IL-33 is constitutively expressed in the nucleus of epithelial cells in tissues exposed to the environment.

<p>A–C: Skin keratinocytes; D–F: Epithelial cells of the mucosal surface in the stomach; G–H: Epithelial cells of the gastric glands in the stomach; I: Epithelial cells of the salivary glands. Immunohistochemical and immunofluorescence staining were performed with IL-33 mAb Nessy-1. For immunofluorescence, IL-33 expression was detected in red and DNA was counter-stained with DAPI (blue). Magnification bars: 70 µm.</p

IL-33 is abundantly expressed in the nucleus of endothelial cells in human tumor tissues.

<p>Expression of IL-33 in the indicated human tumor tissues was analyzed using immunofluorescence staining. Double staining was performed with IL-33 mAb Nessy-1 (red) and anti-CD31 or anti-vWF polyclonal antibodies (green). DNA was counter-stained with DAPI. Magnification bars: A,B,G,H 50 µm; C,D,E,F 20 µm.</p

IL-33 is constitutively expressed in the nucleus of endothelial cells from small blood vessels in normal human tissues.

<p>Expression of IL-33 in the microvasculature was analyzed using immunofluorescence staining. Double staining was performed with IL-33 mAb Nessy-1 (red) and anti-CD31 or anti-vWF polyclonal antibodies (green). DNA was counter-stained with DAPI. Magnification bars: 20 µm.</p

IL-33 is a chromatin-associated nuclear factor constitutively expressed in human secondary lymphoid tissues by HEVs and isolated cells in the interfollicular T cells areas.

<p>A: Immunohistochemical staining of a human tonsil section with IL-33 mAb Nessy-1. B–D: Double staining of human tonsils sections with HEV-specific mAb MECA79 (green) and IL-33 mAb Nessy-1 (B, red) or two distinct IL-33 polyclonal antisera, Cter1 (C, red) or Cter2 (D, red). The follicles (fol) are indicated. E and F: Nuclear staining of HEVs and isolated cells in the T cell areas was abrogated by pre-incubating the IL-33 monoclonal (E) and polyclonal (F) antibodies with IL-33 peptides but not control peptides. G and H: Nuclear accumulation of IL-33 in HEV blood vessels and isolated cells was also observed in lymph node (G, IL-33 mAb, red; CD31, green) and appendix (H, IL-33 mAb, red; vWF, green). I and J: Higher magnification of a human tonsil section double-stained with IL-33 and MECA-79 antibodies and counterstained with the DNA-binding dye DAPI. In the nucleus of both HEVs (arrow, upper panel) and isolated cells (arrowhead, lower panel), IL-33 accumulates in nuclear domains that colocalize with dense regions of DAPI staining (J), indicating association with chromatin. Magnification bars: A, I 10 µm; B, G, H 20 µm; J 5 µm; C, D, E, F 60 µm.</p

The Alarmin Concept Applied to Human Renal Transplantation: Evidence for a Differential Implication of HMGB1 and IL-33

<div><p>The endogenous molecules high mobility group box 1 (HMGB1) and interleukin-33 (IL-33) have been identified as alarmins, capable of mediating danger signals during tissue damage. Here, we address their possible role as innate-immune mediators in ischemia-reperfusion injury (IRI) following human kidney transplantation. We analysed serum and urinary HMGB1 and IL-33 levels, all determined by enzyme-linked immunosorbent assay, in a cohort of 26 deceased renal transplant recipients. Urinary HMGB1 and IL-33 levels were significantly increased as soon as 30 min after reperfusion, as compared to those before treatment. Moreover, both serum and urinary IL-33 (but not HMGB1) increase was positively correlated with cold ischemia time, from 30 min to 3 days post-transplantation. <i>In vitro</i>, human umbilical vein endothelial cells subjected to hypoxia conditions released both HMGB-1 and IL-33, while only the latter was further increased upon subsequent re-oxygenation. Finally, we postulate that leukocytes from renal recipient patients are targeted by both HMGB1 and IL-33, as suggested by increased transcription of their respective receptors (TLR2/4 and ST2L) shortly after transplantation. Consistent with this view, we found that iNKT cells, an innate-like T cell subset involved in IRI and targeted by IL-33 but not by HMGB1 was activated 1 hour post-transplantation. Altogether, these results are in keeping with a potential role of IL-33 as an innate-immune mediator during kidney IRI in humans.</p></div

Hypoxia/re-oxygenation-induced release <i>in vitro</i> of HMGB1 and IL-33.

<p>Confluent (≈95%) monolayer HUVEC were exposed to sixteen hours hypothermia/hypoxia in UW solution (H16h) followed by 1 hour (R1h), or 3 hours (R3h) of re-oxygenation in a new culture medium (Medium 200) at 37°C in 20% O₂. Confluent (≈95%) monolayer HUVEC were used as controls (Ctl). Early release of HMGB1 and IL-33 by HUVEC in response to in vitro hypoxia/re-oxygenation (A–B). HMGB1 (A) and IL-33 (B) in cell culture supernatants were quantified by ELISA. Increase of IL-33 but not HMGB1 mRNAs in HUVEC in response to <i>in vitro</i> hypoxia/re-oxygenation (C–D). Total RNA was extracted from monolayer HUVEC at the indicated time points and expression of HMGB1 (C) and IL-33 (D) mRNAs was quantified by RT-qPCR. Data are expressed as means ± SEM or of fold change relative to D0 and are representative of three separate experiments. *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> Ctl by Mann-Whitney test.</p

Correlation of serum and urinary alarmin levels with cold ischemia time.

<p>POD: Post Operative Day. The correlation coefficient (<i>r</i>) is calculated by the non-parametric Spearman’s rank correlation test. A p-value<0.05 was considered significant.</p

Early activation of iNKT cells after renal IRI: a potential role for IL-33.

<p>(A, B) PBMCs from kidney graft recipients were recovered before transplantation (D0), and 3 hours (H3) and day 3 (POD3) after transplantation. They were membrane-labelled with anti-CD3-FITC, anti-iNKT-PE 6B11 clonotype, and anti-CD69-PerCP/Cy5.5. CD69 analysis was performed by flow cytometry gating on CD3(+)6B11(+) cells, defined as iNKT cells: (A) Flow cytometry plot showing expression profiles of surface marker CD69 on iNKT cells <i>ex vivo</i> from one representative patient at D0 (filled histogram), H3 (bold line) and POD3 (dotted line). Numbers indicate MFI of CD69 expression on iNKT lymphocytes. (B) Mean Fluorescence Intensity (MFI) of CD69 expression on iNKT lymphocytes from the patient cohort (n = 16 at D0, H3, and POD3). (C, D) PBMCs from healthy adult donors (n = 6) were cultured with (black columns) or without (white columns) HMGB1(C) or IL-33 (D) for 3, 6 or 24 hours of culture. MFI of CD69 expression on iNKT lymphocytes was analysed by Flow cytometry as described in (A, B). Data are expressed as means ± SEM. **p<0.01, ***p<0.001 by Wilcoxon test.</p

Increased levels of HMGB1, IL-33 and sST2 in serum and urine shortly after renal IRI.

<p>HMGB1, IL-33 and sST2 levels were quantified by ELISA in serum and urine of kidney graft recipients (n = 26) before transplantation (D0) as control time, and 30 minutes (H0.5), 3 hours (H3), day 1 (POD1) and day 3 (POD3) after transplantation. Serum, urine and urinary molecule/creatinine ratio levels for HMGB1 (A–C), IL-33 (D–F) and sST2 (G–I). Note that serum samples from only 6 out of 26 transplanted patients contained measurable amounts of IL-33. Data are expressed as means ± SEM. *p<0.05, **p<0.01, ***p<0.001 by Wilcoxon or Mann-Whitney test, as appropriate. ns, no significant.</p

Baseline demographic and clinical characteristics of recipients and donors.

(1)<p>BMI: Body Mass Index.</p>(2)<p>ECD: Expanded Criteria Donors.</p>(3)<p>DGF: Delayed Graft Function defined as a need for dialysis within the first week after transplantation.</p