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Comparative Study
. 2000 Aug 1;19(15):4046-55.
doi: 10.1093/emboj/19.15.4046.

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family

I N Holcomb  1 , R C Kabakoff, B Chan, T W Baker, A Gurney, W Henzel, C Nelson, H B Lowman, B D Wright, N J Skelton, G D Frantz, D B Tumas, F V Peale Jr, D L Shelton, C C Hébert
Affiliations
Comparative Study

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family

I N Holcomb et al. EMBO J. .

Abstract

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.

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Figures

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Fig. 1. SDS–acrylamide gel analysis of BALF. Equal volumes (10 µl) of BALF from control mice and BALF obtained from mice with OVA-induced allergic pulmonary inflammation were analyzed under reducing conditions by SDS–PAGE on a Tricine-buffered 16% acrylamide gel. BALF from mice with allergic pulmonary inflammation (lane 2) contains a unique band, co-migrating with an 8.3 kDa molecular weight marker (IL-8, 50 ng, lane 3), which is not present in BALF from control mice (lane 1).
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Fig. 2. Sequences of the FIZZ protein family. (A) The amino acid sequences of murine and human FIZZ proteins. The consensus sequence (Cons.) indicates the position of the conserved residues. Underlined residues represent predicted signal peptide sequences. The corresponding nucleotide sequences are entered in the DDBJ/EMBL/GenBank databases under the following accession Nos: mFIZZ1, AF205951; mFIZZ2, EST AA245405; mFIZZ3, EST W42069; hFIZZ1, EST AA524300; hFIZZ3, AF205952. (B) Amino acid identity (upper right) and homology (lower left) for the five members of the FIZZ gene family (based on PAM250 matrix).
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Fig. 3. mFIZZ1 northern blot. A Clontech adult mouse multiple tissue northern blot was probed with an oligonucleotide corresponding to nucleotides 176–225 of the full-length mFIZZ1 sequence, and exposed for 16 h or 5 days as shown. The same blot, stripped and reprobed with an oligonucleotide for β-actin, and exposed for 7 days, is shown at the bottom of the figure.
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Fig. 4. In situ hybridization of mFIZZ 1 in adult mouse lung. A 33P-labeled mFIZZ1 riboprobe detected patchy expression in bronchial epithelium of control (OVA-challenged, non-immunized mouse) lung after a 4-week exposure (A, C and E). In inflamed lung, a 2-week exposure with the same probe detected diffuse strong expression in bronchial epithelium (B, D and F) and type II pneumocytes (arrows, H and J), while alveolar macrophages (arrowheads, H and J) were negative. Murine FIZZ1 expression was also present in discrete cells in neurovascular bundles in peribronchial interstitium (arrowheads, C, G and I). Dark-field images: A–D, G and H. Corresponding bright-field images: E, F, I and J. Scale bars represent 500 µm (A and B), 50 µm (C–F) or 25 µm (G–J). ar, artery; br, bronchiole; alv, alveolar space.
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Fig. 5. Western blot analysis of BALF and lung homogenates from mice with allergic pulmonary inflammation. Lane 1, BALF sample resolved under reducing conditions and analyzed with pre-immune serum; lane 2, BALF sample resolved under reducing conditions and analyzed with a rabbit anti-FIZZ1 peptide antiserum. Whole-lung homogenate (Hom.) resolved under reducing (lane 3) or non-reducing conditions (lane 4) and analyzed with the same rabbit antiserum. The migration of molecular size markers (kDa) is indicated on the left.
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Fig. 6. Immunohistochemical detection of mFIZZ1 protein in inflamed and control lung. (A and C) Murine FIZZ1 expression in control (OVA-challenged, non-immunized mouse) lung is limited to small patches of bronchial epithelial cells. (B and D) In the inflamed lungs of immunized, OVA-challenged mice, mFIZZ1 protein expression in bronchial epithelium is both more diffuse and more intense. In addition, expression is seen in alveolar epithelial cells with granular cytoplasm, consistent with type II pneumocytes (arrows, D). Alveolar macrophages (arrrowheads, D) and stromal cellular components stain reproducibly, but weakly with anti-FIZZ antibody, despite being negative for FIZZ mRNA by in situ hybridization (Figure 4H and J). Scale bars represent 100 µm (A and B), 10 µm (C) or 25 µm (D).
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Fig. 7. Expression of mFIZZ1 mRNA in colonic submucosal and peribronchial neurovascular bundles. (A and B) In situ hybridization of mFIZZ1 in colon shows strong expression in discrete cells (arrows) in the submucosa after a 4-week exposure. (C–F) Immunohistochemistry of FIZZ1 (C and E) and PGP9.5 (D and F) in serial sections of colonic submucosal (C and D) and peribronchial (E and F) tissue. mFIZZ1-positive cells (arrows) are adjacent to neuronal cell bodies and fibers (arrowheads). Similar results were obtained with antibodies to S-100 and NSE (not shown). Scale bars represent 50 µm (A and B) or 25 µm (C–F). mu, mucosa; sm, submucosa; mp, muscularis propria; ve, venule; ar, arteriole; ag, autonomic ganglion; br, bronchiole; pa, pulmonary artery.
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Fig. 8. Expression of mFIZZ2 and mFIZZ3 mRNA. (A–D) In situ hybridization of mFIZZ2 to a section of colon shows strong signal in crypt epithelium after a 2-week exposure. In (C and D), the ‘jelly roll’ orientation of the colon has artificially brought two segments of colon back-to-back; one segment (arrowhead) is intensely positive for FIZZ2 expression, while the other segment (arrow), several centimeters from the first, is negative. (E and F) In situ hybridization of mFIZZ3 to small bowel and mesentery (4-week exposure). Expression is limited to adipose tissue (arrow) in mesentery. (G and H) In situ hybridization of mFIZZ3 to peritracheal tissue (4-week exposure); expression is limited to adipose tissue (arrow) adjacent to thyroid gland (Th) and trachea (Tr). Dark-field images: A, C, E and G. Corresponding bright-field images: B, D, F and H. Scale bars represent 35 µm (A, B and E–H) or 100 µm (C and D). mu, mucosa; mp, muscularis propria.
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Fig. 9. FIZZ1 inhibits NGF-mediated neuronal survival and gene expression. (A) Surviving rat E14 DRG neurons were counted after 3 days in culture. Recombinant mFIZZ1 at 4 µg/ml inhibited neuronal survival without inducing non-neuronal cell toxicity. (B) NGF induces higher levels of CGRP immunoreactivity in neurons, and mFIZZ1 inhibits the NGF-induced increase in CGRP content. Open bars, no mFIZZ1; hatched bars, 0.4 µg/ml (∼40 nM) mFIZZ1; solid bars, 4 µg/ml (∼0.4 µM) mFIZZ1. mFIZZ1 at 4 µg/ml inhibits the increase in CGRP induced by 1 ng/ml NGF (p <0.002) or 10 ng/ml NGF (p <0.01).
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Fig. 10. SPR biosensor measurements of protein–protein binding interactions. mFizz1, NGF, trkA–IgG or an irrelevant IgG was immobilized on a biosensor chip. At time 0, mFizz1 (A) or NGF (B), each at 0.1 mg/ml, was injected and the response on all four immobilized proteins was recorded. The total response is shown in (A) whereas the net response (RU observed minus RU observed with the irrelevant IgG) is shown in (B).
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