm. (B) IL-33 protein expression is induced in blood vessels from the inflamed colon in mouse during DSS-induced colitis. Sections were double-stained with anti–IL-33 (red) and anti-vWF Abs (green). DNA was counterstained with DAPI (blue). Scale bars, 20 μm.

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X-Gal staining of nonlymphoid tissues from Il-33Gt/Gt adult mice revealed abundant expression of β-galactosidase in several epithelial tissues, including lung alveoli, stratified squamous epithelium of the vagina, and salivary gland ducts (Fig. 4). In contrast, no β-galactosidase activity was observed in the corresponding tissues from wild-type mice, which do not contain the Il-33–LacZ Gt. We concluded that the Il-33 promoter is constitutively active in epithelial barrier tissues. We then analyzed expression of IL-33 at the protein level by immunohistofluorescence with anti–IL-33 Abs (Fig. 5). Constitutive expression of IL-33 was observed in the nuclei of epithelial cells in tissues exposed to the environment, in agreement with previous observations in human tissues (20). The highest levels of IL-33 were observed in the vagina, skin, lung, stomach, and salivary glands. Epithelial staining in all of these tissues was specific, because it was not observed in the corresponding tissues from Il-33–deficient mice (Il-33Gt/Gt). Double staining of tissue sections with the DNA-binding dye DAPI revealed that IL-33 was always localized in the nuclei of the epithelial cells (Fig. 5). No evidence was found for cytoplasmic localization of IL-33 in murine adult tissues. Cytoplasmic staining with the anti–IL-33 Abs was only observed in the lamina propria from the inflamed colon during DSS-induced colitis, but this staining turned out to be nonspecific because it was still present in Il-33–deficient mice (Supplemental Fig. 1). We concluded that IL-33 is a nuclear cytokine constitutively expressed at high levels in epithelial barrier tissues from adult mice.

FIGURE 4.

Constitutive activity of the Il-33 promoter in mouse epithelial barrier tissues. Lung, vagina, and salivary gland tissue sections from Il-33+/+ and Il-33Gt/Gt mice were stained with X-Gal to analyze Il-33 promoter-driven β-galactosidase expression in epithelial barrier tissues. Scale bars are as indicated.

FIGURE 4.

Constitutive activity of the Il-33 promoter in mouse epithelial barrier tissues. Lung, vagina, and salivary gland tissue sections from Il-33+/+ and Il-33Gt/Gt mice were stained with X-Gal to analyze Il-33 promoter-driven β-galactosidase expression in epithelial barrier tissues. Scale bars are as indicated.

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FIGURE 5.

Endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues. IL-33 is constitutively expressed in the nuclei of epithelial cells from mouse tissues exposed to the environment, including stratified squamous epithelia from vagina and skin, stratified cuboidal epithelium from salivary gland, and simple cuboidal epithelium from lung and stomach. Tissue sections from wild-type (Il-33+/+) and Il-33–deficient mice (Il-33Gt/Gt) were stained with anti–IL-33 Abs (red). DNA was counterstained with DAPI (blue). The background staining observed in tissues from Il-33Gt/Gt mice was also observed with the secondary Ab alone (Supplemental Fig. 2). Scale bars are as indicated.

FIGURE 5.

Endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues. IL-33 is constitutively expressed in the nuclei of epithelial cells from mouse tissues exposed to the environment, including stratified squamous epithelia from vagina and skin, stratified cuboidal epithelium from salivary gland, and simple cuboidal epithelium from lung and stomach. Tissue sections from wild-type (Il-33+/+) and Il-33–deficient mice (Il-33Gt/Gt) were stained with anti–IL-33 Abs (red). DNA was counterstained with DAPI (blue). The background staining observed in tissues from Il-33Gt/Gt mice was also observed with the secondary Ab alone (Supplemental Fig. 2). Scale bars are as indicated.

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In addition to lymphoid organs and epithelial barrier tissues, IL-33 was abundantly expressed in the brain and the eye (Fig. 6). X-Gal staining revealed abundant expression of β-galactosidase in the brain from Il-33Gt/Gt adult mice (Fig. 6B, 6D) compared with the brain from wild-type mice (Fig. 6A, 6C). Strong activity of the Il-33 promoter was observed in the corpus callosum, hippocampus (dentate gyrus), thalamus (Fig. 6B), and the cerebellum (granular layer and white matter) (Fig. 6D). In the eye, in addition to the optic nerve, IL-33 protein was abundantly expressed in the nuclei of cells from the retinal inner nuclear layer (Fig. 6E, 6F). This staining was specific because it was not observed in the Il-33Gt/Gt mice lacking IL-33 protein expression (Fig. 6G, 6H). Double staining of the eye sections with Abs against IL-33 and Sox2 revealed that the IL-33+ cells in the retina corresponded to Sox2+ Müller glial cells (Fig. 6I–K). Finally, IL-33 protein was also highly expressed in the nuclei of epithelial cells in the ciliary body, from the ora serrata to the iris (Fig. 6L, 6M).

FIGURE 6.

Endogenous IL-33 is constitutively expressed in the brain and the eye. Il-33 promoter-driven β-galactosidase expression in brain (B) and cerebellum (D) from Il-33Gt/Gt mice. Brain tissue from wild-type (Il-33+/+) mice was used as control (A, C). (EK) Endogenous IL-33 protein is highly expressed in the nuclei of Sox2+ Müller glial cells from the retinal inner nuclear layer. Retinal tissue sections from wild-type (Il-33+/+) (E, F, I–K) and Il-33–deficient (Il-33Gt/Gt) (G, H) mice were stained with anti–IL-33 (red, E–I, K) and anti-Sox2 (green, J, K) Abs. (L and M) Endogenous IL-33 protein is constitutively expressed in the nuclei of epithelial cells from the ciliary body. Tissue sections from wild-type (Il-33+/+) mice were stained with anti–IL-33 (red, L, M) Abs. DNA was counterstained with DAPI (blue, F, H, M). Original magnification ×10 (A–D). Scale bars, 50 μm (E–H, L, M), 10 μm (I –K). cc, corpus callosum; dg, dentate gyrus; Hip, hippocampus; th, thalamus.

FIGURE 6.

Endogenous IL-33 is constitutively expressed in the brain and the eye. Il-33 promoter-driven β-galactosidase expression in brain (B) and cerebellum (D) from Il-33Gt/Gt mice. Brain tissue from wild-type (Il-33+/+) mice was used as control (A, C). (EK) Endogenous IL-33 protein is highly expressed in the nuclei of Sox2+ Müller glial cells from the retinal inner nuclear layer. Retinal tissue sections from wild-type (Il-33+/+) (E, F, I–K) and Il-33–deficient (Il-33Gt/Gt) (G, H) mice were stained with anti–IL-33 (red, E–I, K) and anti-Sox2 (green, J, K) Abs. (L and M) Endogenous IL-33 protein is constitutively expressed in the nuclei of epithelial cells from the ciliary body. Tissue sections from wild-type (Il-33+/+) mice were stained with anti–IL-33 (red, L, M) Abs. DNA was counterstained with DAPI (blue, F, H, M). Original magnification ×10 (A–D). Scale bars, 50 μm (E–H, L, M), 10 μm (I –K). cc, corpus callosum; dg, dentate gyrus; Hip, hippocampus; th, thalamus.

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X-Gal staining in Il-33–LacZ Gt mice revealed constitutive activity of the Il-33 promoter during embryonic development (Fig. 7). β-galactosidase activity was already detected in embryonic day (E)11.5 d postcoitum (dpc) embryo in the caudal part along the anteroposterior axis in Il-33Gt/Gt mice but not in control wild-type mice (Fig. 7A), and it was further increased and restricted at E15.5 to specific tissues of the embryo (Fig. 7B). Strong expression of the Il-33–LacZ reporter was detected in the mesenchyme surrounding the eye, in the nasal cavity, in the grooves separating the lines of vibrissae in the snout (black arrows), the hind limb, and the parotid gland (Fig. 7B). Constitutive expression of IL-33 in embryos was confirmed at the protein level. Analysis of embryonic tissue sections by immunohistofluorescence revealed constitutive expression of IL-33 in the olfactory epithelium and mesenchyme (Fig. 7C). Double staining with DAPI indicated that the endogenous IL-33 protein accumulates in the nuclei of the embryonic cells.

FIGURE 7.

Endogenous IL-33 is constitutively expressed in mouse embryonic tissues. (A and B) Constitutive activity of the Il-33 promoter in mouse embryonic tissues. Whole-mount from Il-33+/+ (A) and Il-33Gt/Gt (A, B) E11.5 or E15.5 dpc embryos were stained with X-Gal to analyze Il-33 promoter-driven β-galactosidase expression in embryonic tissues, black arrows indicate areas of strong expression. Scale bars, 1 mm. (C) IL-33 protein is constitutively expressed in the nuclei of embryonic cells, in the olfactory epithelium, and in mesenchyme from thoracic dorsal part. Tissue sections from E15.5 dpc wild-type embryos were stained with anti–IL-33 Abs (red). DNA was counterstained with DAPI (blue). The last row of images represents higher magnification of the areas boxed upstream. Scale bars, 1 mm (A, B); others are as indicated.

FIGURE 7.

Endogenous IL-33 is constitutively expressed in mouse embryonic tissues. (A and B) Constitutive activity of the Il-33 promoter in mouse embryonic tissues. Whole-mount from Il-33+/+ (A) and Il-33Gt/Gt (A, B) E11.5 or E15.5 dpc embryos were stained with X-Gal to analyze Il-33 promoter-driven β-galactosidase expression in embryonic tissues, black arrows indicate areas of strong expression. Scale bars, 1 mm. (C) IL-33 protein is constitutively expressed in the nuclei of embryonic cells, in the olfactory epithelium, and in mesenchyme from thoracic dorsal part. Tissue sections from E15.5 dpc wild-type embryos were stained with anti–IL-33 Abs (red). DNA was counterstained with DAPI (blue). The last row of images represents higher magnification of the areas boxed upstream. Scale bars, 1 mm (A, B); others are as indicated.

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We then used the Il-33–LacZ Gt reporter strain to analyze IL-33 expression during inflammation. LPS-induced endotoxin shock is characterized by a systemic inflammatory response associated with dysfunction of multiple organs, including the liver and the lung. This LPS-induced systemic inflammatory response is substantially decreased in Il-33–deficient mice (18). Interestingly, we found that expression of endogenous IL-33 in the liver, which is not observed in the absence of inflammation, is highly induced around blood vessels during LPS-induced endotoxin shock (Fig. 8A). We then analyzed a model of lung inflammation (papain-induced airway inflammation), which was shown to be dependent on endogenous IL-33 (18). Papain is a protease allergen considered a cause of occupational asthma, which induces innate-type allergic airway inflammation in mouse (28). Papain-induced airway inflammation (eosinophilia) is profoundly impaired in Il-33–deficient mice (18). In contrast to the upregulation of IL-33 expression in the liver during LPS-induced endotoxin shock, we observed that expression of endogenous IL-33 in the lung alveoli, which is already very high under basal conditions (Fig. 8B, see also Fig. 4), is not increased further during papain-induced airway inflammation. Together, these results indicated that the Il-33–LacZ Gt mouse is a useful tool to analyze IL-33 expression in inflamed tissues.

FIGURE 8.

Expression of the Il-33-LacZ reporter in inflamed tissues. (A) Il-33 promoter-driven β-galactosidase expression in the liver after LPS-induced endotoxin shock. Whole-mount and liver tissue sections from Il-33Gt/Gt mice (control or LPS treated) were stained with X-Gal to analyze Il-33 promoter activity in the inflamed liver. (B) Il-33 promoter-driven β-galactosidase expression in the lung after papain-induced allergic airway inflammation. Whole-mount and lung tissue sections from Il-33+/+ and Il-33Gt/Gt mice (control or papain treated) were stained with X-Gal to analyze Il-33 promoter activity in the inflamed lung. Results are representative of three independent experiments. Scale bars are as indicated.

FIGURE 8.

Expression of the Il-33-LacZ reporter in inflamed tissues. (A) Il-33 promoter-driven β-galactosidase expression in the liver after LPS-induced endotoxin shock. Whole-mount and liver tissue sections from Il-33Gt/Gt mice (control or LPS treated) were stained with X-Gal to analyze Il-33 promoter activity in the inflamed liver. (B) Il-33 promoter-driven β-galactosidase expression in the lung after papain-induced allergic airway inflammation. Whole-mount and lung tissue sections from Il-33+/+ and Il-33Gt/Gt mice (control or papain treated) were stained with X-Gal to analyze Il-33 promoter activity in the inflamed lung. Results are representative of three independent experiments. Scale bars are as indicated.

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In this study, we show that endogenous IL-33 is constitutively expressed at high levels in mouse adult tissues and embryos with a very refined expression pattern. Using a novel Il-33–LacZ Gt reporter strain (Il-33Gt/Gt), we demonstrate that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs (lymph node, spleen), epithelial barrier tissues (lung, skin, vagina), brain, and embryos. Strong expression of the Il-33–LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. IL-33–producing cells in tissues were further characterized by indirect immunofluorescence staining with anti–IL-33 Abs, using Il-33Gt/Gt mice (Il-33–deficient mice) as control to validate the specificity of the staining. We found that the IL-33 protein is highly expressed in the nuclei of epithelial cells from mouse epithelial barrier tissues, such as the vagina, skin, lung, stomach, and salivary glands. The IL-33 protein was also strongly expressed in the nuclei of FRCs from mouse lymphoid organs but was not found in HEVs. Strikingly, IL-33 expression was also not detected in blood vessels from other mouse tissues during homeostasis, revealing the existence of important species-specific differences between humans and mice. In addition to epithelial barrier tissues and lymphoid organs, we identified the eye as another major site of IL-33 expression in mouse tissues. Nuclear expression of the IL-33 protein was found in Sox2+ Müller glial cells from the retina and epithelial cells from the ciliary body. Finally, strong expression of the IL-33 protein was also observed in olfactory epithelium and some mesenchymal cells from E15.5 embryonic tissues. Interestingly, in some organs (i.e., lymph nodes, salivary glands), IL-33 was constitutively expressed from late-gestation embryo to adult life. Importantly, IL-33 protein was always localized in the nucleus of producing cells, with no evidence for cytoplasmic localization. Together, our data indicate that endogenous IL-33 is a nuclear cytokine constitutively expressed at high levels in mouse epithelial barrier tissues, lymphoid organs, brain, eye, and embryo.

We believe that the findings reported in this article support the alarmin function of IL-33, which we initially proposed based on the high levels of constitutively expressed IL-33 in human blood vessels and epithelial barrier tissues (20). Like in humans, IL-33 is highly expressed in the nuclei of epithelial cells of mouse tissues in contact with the environment (lungs, stomach, skin), where pathogens, allergens, and other environmental agents are frequently encountered. Thus, IL-33, as an epithelial alarmin, could play important roles in the response to tissue injury or infection. IL-33 is likely to be a very good alarm signal: it is constitutively expressed at high levels in tissues in vivo [this study, (20)]; it can be released after cellular injury or necrosis (23, 24); it is active as a full-length molecule that does not require maturation for biological activity (2325); and it has the capacity to activate many actors of the innate immune system (4, 5, 29). Mast cells (30) and type 2 innate immune cell populations, such as natural helper cells (13, 14), nuocytes (15), and innate helper 2 cells (16), are likely to play major roles in the response to the IL-33 alarm signal in vivo. Indeed, IL-33 signaling through the ST2 receptor was recently shown to play an important role in the activation of these type 2 innate immune cell populations, after helminth infection in the intestine (13, 15, 16) or influenza virus infection in the lungs (14, 17).

We previously proposed that IL-33, which is abundantly expressed in human blood vessels along the vascular tree (1, 2, 20, 21), may play important roles in immune surveillance in humans as an endothelial alarmin responsible for alerting the immune system of blood vessel damage (23). Surprisingly, we discovered that IL-33 is not constitutively expressed in endothelial cells from blood vessels in mouse tissues. Particularly striking was the absence of IL-33 in HEVs from mouse lymphoid organs, because we discovered IL-33/NF from HEVs based on its abundant expression in human HEVs (1, 2). These species-specific differences in IL-33 expression along the vascular tree indicate that the endothelial alarmin function of IL-33 may be absent in mice. Although constitutive expression of IL-33 in blood vessels was not detected in mouse tissues, we observed inducible expression of IL-33 in the nuclei of endothelial cells from the inflamed colon during DSS-induced colitis. Expression of IL-33 in blood vessels from inflamed mouse tissues was also reported in experimental atherosclerosis (31) and mouse liver fibrosis (32). Differences in the regulation of endothelial cell gene expression of IL-33 between humans (constitutive) and mice (inducible) will need to be carefully considered when extrapolating results obtained in mouse models to humans.

We found that the Il-33 promoter is highly active in the T cell areas from mouse lymphoid organs and that the IL-33 protein is constitutively expressed in the nuclei of α-SMA+ FRCs from lymph nodes and spleen. T-zone FRCs produce high levels of cytokine IL-7 (33) and chemokines CCL21 and CCL19 (34), which are essential for lymphocyte survival and migration, respectively. Our results indicate that T-zone FRCs also constitute the major cellular sources of IL-33 in mouse lymphoid organs. FRC-derived IL-33 may play important roles in the activation of nuocytes and innate helper 2 cells, which are found in lymph nodes and spleen (15, 16). Because IL-33 expression was not detected in fibroblasts from nonlymphoid tissues, the constitutive expression of IL-33 in T-zone FRCs is likely to be linked to their myofibroblastic features, including the expression of α-SMA, which is associated with increased generation of contractile forces (35). Interestingly, IL-33 expression was also reported in α-SMA+ myofibroblasts associated with tissue fibrosis and/or inflammatory diseases, including cardiac myofibroblasts in cardiac hypertrophy and fibrosis (36), activated hepatic stellate cells in fibrotic liver (32), pancreatic myofibroblasts in chronic pancreatitis (37, 38), and ulceration-associated myofibroblasts in ulcerative colitis (39, 40). Thus, α-SMA+ myofibroblasts may represent a major cellular source of IL-33 in vivo.

Our in situ observations suggest that IL-33 may play important roles in the eye and the brain. We found that endogenous IL-33 protein is abundantly expressed in the eye, in the nuclei of Sox2+ Müller glial cells in the retinal intergranular layer, and in epithelial cells from the ciliary body. We also observed high levels of Il-33 promoter activity in corpus callosum, hippocampus, thalamus, and cerebellum. Interestingly, the IL-33 gene has been identified as a candidate gene for Alzheimer’s disease (41), and expression of IL-33 was shown to be induced in glial cells from the brain (glial fibrillary acidic protein+ astrocytes) after treatment with pathogen-associated molecular patterns (LPS or dsRNA) (42). IL-33 may play critical roles in innate immune responses in the brain and the eye, and it will be important to further characterize these roles in future studies.

In conclusion, we believe that the Il-33–LacZ Gt reporter strain is a useful tool to characterize the cellular sources of endogenous IL-33 in vivo during homeostasis and inflammation. Our data indicate that epithelial cells from barrier tissues constitute the major cellular sources of IL-33 in normal mouse tissues. Myofibroblasts and endothelial cells may also be important sources of IL-33 in inflamed mouse tissues. Other cell types have been proposed to produce high levels of IL-33 in inflamed tissues, including alveolar macrophages (14), and the Il-33–LacZ Gt reporter strain will represent an important tool to validate and determine the relative importance of these additional cellular sources of IL-33 during inflammation. In our analyses, we observed induction of the Il-33–LacZ reporter in the liver during LPS-induced endotoxin shock but no upregulation in the lung during papain-induced airway inflammation. This observation, which is likely explained by the fact that expression of endogenous IL-33 in the lung alveoli is already very high under basal conditions, suggests that papain may act by modulating IL-33 release or bioactivity rather than IL-33 expression in the inflamed airways. The Il-33–LacZ Gt reporter strain will also be very useful to investigate the localization of endogenous IL-33 protein in vivo. For instance, although cytoplasmic expression of IL-33 protein was reported in some studies (43), we observed that cytoplasmic staining with anti–IL-33 Abs in the inflamed colon during DSS-induced colitis was still present in the Il-33–LacZ Gt reporter strain, which is Il-33 deficient.

The fundamental mechanisms of IL-33 synthesis, localization, and release during inflammation or infection remain to be fully characterized, and we are confident that the Il-33–LacZ Gt reporter strain will be a critical tool to answer these important questions.

We thank the Anexplo-Institut de Pharmacologie et de Biologie Structurale zootechny, transgenesis, and histology facilities. We are grateful to the Texas A&M Institute for Genomic Medicine for providing ES cells, Dimitri Marsal for help with animal experiments, Elisabeth Bellard (Toulouse Réseau Imagerie-Institut de Pharmacologie et de Biologie Structurale) for help with imaging, and Marc Le Bert (Centre National de la Recherche Scientifique Orléans) for advice on ES cell manipulation.

This work was supported by grants from the Ligue Nationale Contre le Cancer (Equipe Labellisée Ligue 2009 to J.-P.G.) and the Association pour la Recherche sur le Cancer (Ph.D. fellowship to E.L.; Programme Association pour la Recherche sur le Cancer no. SL220110603471; Equipment Association pour la Recherche sur le Cancer no. ECL2010R00650).

The online version of this article contains supplemental material.

Abbreviations used in this article:

dpc

days postcoitum

DSS

dextran sulfate sodium

E

embryonic day

ES

embryonic stem

FRC

fibroblastic reticular cell

Gt

gene trap

HEV

high endothelial venules

α-SMA

α-smooth muscle actin

vWF

von Willebrand factor.

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The authors have no financial conflicts of interest.