QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 19 April 2005
doi: 10.1242/jcs.02316

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Habtezion, A. Articles by Omary, M. B. Search for Related Content PubMed PubMed Citation Articles by Habtezion, A. Articles by Omary, M. B.

Keratin-8-deficient mice develop chronic spontaneous Th2 colitis amenable to antibiotic treatment

¹ Department of Medicine, Palo Alto VA Medical Center, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304, USA
² Department of Pathology, Palo Alto VA Medical Center, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304, USA
³ Stanford University School of Medicine Digestive Disease Center, Stanford, CA 94305, USA

View larger version (90K): [in a new window]   Fig. 1. Immunohistochemical staining of K8+/+ and K8–/– colons with fluorescent antibodies to T cell surface markers; the original color staining is depicted in black and white. Frozen sections from 6-month-old K8+/+ and K8–/– colons were triple-stained with fluorophore-conjugated antibodies directed to: CD8 (PE, red), CD4 (FITC, green), and TCRß (APC, blue). A dramatic increase in the number of TCRß and CD4+ cells are present within the LP and mucosa of the K8–/– (d-f) as compared to the K8+/+ (a-c) colons. L, lumen; M, muscle layer. Scale bar: 50 µm.

View larger version (11K): [in a new window]   Fig. 2. Colon lamina propria lymphocytes stained with surface markers and analyzed by flow cytometry. LPL were isolated from K8+/+ and K8–/– colons, stained with the indicated markers then analyzed as described in Materials and Methods. Markedly higher numbers of TCRß and CD4+ T cells are recovered from the K8–/– compared to K8+/+ mouse colons in an age-dependent manner. The data is presented as mean cell number±s.e.m. (n=4). *P<0.05 when comparing K8–/– with K8+/+.

View larger version (96K): [in a new window]   Fig. 3. Colons from 2-week (wk), 4-wk and 6-month (mo)-old K8+/+ and K8–/– mice analyzed by histology and immunohistochemistry. (A) Hematoxylin and Eosin staining of colons from 2-wk and 4-wk-old K8–/– mice show increased areas of inflammation (d,f, arrows). Frozen colon sections from 2-wk-old K8+/+ and K8–/– mice were stained with anti-CD4 FITC (b,e). K8–/– colon LP shows increased CD4+ cells (e, arrows). L, lumen; M, muscle layer. Scale bar: 10 µm (a,c,d,f); 30 µm (b,e). (B) Frozen sections from 2-wk and 6-month-old K8+/+ and K8–/– colons were stained with anti-EPCAM (an epithelial cell marker, red), anti-I-A/I-E (MHC class II antigens, green), and a nucleus-staining dye (Toto-3, blue). Note the overexpression of MHC class II antigens (arrows) in colonocytes of K8–/– mice. Scale bar: 10 µm.

View larger version (21K): [in a new window]   Fig. 4. Cytokine production by K8+/+ and K8–/– colon LP cells. (A) Cells were stimulated with PMA/ionomycin and stained for surface CD4 and intracellular cytokines (IL-5, IL-10, TNF, IL-4, IFN or IL-13) or isotype control antibodies. Samples were analyzed by FACS with gating on CD4+ T cells. To confirm specificity of the IL-4 and IL-13 staining, stimulated cells were pre-incubated with excess unlabeled recombinant (r) cytokines (rIL-4 or rIL-13). The frequency of cytokine-producing CD4+ T cells is indicated in the quadrants as percentages (e.g. 1% for IL-5 and 1.8% for IL-10 for K8+/+ CD4+ T cells). (B) Summary of the result shown in A. CD4+ T cells from K8–/– colons produced higher Th2 cytokines (IL-5, IL-4, and IL-13), and IL-10. Data is presented as mean±s.e.m. (n=4). (C) Cytokine production by K8+/+ and K8–/– colon LP cell culture. Cells were cultured in plates coated with anti-CD3 and soluble anti-CD28 (Stimulated) or in the presence of medium alone (Unstimulated) for 48 hours. Culture supernatants from the stimulated cells were analyzed by ELISA for IFN, TNF, IL-10, IL-5 and IL-4 production. Note that K8–/– cultures produced higher levels of Th2 cytokines. Data are presented as mean±s.e.m. (n=3). *P<0.05 when comparing K8–/– with K8+/+.

View larger version (115K): [in a new window]   Fig. 5. Expression of 4ß7 and endothelial markers by K8+/+ and K8–/– colons. (A) Frozen sections from 3-month-old K8+/+ (a) and K8–/– (b) colons were stained with anti-4ß7. Sections from 6-month-old K8+/+ (c,e) and K8–/– (d,f) colons were stained with anti-MAdCAM-1. Increased 4ß7+ cells (b) and MAdCAM-1+ venules (d,f, arrows) were seen in K8–/– colons. L, lumen; M, muscle layer. Scale bars: (a-d) 50 µm; (e,f) 30 µm. (B) Colons from K8+/+ (a,c) and K8–/– (b,d) mice were double-stained for the endothelial markers anti-PECAM/CD31 (green) and the vascular adhesion molecule PNAd (red). Merged images are shown in e and f. Inserts show a magnified view of a positive double-stained venule. Note the aberrant expression of PNAd in K8-null colon (compare c and d). Arrows in f indicate areas of co-localization. Scale bar: 50 µm.

View larger version (83K): [in a new window]   Fig. 6. K8–/– colon histology following treatment with broad-spectrum oral antibiotics. (A) Hematoxylin and Eosin staining of proximal colons from K8–/– mice that were given normal drinking water for 8 weeks (a) or water containing vancomycin and imipenem at 50 mg/kg body weight (b). Significant inflammation (arrows) is present in the non-treated K8–/– colon. L, lumen; M, muscle layer. Scale bar: 10 µm, (B) Summary of the colon inflammation histology score (as described in Materials and Methods) is shown for the non-treated (–Antibiotics) and treated (+Antibiotics) groups. Data is presented as mean±s.e.m. (n=4). *P<0.005 when comparing untreated K8–/– with K8+/+, **P<0.01 when comparing untreated with treated K8–/–.

View larger version (68K): [in a new window]   Fig. 7. Effect of oral antibiotic treatment on AE1/2 ion transporter localization. Proximal colon from non-antibiotic-treated K8+/+ (a), K8–/– (b), and antibiotic-treated K8–/– (c) mice were fixed in formalin, paraffin embedded and sectioned, then double-stained with anti-AE1/2 (red) and nuclear dye (blue). Note the reversal and normalization of the brighter and supranuclear AE1/2 staining in K8–/– colon (b, arrows) following antibiotic treatment (c), to resemble the staining of non-antibiotic treated K8+/+ colon (a). Scale bar: 50 µm.

View larger version (18K): [in a new window]   Fig. 8. A model depicting the effects of mechanical and non-mechanical stresses on intestinal epithelial cells, depending on the presence or absence of keratin intermediate filaments.