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Neonatal Fc receptor for IgG regulates mucosal immune responses to cquired immunity
Research article Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria Masaru Yoshida,1 Kanna Kobayashi,1 Timothy T. Kuo,1 Lynn Bry,2 Jonathan N. Glickman,2 Steven M. Claypool,1 Arthur Kaser,1 Takashi Nagaishi,1 Darren E. Higgins,3 Emiko Mizoguchi,4 Yoshio Wakatsuki,5 Derry C. Roopenian,6 Atsushi Mizoguchi,4 Wayne I. Lencer,7,8 and Richard S. Blumberg1,8 1Gastroenterology Division, Department of Medicine, Brigham and Women's Hospital, 2Department of Pathology, Brigham and Women's Hospital, and 3Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA. 4Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA. 5Division of Clinical Bioregulatory Science, Kyoto University, Kyoto, Japan. 6The Jackson Laboratory, Bar Harbor, Maine, USA. 7Gastrointestinal Cell Biology, Department of Medicine, Children's Hospital, Boston, Massachusetts, USA. 8Harvard Digestive Disease Center, Boston, Massachusetts, USA. The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. Selective expression of FcRn in the epithelium is shown here to be associated with secretion of IgG into the lumen that allows for defense against an epithe- lium-associated pathogen (Citrobacter rodentium). This pathway of host resistance to a bacterial pathogen as mediated by FcRn involves retrieval of bacterial antigens from the lumen and initiation of adaptive immune responses in regional lymphoid structures. Epithelial-associated FcRn, through its ability to secrete and absorb IgG, may thus integrate luminal antigen encounters with systemic immune compartments and as such provide essential host defense and immunoregulatory functions at the mucosal surfaces. Introduction out life in the circulation through its expression on endothelia and Secretory Igs such as IgA, IgM, and IgG that are present in mucosal potentially other cell types (7). It has been recently appreciated that surfaces potentially provide a first line of defense against microor- significant levels of FcRn are also constitutively expressed by epithe- ganisms (1–3). Secretory IgA (sIgA) is well known to be transported lial cells throughout life in human intestine, lung, and kidney (8, 9). across epithelial cells into the lumen through an active unidirec- This is in contrast to expression of FcRn in the intestinal epithelia of tional process by the polymeric Ig receptor (pIgR) (4). In addition rodents, which is developmentally regulated — in that FcRn is highly to sIgA, significant quantities of IgG can also be secreted into the expressed at birth with a dramatic decrease of expression occurring intestinal lumen of adult humans and rodents. It has been reported after 2 weeks of life at the time of weaning. The recent identification that nasal secretions contain 300 mg/ml IgG (4), and approximate- of FcRn expression in numerous epithelial cell types in humans dur- ly 800 mg/ml IgG can be detected in the human rectum (5). Like ing adult life and more recently in other mammals such as nonhu- sIgA, which has been well documented as actively participating in man primates (10), together with the recognition that FcRn medi- the defense against some pathogens (3, 6), mucosally associated ates the bidirectional transport of IgG (from basolateral to apical IgG has also been recently suggested to contribute to host defense as well as from apical to basolateral surfaces) in vitro and in vivo (1, 2). However, despite all that is known about IgA transport and across epithelial barriers, has directed attention to other potential its relation to mucosal host defense, the role of intestinal luminal extensions of FcRn function in immunity beyond the passive trans- IgG in defending against enteric bacteria and the mechanisms by fer of IgG and the protection of IgG from catabolism (2, 8, 11–13). which this is achieved remains to be established. Specifically, this bidirectional transport of IgG confers a unique It has been previously shown that IgG can be transported across ability on FcRn to retrieve intestinal luminal antigens as a complex intact epithelial barriers through the placenta in humans and neo- with IgG and deposit them into the intestinal mucosa, where the natal intestine in rodents for the passive transfer of immunity from antigen/IgG complexes can be captured by DCs for subsequent mother to fetus or into the neonatal host, respectively. The receptor presentation to CD4+ T cells (2). These properties of FcRn define a responsible for mediating this transport is the neonatal Fc receptor unique mechanism by which absorptive epithelia, which covers the for IgG (FcRn), a b2-microglobulin–associated (b2m-associated), majority of the surface of the intestines, can specifically acquire and major histocompatibility complex class I–related molecule that is transport antigens into the lamina propria. Consistent with this, also responsible for the protection of IgG from catabolism through- recent studies have indicated that intestinal bacterial antigens are required to direct the maturation of immune responses (14) and that such immune responses are induced throughout the intestine Nonstandard abbreviations used: FcRn, neonatal Fc receptor for IgG; GALT, rather than within restricted regions such as Peyer's patches (15). gut-associated lymphoid tissue; IFABP, intestinal fatty acid–binding protein gene promoter; b2m, b2-microglobulin; m-, mouse; MLN, mesenteric lymph node. Therefore, these recent observations have raised a potential possibil- Conflict of interest: R.S. Blumberg and W.I. Lencer have an equity interest in Synto- ity that epithelial cell–mediated sampling of luminal bacterial anti- nix Pharmaceuticals Inc., which is developing FcRn-related therapeutics. gens throughout the intestinal surface contributes to the regulation Citation for this article: J. Clin. Invest. 116:2142–2151 (2006). doi:10.1172/JCI27821. of mucosal and systemic immune responses. 2142 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 research article Figure 1 Absence of intestinal luminal IgG in FcRn–/– mice. (A) The construct for the Tg IFABP-mFcRnTg/mb2mTg mouse, designed to express mFcRn and mb2m under the control of the IFABP. (B) Increased mFcRn expression in epithelial cells of IFABP-mFcRnTg/mb2mTg (Tg) mouse. RNA was extracted from epithelial cells of upper and lower small intestines (USI and LSI, respectively) and cecum in 6-week-old IFABP-mFcRnTg/ mb2mTg founder BALB/c mice and littermate WT BALB/c mice and subjected to RT-PCR. (C and D) Immunohistochemical analysis of lower small intestine in WT (C) and IFABP-mFcRnTg/mb2mTg mice (D). Arrows indicate staining of FcRn. (E–H) The levels of Igs secreted into the intestinal lumen. Secretory IgM (E), dimeric IgA (F), IgG1 (G), and IgG2a (H) were measured by ELISA. The mean ± SD are shown for each group (n = 8). *P < 0.05. (I) The levels of Igs (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, and IgE) secreted into the lumen of the indicated mouse strains on a C57BL/6 background were measured by a cytometric bead array. We therefore investigated the role of FcRn within the intestinal the eradication of this pathogen through a pathway that involves epithelia in host defense by examining the response to Citrobacter its transport functions and ability to direct antigens to inductive rodentium. This bacterium is normally restricted in its localiza- sites associated with mucosal tissues. tion to the epithelium, and its eradication is highly dependent upon CD4+ T cell responses and IgG (16–18). The separation of Results the antigens associated with this bacterium within the epithelium Establishment of intestine-specific mouse FcRn Tg mice. To study the from the other mucosal and systemic tissues implies that eradica- role of mouse FcRn (mFcRn) in intestinal epithelium in medi- tion of this organism must require an immunologic pathway that ating antimicrobial immunity, since the expression of FcRn has integrates and regulates the function of these compartments. We been shown to decrease at the time of weaning in rodents (19), therefore reasoned that FcRn provides this integrating function by we established FcRn Tg mouse lines in which mFcRn and mb2m virtue of its transport properties and, in so doing, would confirm a were specifically expressed by intestinal epithelial cells (Figure 1A) unique role for FcRn in host defense. To confirm this hypothesis, using the intestinal fatty acid–binding protein gene promoter we generated a mouse model in which FcRn is expressed consti- (IFABP; ref. 20) to create IFABP-mFcRnTg/mb2mTg mice (20). tutively and solely by intestinal epithelial cells in adult life. Using We expressed b2m in this manner to ensure that it was not sub- this mouse model we dissected the biological roles of intestinal strate limiting in expression of the FcRn transgene. Two founder epithelial cell–associated FcRn during infection with an epithelial lines that expressed FcRn in the epithelium at the highest levels cell–associated pathogen and showed that FcRn participated in were selected in this study. RT-PCR analysis showed that the Tg The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 2143 research article Figure 2 IgG transport into the lumen of IFABP-mFcRnTg/mb2mTg/FcRn–/– mice. (A) Quantitative PCR of cDNA using PCR primers originating in exon 2 in variety of tissues in IFABP-mFcRnTg/mb2mTg/FcRn–/– (Tg/FcRn–/–) mice. IEC, intestinal epithelial cells. (B) Serum rabbit IgG levels in IFABP-mFcRnTg/mb2mTg/FcRn–/– (black bars) and lit- termate control FcRn–/– mice (white bars) at 24, 48, and 120 hours after injection of rabbit IgG. (C) Serum rabbit IgG levels in IFABP- mFcRnTg/mb2mTg/FcRn–/– mice measured at 12 hours after injection of rabbit IgG (n = 4). (D) Secretory rabbit IgG levels in feces of IFABP- mFcRnTg/mb2mTg/FcRn–/– mice measured at 12 hours after injection of rabbit IgG as ng per mg of feces (n = 4). *P < 0.05. termate controls and FcRn–/– mice on the same genetic background (Figure 1I). No IgG was detected in the feces of FcRn–/– C57BL/6 mice, whereas low levels of IgGs were detectable in the feces of WT control C57BL/6 mice, which express low levels of FcRn in the epi- thelium (Figure 1B). This latter observation is notable because our studies indicate that FcRn expression (Figure 1B) and function (Figure 1I) are not completely extinguished in adult life, but are just markedly diminished relative to neonatal rodents. This is consistent with previous predictions by Ward and colleagues (19) that FcRn is decreased 1,000-fold in adult mice after weaning relative to neonatal mice, which express extremely high levels of FcRn for passive acquisi- tion of IgG from maternal milk. More significantly, increased quanti- ties of secretory IgG1, IgG2b, and IgG3 were detected in the feces of IFABP-mFcRnTg/mb2mTg C57BL/6 mice, in which intestinal epi- thelial FcRn expression is enhanced compared with WT and especial- ly FcRn–/– mice (Figure 1I, top panels). In contrast, after the culture of mesenteric lymph node (MLN) cells from these mouse groups with- out any in vitro stimulation for 2 hours at 37°C, similar levels of IgGs were detected in the culture medium. This indicates that the absence of intestinal IgG secretion in FcRn–/– mice was not due to decreased lines most strongly expressed FcRn in epithelial cells of the upper IgG production (Figure 1I, bottom panels). The absence of detect- and lower intestine and cecum at 6 weeks of age compared with able IgG2a was due to the genetic background of the mice examined, control BALB/c mice, which weakly expressed mFcRn (Figure 1B). C57BL/6, as these mice do not express this isotype of IgG. In contrast, the levels of mb2m expression were indistinguishable Given the low-level expression of FcRn observed in intestinal between Tg and WT mice. These findings are consistent with a epithelia of WT mice by RT-PCR (Figure 1B), IFABP-mFcRnTg/ decrease in FcRn, but not b2m, expression in mice after wean- mb2mTg mice were further backcrossed onto a FcRn –/– back- ing. To further confirm the RT-PCR results at the protein level, ground to generate IFABP-mFcRnTg/mb2mTg/FcRn–/– mice in immunohistochemical analysis was also performed. An increase order to limit FcRn expression and function to the epithelium. in FcRn was detected in the epithelial cells of lower small intestine This would allow for comparison with mice in which FcRn expres- (Figure 1D) and cecum (data not shown) of IFABP-mFcRnTg/ sion was extinguished in the intestinal epithelium, as observed in mb2mTg mice compared with WT littermate controls, consistent FcRn–/– mice. Figure 2A shows an analysis of FcRn expression as with the activity of this promoter (Figure 1C) (20). These data indi- defined by quantitative PCR analysis of a variety of tissues from cate that IFABP-mFcRnTg/mb2mTg mice express increased FcRn IFABP-mFcRnTg/mb2mTg/FcRn–/– mice compared with WT and protein as well as mRNA in the epithelium of the small intestine FcRn–/– mice. As previously reported (20–22), FcRn expression in and cecum in adult mice relative to WT littermate controls. the IFABP-mFcRnTg/mb2mTg/FcRn–/– mice was mainly observed FcRn-mediated transport of IgG across the epithelial barrier. Since FcRn in the intestinal epithelium of the small intestine. In addition, sig- has been demonstrated to be involved in the secretion of IgG from nificant expression of FcRn was also observed in the ovaries and tissue spaces into the lumen through epithelial cells in a mouse bladders of IFABP-mFcRnTg/mb2mTg/FcRn–/– mice. Low-level expressing a human transgene under the control of its endogenous FcRn expression was detected in the colon, brains, lungs, livers, promoter (2), we next examined the secretory levels of Igs including kidneys, testes, and peripheral blood but not the spleens, uteri, IgA, IgM, and IgG within the intestinal lumen of IFABP-mFcRnTg/ bone marrow, thymi, or MLNs of IFABP-mFcRnTg/mb2mTg/ mb2mTg and WT littermate control BALB/c mice by ELISA. IFABP- FcRn–/– mice. Importantly, there was no detectable FcRn expression mFcRnTg/mb2mTg mice exhibited significant secretion of IgG1 in intestinal epithelia of FcRn–/– mice, as predicted (23), but low- and IgG2a, but not IgA or IgM, into the intestinal lumen (Figure 1, level FcRn was detected in WT mice, consistent with the RT-PCR E–H). This finding was further confirmed by an alternative approach results shown in Figure 1B. These studies show that Tg expression using a cytometric bead array with IFABP-mFcRnTg/mb2mTg mice of FcRn under the control of the IFABP reconstitutes FcRn expres- on a different genetic background (C57BL/6) compared with WT lit- sion levels in the intestinal epithelium in FcRn–/– mice. 2144 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 research article Figure 3 Susceptibility to C. rodentium infection in the presence of FcRn. (A and B) Susceptibility to infection with 1 × 109 CFU of C. rodentium in FcRn–/– BALB/c mice. (A) Body weight changes in FcRn–/– and FcRn+/– mice with C. rodentium infection. (B) CFU of C. rodentium in feces of FcRn–/– and FcRn+/– mice 21 days after infection. Mean ± SD are shown for each group (n = 6). (C–E) Susceptibility to infection with C. rodentium in FcRn–/– C57BL/6 mice. Survival rate (C) and body weight changes (D) in FcRn–/– and FcRn+/– mice with C. rodentium infection. (E) CFU of C. rodentium in feces of FcRn–/– and FcRn–/+ mice 21 days after infection. Mean ± SD are shown for each group (n = 8). (F) Immunohistochemical analysis of the colon to detect intimin in mice with C. rodentium infection. Colonic tissues were collected at day 7 from selected mice on a C57BL/6 background. Sections were stained for intimin using a polyclonal rabbit anti–C. rodentium intimin antibody (red) and nuclei (blue) and were examined by confocal microscopy. Magnification, ×400. Macroscopic findings (G) and the length of colon (H) in FcRn–/– and FcRn+/– C57BL/6 mice, uninfected or infected with C. rodentium, at 21 days after infection. (I) Histological findings of colon in FcRn–/– and littermate FcRn+/– C57BL/6 mice with or without C. rodentium infection (21 days after infection). Magnification, ×100. (J) Histological score of colonic tissue in the mice with or without C. rodentium infection at day 21. *P < 0.05. The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 2145 research article Figure 4 Induction of immune response to C. rodentium–derived antigen in the presence of circulating IgG and FcRn in intestinal epithelial cells. (A and B) The effect of FcRn-mediated IgG transport into the intestinal lumen in C. rodentium infection. Body weight changes (A) and CFU of C. rodentium in feces 21 days after infection (B) in IFABP-mFcRnTg/mb2mTg/FcRn–/– and FcRn–/– C57BL/6 mice with i.v. injection of anti–C. rodentium IgG or control IgG. Mean ± SD are shown for each group (n = 6). (C) Establishment of a genetically engineered C. rodentium strain that constitutively produces an OVA fragment. The immunoblot confirms the expression of the OVA fragment by C. rodentium. (D) Summary of the experimental pro- tocol, with the inoculation of C. rodentium–OVA or control bacteria, the injection of anti–C. rodentium IgG or control IgG, and the adoptive transfer of CD45.1+CD4+OVA-specific T cells from CD45.1+OT-II mice. (E) The number of OVA-specific CD4+ T cells in the MLNs in IFABP-mFcRnTg/ mb2mTg/FcRn–/– and FcRn–/– mice increased in the presence of anti–C. rodentium IgG or control IgG (n = 3). Arrows indicate increasing rounds of cell division. (F and G) Cytokine production in OVA-specific CD4+ T cells purified from the MLNs and cultured with OVA for 48 hours in vitro. Cytokine production of IFN-g (F) and IL-4 (G) was measured by ELISA. Mean ± SD are shown for each group (n = 4). *P < 0.05. We next directly examined whether FcRn in intestinal epithe- lized differently in the presence of FcRn expression within the intes- lial cells transports IgG into mucosal secretions, as we previously tinal epithelium of FcRn–/– mice by virtue of FcRn expression driven showed using human FcRn Tg mice (2). To determine whether i.v. by the IFABP. These studies showed that the rabbit IgG concentra- injected IgG could be transported into the lumen across the epi- tions were identical in the IFABP-mFcRnTg/mb2mTg/FcRn–/– and thelial barrier by FcRn-dependent transcytosis, IFABP-mFcRnTg/ FcRn–/– mice over this time period, suggesting that the expression mb2mTg/FcRn–/– mice were examined for their ability to transmit of FcRn under control of the IFABP contributes little to protect- rabbit IgG into the lumen. Since mFcRn can bind rabbit IgG as well ing IgG from catabolism (Figure 2B). Therefore, rabbit IgG was as mouse IgG (24), rabbit IgG was injected i.v. into IFABP-mFcRnTg/ injected i.v. into IFABP-mFcRnTg/mb2mTg/FcRn–/– and FcRn–/– mb2mTg/FcRn–/– or littermate control FcRn–/– mice, and the levels mice, and IgG levels were examined at 12 hours after injection. of IgG in the serum and feces were examined. In the first group of Although there were no differences in the levels of rabbit IgG in the studies, rabbit IgG was examined in the serum after i.v. injection sera at this time point (Figure 2C), rabbit IgG was detected at sig- over a period of 120 hours to determine whether IgG was catabo- nificantly higher levels in the feces of IFABP-mFcRnTg/mb2mTg/ 2146 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 research article Figure 5 Luminal bacterial antigens transported as an immune complex across intestinal epithelial cells via FcRn in vivo. (A and B) Flow cytom- etry of rabbit anti–E. coli IgG and control IgG followed by PE-conjugated goat anti-rabbit IgG (A) and FITC-conjugated E. coli (B). (C–F) Con- focal microscopy analysis of transport of bacte- rial antigens across intestinal epithelial cells. Sections were stained for actin (phalloidin; red) and nuclei (blue). Magnification, ×400. Arrow in F illustrates transported FITC-conjugated E. coli in intestinal epithelial cells in the pres- ence of rabbit anti–E. coli IgG. (G) Presence of FITC-conjugated E. coli in CD11c+ cells of the MLNs of IFABP-mFcRnTg/mb2mTg/FcRn–/– mice 5 hours after FITC-conjugated E. coli admin- istration in the presence of rabbit anti–E. coli IgG. Mean fluorescence intensity (MFI) on gated CD11c+ cells is shown. The mean ± SD was shown for each group (n = 4). *P < 0.05. FcRn–/– mice compared with littermate FcRn–/– mice (Figure 2D). successfully cleared C. rodentium from their feces by 6 weeks after These results indicate that FcRn expression solely in intestinal epi- inoculation (data not shown). Increased amounts of C. rodentium thelial cells is involved in the transport of IgG into mucosal secre- were also detected within epithelial and subepithelial tissues of tions and is the major means by which IgG enters the lumen. colon in FcRn–/– mice at 7 days after infection (Figure 3F, right FcRn-deficient mice are susceptible to infection with an epithelial cell– panel), without evidence of C. rodentium in the MLNs and spleen specific pathogen, C. rodentium. C. rodentium is a bacterial pathogen (data not shown). In contrast, C. rodentium infection was limited that causes a murine infectious colitis equivalent to enterohemor- to the surface of the colonic intestinal epithelium in FcRn+/– mice rhagic and enteropathogenic Escherichia coli infection in humans. (Figure 3F, middle panel). No intimin staining was observed in the This pathogen causes a primary infection that is associated with uninfected colon (Figure 3F, left panel). At the same time, there the apical surface of the gut epithelium. Importantly, CD4+ T cells, were no detectable differences in C. rodentium quantities in the B cells, and IgG, but not secretory IgA or IgM, have been shown feces at day 7 (FcRn–/–, 3.4 × 108 ± 9.2 × 107 CFU/mg feces; FcRn+/–, to play a critical role in prevention against this infection (16–18). 3.2 × 108 ± 5.9 × 107 CFU/mg feces; n = 5; P = 0.67). This suggests Therefore, to test the pathophysiological role of FcRn-mediated that one of the earliest events to occur in the FcRn-deficient state IgG secretion into intestinal lumen in the C. rodentium infection, is penetration of C. rodentium into the epithelial and subepithelial FcRn–/– and littermate control (FcRn+/–) mice were orally inocu- tissues. Notably in this regard, FcRn+/– mice, but not FcRn–/– mice, lated with 1 × 109 of C. rodentium at 3 weeks of age. To exclude contained significant levels of endogenous IgGs in the serum that the possibility that the pathological changes in C. rodentium infec- could react serologically with C. rodentium before infection and at tion was directly related to the absence of FcRn and not to other day 7 (Supplemental Figure 1; available online with this article; alterations imposed by the knockout mouse model, both BALB/c doi:10.1172/JCI27821DS1). These endogenous IgGs in naive ani- and C57BL/6 background strains were used in this study. Previ- mals, which presumably heterologously bind to C. rodentium but ous studies have shown that both strains of mice exhibit differing are directed at other bacteria, may be important to managing sensitivities to C. rodentium infection, with greater clinical evidence C. rodentium within epithelial and subepithelial tissues in the pres- of disease and a fecal burden of bacteria in C57BL/6 mice com- ence of FcRn expression within the epithelium. pared with BALB/c mice (25). Interestingly, FcRn–/– mice exhibited At later time points (Supplemental Figure 1), a significant more body weight loss (Figure 3, A and D) and higher bacterial increase in presumably specific anti–C. rodentium IgGs was concentrations in the feces at 21 days after infection (Figure 3, detected in the serum of FcRn+/– mice at levels higher than those B and E) than did FcRn+/– mice, regardless of the genetic back- observed in FcRn–/– mice. This diminished serologic response in grounds, with more severe disease and increased levels of fecal bac- FcRn–/– mice, in association with an inability to secrete IgGs into teria in C57BL/6 mice as previously noted (25). Consistent with the epithelium and lumen in the absence of FcRn expression in the this finding, FcRn–/– C57BL/6 mice receiving C. rodentium orally epithelium, presumably limited the ability of FcRn-deficient mice demonstrated a decreased median survival rate, with only 75% of to properly manage C. rodentium infection. Indeed, macroscopic mice surviving at 30 days (Figure 3C), although the surviving mice and microscopic injury was greater in FcRn–/– than in FcRn+/– mice The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 2147 research article at day 21 after infection. The colons of FcRn–/– mice were charac- These results indicate that the OVA fragment produced by C. terized by severe shortening and thickening compared with those rodentium can be recognized by OVA-specific CD4+ T cells. To test of FcRn+/– mice (Figure 3, G and H). Consistent with these mac- whether FcRn-mediated delivery of IgG to host cells is required for roscopic changes, FcRn–/– C57BL/6 mice that were infected with the induction of immune responses to pathogens in vivo, IFABP- C. rodentium exhibited increased mononuclear cell and neutrophil mFcRnTg/mb2mTg/FcRn–/– and FcRn–/– mice were infected with infiltration into the tissues and significantly increased epithelial C. rodentium–OVA at 3 weeks of age. Anti–C. rodentium IgG or con- injury compared with that observed in FcRn+/– mice (Figure 3I). A trol IgG was injected at day 4 after infection, followed by adop- quantitative evaluation of histological findings also confirmed the tive transfer of CFSE-labeled OT-II T cells at day 5. Mononuclear increased injury in the FcRn–/– mice (Figure 3J). These results indi- cells from the MLNs were isolated at day 7 and examined by flow cate that FcRn–/– mice, which show an absence of FcRn expression cytometry (Figure 4D). Injection of anti–C. rodentium IgG led to a in the intestinal epithelium and intestinal luminal IgG (Figure 1I), significant increase in the number of CD4+ OT-II cells, as demon- are more susceptible to C. rodentium–induced colitis. strated by the multiple cell divisions in CFSE-loaded OVA-specific Anti–C. rodentium IgG improves infection via mFcRn when expressed CD4+ T cells in the MLNs (Figure 4E, arrows in right panel) of the on intestinal epithelial cells. The above studies show that defense IFABP-mFcRnTg/mb2mTg/FcRn–/– mice infected with C. roden- against an epithelial-associated pathogen is dependent upon FcRn tium–OVA. In contrast, even in the presence of C. rodentium–OVA expression. Since FcRn-deficient mice lack both FcRn expression infection, no significant increase in the number of OVA-specific in epithelia and high levels of serum IgG due to a lack of FcRn CD4+ T cells was detected in the MLNs of either IFABP-mFcRnTg/ protection function, it was possible that the inability of these mice mb2mTg/FcRn–/– mice that received control IgG or FcRn–/– mice to resist C. rodentium infection was due to either or both of these that received anti–C. rodentium IgG. Furthermore, injection of deficiencies. Therefore, to test for the role of IgG and/or FcRn anti–C. rodentium IgG into IFABP-mFcRnTg/mb2mTg/FcRn–/– expression in epithelia, IFABP-mFcRnTg/mb2mTg/FcRn–/– mice mice infected with C. rodentium–OVA led to increased production were compared with FcRn–/– mice for their ability to eradicate of IFN-g (Figure 4F) and IL-4 (Figure 4G) by MLN cells after in C. rodentium in the presence and absence of anti–C. rodentium IgG. vitro OVA stimulation. These results indicate that epithelial FcRn To test whether specific IgG for bacteria is required for the regula- can induce effective CD4+ T cell responses systemically to patho- tion of C. rodentium infection through FcRn-mediated transport gen-derived antigens associated with the lumen and/or intestinal of IgG, C. rodentium was orally inoculated into 3-week-old IFABP- epithelium when they are retrieved as antigen/IgG complexes. mFcRnTg/mb2mTg/FcRn–/– and FcRn–/– mice that had received Intestinal bacterial antigens can be transported from the lumen as an i.v. administration of either a polyclonal anti–C. rodentium anti- immune complex into the lamina propria by mFcRn in vivo and received body or a nonimmune control antibody. Whereas the control by CD11c+ cells. Since normal enteric bacteria have recently been antibody had no effect on C. rodentium eradication in FcRn–/– or Tg demonstrated to direct the maturation of the host immune mice, the preadministration of specific antibody resulted in less system (14), we next established a model system to determine body weight loss (Figure 4A) and lower bacterial concentrations in whether antibody in the serum could retrieve a physiologic bacte- the feces (Figure 4B) in IFABP-mFcRnTg/mb2mTg/FcRn–/– mice rial antigen from the lumen into mucosal tissues when FcRn was compared with FcRn–/– mice. Decreased quantities of C. rodentium expressed in the intestinal epithelium. To do so, rabbit anti–E. coli were also detected within the epithelial and subepithelial tissues IgG (Figure 5A) or control IgG was injected i.v. into IFABP-mFcRn- of mice in the presence of both FcRn expression in epithelia and Tg/mb2mTg/FcRn–/– mice or FcRn–/– mice, and FITC-labeled anti–C. rodentium IgG, as defined by immunohistochemistry (data E. coli (Figure 5B) was orally administered 12 hours after the i.v. not shown). These results indicate that FcRn expression in intes- injection. Two hours after oral administration, a FITC signal was tinal epithelial cells contributes to protecting against C. rodentium detected in epithelial cells in the small intestine of the IFABP- infection, but only in the presence of specific IgG antibodies. mFcRnTg/mb2mTg/FcRn–/– mice in the presence of anti–E. coli Antibacterial IgG affects antigen-specific CD4+ T cell responses to C. IgG (Figure 5F), but not in the presence of control IgG (Figure 5E) rodentium when FcRn is expressed in intestinal epithelial cells. Both or in the absence of FcRn (Figure 5, C and D). To examine whether innate and acquired immune responses are involved in the patho- the bacterial antigen/IgG complexes that were transported across genesis of infectious colitis (16–18). We have recently demonstrat- epithelial cells by FcRn were taken up by DCs in gut-associated ed that FcRn can retrieve luminal IgG/oral antigen (OVA) com- lymphoid tissue (GALT), as we have previously shown (2), cells plexes into the intestinal mucosa, which activate antigen-specific were obtained from the MLNs 4 hours after the oral inoculation of immune responses systemically by transport of immune com- FITC–E. coli and examined by flow cytometry. FITC–E. coli uptake plexes to regional lymphatic tissues via DCs. To examine whether by DCs in the MLNs was evident, as demonstrated by increased FcRn also plays a role in infection-induced acquired immune mean fluorescence intensity in the CD11c+ population within the responses by delivering bacteria-derived antigens coupled to spe- MLNs obtained from IFABP-mFcRnTg/mb2mTg/FcRn–/– mice in cific IgG into mucosal immune cells, a genetically engineered C. the presence of anti–E. coli IgG (Figure 5G). In contrast, the mean rodentium strain was created that constitutively expresses an OVA fluorescence intensity in CD11c+ cells from the MLNs of FcRn–/– fragment (residues 139–386 of chicken OVA) containing the pep- mice that received anti–E. coli IgG was not significantly different tides recognized by OT-II Tg T cells (residues 323–339) (C. roden- from that observed in the CD11c+ cells from FcRn–/– mice that tium–OVA) (see Methods). Immunoblot analysis confirmed the received control IgG (Figure 5G). No evidence of E. coli was evident expression of OVA in the cell sonicates of C. rodentium–OVA but in the MLNs, indicating that the fluorescence signal identified was not control C. rodentium (Figure 4C). The bacterial sonicates from not due to the presence of live bacteria (data not shown). These C. rodentium–OVA were also able to stimulate OVA-specific T cells data indicate that FcRn in the epithelium delivers luminal bacte- from OT-II mice in in vitro culture with mitomycin C–treated rial antigens to GALT-associated CD11c+ cells in the presence of antigen-presenting cells from C57BL/6 mice (data not shown). circulating antibacterial IgG. 2148 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 research article Discussion lial infections. Importantly, no increase in the number of CFSE- The recent recognition that FcRn can be expressed by mucosal epi- labeled CD4+ T cells was observed in FcRn Tg mice in the absence thelial cells in adult humans and that FcRn expression mediates of specific IgG, indicating an important role for FcRn-mediated an important physiological process that is characterized by bidi- antibody transport in regulating adaptive immune responses. rectional transport of IgG (11, 12) has led to the notion that the In addition to infection, these results may have important in vivo function of FcRn is broader than the simple acquisition of implications for the dysregulated host/microbial interactions passive immunity. This is further supported by the recent demon- that underlie the development of chronic intestinal inflammation stration that human FcRn, when expressed as a transgene in mice associated with inflammatory bowel disease. Inflammatory bowel under the control of its endogenous human promoter, can medi- disease is notable for the presence of highly activated pathogenic ate the transport of a model antigen, OVA, from the lumen (2). CD4+ T cell populations (27). An increase in the production of IgG This raises the possibility that a significant physiological function specific for luminal bacteria is also associated with the develop- for FcRn is in mediating intestinal transport of bacterial antigens, ment of experimental colitis (28, 29). The bacterial antigens, which given the intestine's constitutive exposure to a wide spectrum of are recognized by such IgGs, are often identical to the antigens enteric commensal and pathogenic bacteria. Therefore, this study that drive the pathogenic T cell responses, as was recently shown was designed to establish and test an appropriate animal model for bacterial flagellins (30). Therefore, it is also possible that FcRn- in order to define the role of intestinal epithelial cell–associated mediated retrieval of enteric bacterial antigens as IgG immune FcRn in directing immune responses toward epithelial- and lumi- complexes may be — in some situations — involved in the patho- nal-associated bacteria. To do so, we established what we believe genesis of chronic colitis by activating harmful acquired immune to be a novel mouse model (IFABP-mFcRnTg/mb2mTg/FcRn–/–), responses. Consistent with this, FcRn has recently been implicat- in which mFcRn expression was genetically engineered to be spe- ed in the pathogenesis of rheumatoid arthritis (31) and bullous cifically restricted to intestinal epithelial cells, and examined the pemphigoid (32) by virtue of FcRn's ability to regulate IgG levels response of these mice to luminal (E. coli) and epithelial cell–asso- including pathogenic IgGs. ciated (C. rodentium) bacteria. Enteric bacterial antigens are also involved in generating the We herein demonstrate that intestinal luminal secretion of IgG normal structure and function of the GALT (14). The transport of was abrogated in FcRn–/– mice. Importantly, the absence of FcRn intestinal antigens is tightly regulated by the epithelial layer, which was shown to enhance the susceptibility to infection of an intesti- anatomically provides an important barrier to separate the intes- nal pathogen, C. rodentium. However, selective restoration of FcRn tine from the luminal contents. M cells, a specialized epithelial cell expression in intestinal epithelial cells led to a reduction in sus- type present in the follicle-associated epithelium, have been previ- ceptibility to this infection, but only in the presence of circulat- ously considered as the major "professional" antigen-sampling cell ing IgG specific for this pathogen. These findings indicate that type that is capable of delivering luminal antigens into the lamina intestinal epithelial cell–associated FcRn is actively involved in the propria (33). In addition, intestinal DCs have been demonstrated to inhibition of an epithelial cell–associated pathogen, C. rodentium, directly capture antigens or bacteria present in the intestinal lumen by delivering IgG into the epithelial cell and/or intestinal lumen. by opening the tight junctions between epithelial cells and sending Indeed IgG, but not secretory IgA or IgM, has been shown to play dendrites into the lumen for antigen capture and retrieval (34, 35). a critical role in the prevention against infection by the attach- We have recently demonstrated a third pathway, in which intesti- ing and effacing pathogen C. rodentium (16–18). Mechanistically, nal epithelial cell–associated FcRn is involved in sampling orally it may be predicted that intestinal luminal IgG delivered from the administrated antigens (such as food antigens) by retrieving the lamina propria by FcRn-mediated transport directly contributes antigen as an IgG immune complex through absorptive epithelial to the suppression of C. rodentium infection. cells (2). In the present study, by showing that commensal bacterial There are 2 mechanisms by which FcRn in the epithelium may antigens (E. coli) could also be sampled from the intestinal lumen play a role in such a host defense function. The first possibility is by an FcRn-mediated transport pathway and delivered into DCs that FcRn inhibits the adhesion and/or invasion of the bacterium within MLNs, our studies may have important implications for by directing IgG into the location of the invading pathogen with or establishment of the normal GALT. It is presumed that these pro- without the fixation of complement. The second possibility is that cesses occur in situations wherein the host has acquired IgG either FcRn-mediated transcytosis of IgG activates C. rodentium–specific passively (e.g., neonatal rodent or human) or actively (e.g., after a acquired immune responses. Such responses may be induced by primary immune response). In this context, the broad expression DCs, which capture IgG/antigen complexes that are retrieved from of FcRn in absorptive epithelia would allow for transport of lumi- the intestinal lumen through epithelial-associated FcRn. Indeed, nal antigen over a wide surface area, allowing for broad flexibility we demonstrate here that intestinal epithelial cell–associated FcRn in extending an opportunity for the host to respond to luminal retrieved luminal bacterial antigens as a complex with IgG across antigen. In addition, recent studies have suggested that the man- epithelial barriers into the lamina propria in vivo. Importantly, ner in which antigen is taken up by a DC confers distinct functions the retrieved bacterial antigens were efficiently captured by DCs on the DC (26). Interestingly, Fcg receptor IIB–mediated (FcgRIIB- that were able to subsequently activate antigen-specific acquired mediated) capture of immune complexes are involved in the gen- immune responses by CD4+ T cells within the MLNs. The effi- eration of DC-mediated immune tolerance through the activation cient capture of IgG/antigen complexes by DCs is consistent with of inhibitory motifs of these receptors (36). In addition, Igs have recent studies showing uptake of immune complexes by DCs (26). been proposed to suppress intestinal inflammation by facilitating Since CD4+ T cells have been shown to participate in prevention phagocytosis of pathogenic antigens (37), and IgG in the lumen against C. rodentium infection (16, 18), FcRn may be an important of the lung has been suggested to reduce allergen-induced asthma and essential means to stimulate CD4+ T cell–mediated acquired (38). Therefore, it is possible that the sampling of intestinal lumi- immune responses to inhibit this, and potentially other, epithe- nal antigens by an FcRn-mediated process contributes to unique, The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 2149 research article broadly expressed, and highly flexible functions of mucosal effector Kingdom) followed by Alexa564-conjugated anti-rabbit IgG (Molecular and/or regulatory pathways. These may be involved in the mainte- Probes) and nuclei and examined under confocal microscopy (MRC1024 nance of mucosal tolerance characteristic of this compartment or laser scanning confocal system; Bio-Rad). the development of intestinal inflammation, as might occur if the Detection of serum and secretory mouse and rabbit Ig. Secretory mouse and transported antigen has adjuvant qualities. Such properties may rabbit Igs present in the intestinal lumen were assessed by ELISA as previ- have important consequences for establishing GALT structure dur- ously described (2). The levels of Igs (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, ing development and in response to pathogenic exposures. and IgE) secreted into the lumen were also examined by an alternative In this study, we provide what we believe are novel insights into method, a cytometric bead array performed according to the manufactur- the role of FcRn when associated with the intestinal epithelium. er's instruction (BD Biosciences — Pharmingen). The collected feces were FcRn in the epithelium is the primary means by which IgG reaches resuspended in PBS and incubated with the fluorescent beads at concentra- the lumen, as shown by the absence of luminal IgG in the context tions that were normalized for the quantities of protein. After incubation of FcRn deficiency. Moreover, FcRn may play a significant role in with anti-mouse k detector antibodies, the beads were analyzed by flow defending against epithelial-associated pathogens, as shown by cytometry. To examine IgG production by the MLNs, mononuclear cells increased sensitivity to C. rodentium in the absence of FcRn expres- were collected from the MLNs of FcRn–/–, IFABP-mFcRnTg/mb2mTg, and sion and by restoration of host resistance to C. rodentium in the WT control mice (C57BL/6). Cells (1 × 107/ml) in PBS (pH 7.4) were cul- context of Tg FcRn reconstitution, specifically within the intes- tured without any in vitro stimulation for 2 hours on 37°C, after which the tinal epithelium. We specifically associate this observation with Ig levels were evaluated by flow cytometric analysis as described above. FcRn's ability to provide this immune defense function in the Preparation of cell suspensions. Antigen-presenting cells were isolated using presence of circulating IgG and to direct IgG into the lumen and a digestion buffer, as described previously (2), with a modification. Cells antigen into DCs, whereupon adaptive immunity is enhanced. were carefully collected from the interface, washed with PBS, and used. FcRn within the epithelium may thus be able to link microbial Protocol for induction of colitis by C. rodentium infection. C. rodentium strain antigen encounters within the lumen and/or epithelium with DBS100 (catalog no. 51459; ATCC) was used. 3-week-old mice were orally systemic immune compartments. The ability of FcRn to provide infected as described previously (16). OVA-expressing C. rodentium was cre- these functions over a broad surface area may have important ated by electroporating plasmid DH136 into C. rodentium strain DBS100. implications for understanding the development of GALT and DH136 contains a cassette of the pTR(lac) promoter driving expression of its dysregulation in inflammation. residues 139–386 of chicken OVA, cloned into the EcoRV site of the low-copy plasmid pACYC184 (chloramphenicol selection). pTR(lac) drives constitu- Methods tive expression of OVA. After electroporation with a Bio-Rad Gene Pulser II, Animals. A Tg mouse strain in which both mFcRn and mb2m are constitu- chloramphenicol-resistant transformants were verified to retain normal pro- tively and specifically expressed in the intestinal epithelial cells under the duction of LPS and to constitutively express the 139–386 fragment of OVA. control of an IFABP (kindly provided by J. Gordon, Washington University, To ensure maintenance of the plasmid during in vivo infection, 500 mg/ml St. Louis, Missouri, USA) (20–22) was generated and further backcrossed chloramphenicol was added to the drinking water. All methods and proce- to FcRn–/– mice (23) in order to eliminate FcRn expression in other cell dures with infected animals were performed in BL-2 safety cabinets. Colonic types (IFABP-mFcRnTg/mb2mTg/FcRn–/–). These mice were backcrossed tissues with H&E stain were evaluated by a pathologist (J.N. Glickman) in a 6 generations onto either BALB/c or C57BL/6 (CD45.2+). For OVA studies, blinded fashion, and results were represented as a total score (43). OT-II mice were used (39), which express a restricted Tg T cell receptor that Adoptive transfer, CFSE labeling of OVA-specific OT-II CD4 + T cells, and anti- recognizes a peptide fragment of OVA (residues 323–339) in the context of genic challenge of C. rodentium–OVA. IFABP-mFcRnTg/mb2mTg/FcRn–/– or I-Ab (CD45.1+C57BL/6). These studies were approved by the Standing Com- FcRn–/– C57BL/6 mice (3 weeks old) were orally infected with 1 × 109 CFU mittee on Animals at Harvard Medical School. of C. rodentium–OVA or WT C. rodentium. The mice received 2 mg of anti– RT-PCR. Epithelial cells were nonenzymatically isolated as described C. rodentium IgG or control IgG i.v. at day 4 after the bacterial inoculation previously (40), and total RNA was extracted using TRIzol reagent and subsequently received CFSE-labeled CD45.1 +CD4+OT-II T cells for (Invitrogen). RT-PCR reaction was carried out using specific primer sets the detection of cell division or nonlabeled CD45.1+CD4+OT-II T cells for for mFcRn, mb2m, and mb-actin as described previously (41). examination of antigen-specific cytokine production by adoptive transfer Quantitative PCR. Tissues were collected into RNAlater (Ambion), and at day 5. Cytokine measurements were made with CD4+ T cells prepared total RNA was extracted using TRIzol reagent (Invitrogen). For the from MLNs and stimulated with C57BL/6 splenocytes pulsed with OVA expression analysis of the mFcRn transgene, specific primer sets for the as antigen-presenting cells. On day 7, MLN cells were subjected to flow mFcRn transgene (Primer 1, 5′-GCCCTCAGCCTCTTGTTGG-3′; Primer cytometric analysis for the evaluation of CFSE intensity (indicating cell 2, 5′-CCCGTAGATGGGTTTGACACA-3′) were used for amplification division) of the CD4+ T cells gated on CD45.1+ cells. with detection of a 102-bp cDNA fragment. Quantitative PCR was carried Preparation of FITC–E. coli and in vivo transcytosis of antigen/IgG complexes. out with 1 ml cDNA in the presence of iQ SYBR Green Supermix (Bio- E. coli (DH5a; Invitrogen) was labeled with 1 mg/ml of FITC solution in Rad) and an iQ5 Real-Time PCR Detection System (Bio-Rad) for real-time PBS at pH 7.4 for 15 minutes. To analyze the transcytosis of bacterial anti- detection. mFcRn transgene expression levels were determined via the gen/IgG complexes, 2 mg rabbit anti–E. coli IgG (Dako) or control IgG was comparative cycle threshold (2–DDCt) methods (Applied Biosystems User injected i.v. into mice. FITC-conjugated E. coli (1 × 109 cells) was adminis- Bulletin 2) and normalized to mb-actin. tered intragastrically 12 hours after the i.v. injection of IgG, and tissue from Immunohistochemistry. For the detection of mFcRn, tissues were snap-fro- the mid-small intestine was collected 2 hours after the oral inoculation. Tis- zen and subjected to immunohistochemical staining (42) using rabbit anti- sue sections were subjected to staining for detection of actin (phalloidin; FcRn antibodies (kindly provided by N.E. Simister, Brandeis University, red) and nuclei (blue) and examined by confocal microscopy as previously Waltham, Massachusetts, USA). For the detection of C. rodentium, sections described. MLN cells were isolated 5 hours after oral inoculation, stained were stained for intimin using the polyclonal rabbit anti–C. rodentium inti- with PE-conjugated anti-CD11c and 7-amino-actinomycin D (7-AAD) to min sera (kindly provided G. Frankel, Imperial College, London, United exclude dead cells, and examined by flow cytometry. 2150 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 8 August 2006 research article Statistics. Statistical significance was determined by a 2-tailed Student's t Received for publication January 3, 2006, and accepted in revised test. P values less than 0.05 were considered significant. form May 16, 2006. Acknowledgments Address correspondence to: Richard S. Blumberg, Gastroenterology This work was supported in part by Research Fellowships from Division, Department of Medicine, Brigham and Women's Hospi- Crohn's and Colitis Foundation of America (M. Yoshida, T. tal and Harvard Medical School, 75 Francis Street, Boston, Massa- Nagaishi, and A. Kaser) and by NIH grant DK53056 (R.S. Blum- chusetts 02115, USA. Phone: (617) 732-6917; Fax: (617) 264-5185; berg and W.I. Lencer), Harvard Digestive Diseases Center grant E-mail: firstname.lastname@example.com. P30 (R.S. Blumberg and W.I. Lencer), and NIH grants DK51362 and DK44319 (R.S. Blumberg). A. Mizoguchi was supported by Masaru Yoshida's present address is: Frontier Medical Science in NIH grant DK64351, and E. Mizoguchi was supported by NIH Gastroenterology, International Center for Medical Research and grant DK64289. Treatment, Kobe University School of Medicine, Kobe, Japan. 1. Robert-Guroff, M. 2000. IgG surfaces as an impor- cell-dependent serum antibody, but not the gut- plasmic antibodies in T-cell receptor alpha-defi- tant component in mucosal protection. Nat. Med. associated lymphoid tissue, for surviving acute cient mice with chronic colitis. 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Dog Abandoned On Street- Caught On Cam
Henderson, Nevada (CNN) - If you are a dog lover, what this security camera caught on tape will probably anger you. It's clear that the dog owner didn't want the pet. The car is seen driving slowly while the dog is pushed out. Scott Daniels reports.
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According to the Tribune News Service, Egypt's presidential spokesman said that the owners of the Red Sea ferry that sank last week, causing about 1,000 people to drown, did not inform the government of the disaster for nearly six hours. Suleiman Awad said the government first heard from owner Al Salaam Maritime Transport Co. that the ship was in danger at 7 a.m. Friday. Reports indicate that the Al-Salaam Boccaccio 98 sank no later than 2 a.m.
Supertankers Pose No Danger to Bay of Fundy
Proponents of a controversial liquefied natural gas plant in Maine say a study ordered by the Canadian government has found there is no reason to ban supertankers from the Bay of Fundy, as Ottawa has threatened to do if the U.S. approves the project. Downeast LNG said the environmental study found there were no immediate risks of having LNG supertankers go through the Bay of Fundy, part of which is split by the New Brunswick-Maine border, to the proposed plant.
400 Jobs on the Line
According to a March 12 report from Inquirer.net, about 400 workers of a ship repair company in Lapu-Lapu City are in danger of losing their jobs as the management decides to focus resources on shipbuilding. According to Inquirer.net, Keppel Cebu Shipyard Inc. president David Loh said in a memorandum to the workers that the company has to reduce its workforce due to continuing market decline. (Source: Inquirer.net)
Storm Damages Sunken Frigate
The storm that lashed the lower North Island has caused wreckage from an old navy frigate sunk near Wellington last year, to be washed ashore. The former HMNZS Wellington was sold to the Sink F69 Trust, which sunk it about 500 meters off Island Bay – virtually in Cook Strait – on November 14. Prior to the sinking Island Bay man, some warned that the lightly built ship would break up very quickly and become a danger to divers, not the tourist and diving area the trust claimed it would be
Ship Grounded off Norfolk Coast
A rescue operation has been launched after a large container ship ran aground off Haisboro Sands. The vessel, The City of Sunderland, is carrying a cargo of 642 Nissan cars and is currently lying on a sand bar eight miles off the north Norfolk coast. There are 13 crew members on board. The 9,576 ton ship became stuck at approximately 2.20am this morning while on a voyage from the port of Zeebrugge to Tees Port.
Russian PM Out of Tune With Arctic Protesters
Medvedev has proposed toughening punishment for illegal entry to fuel and energy complex facilities, reports Arctic info (Russia). This initiative was proposed by the head of government at a recent meeting on problems of land use and thetransportation of hydrocarbons in the Caspian Sea. Commenting on the recent arrest of Greenpeace activists protesting against oil production in the Arctic at the Prirazlomnaya platform
USCG Searching for 2 Trapped Under Sunken Barge
The Coast Guard, along with the Arkansas Game and Fish Commission and the Mississippi County, Ark. Sheriff's Department, are searching for two people in the water near a sunken barge in Hickman Landing Wednesday. Watchstanders at Coast Guard Sector Lower Mississippi River received a report at 4:15 p.m. Tuesday that two people became trapped beneath a barge that flipped and sank in the Mississippi River. Sector Lower Mississippi River directed the launch of a 25-foot Response Boat —
Making Sailors 'SAFFiR'
Navy Unveils Firefighting Robot Prototype at Naval Tech EXPO. Scientists unveiled a firefighting robot prototype Feb. 4 at the Naval Future Force Science & Technology EXPO, revealing details about its successful demonstrations last fall.
USCG Warns Against Ice Dangers On Lake Champlain
Coast Guard crews at Station Burlington, Vermont, are urging Lake Champlain locals and visitors to use extreme caution on the lake and surrounding waters this weekend. With more snow forecasted, unsafe ice may be hidden from view and poses greater danger than normal as the added weight
Greek Banks Mull Cutting Shipping Portfolios
Greece's leading banks are considering offloading part of their portfolios of shipping debt worth billions of dollars in a bid to shore up their capital, according to banking and ship financing sources. Greece has secured an extension to its bailout from its European partners
Satellite Images Reveal Ocean Acidification
Ocean acidification can now be seen from space, highlighting an ongoing danger of climate change and revealing the regions most at risk. Pioneering techniques that use satellites to monitor ocean acidification are set to revolutionize the way that marine biologists and climate
McDonald's Japan Rations Fries as U.S. Port Labor Dispute Continues
McDonald's Holdings Co (Japan) Ltd has embarked on the emergency measure of only offering small-sized french fries to customers as a protracted labour dispute at U.S. West Coast ports has contributed to long delays in imports. The fast-food chain said it was importing more than 1
Moore Stephens: Shipping Must Adopt Can-do Attitude
International accountant and shipping adviser Moore Stephens says shipping needs to adopt a can-do attitude in order to successfully meet the challenges which are likely to come its way in 2015. Moore Stephens shipping partner Richard Greiner says
UKHO Launches New H-Note App
The United Kingdom Hydrographic Office (UKHO) has unveiled a new digital application that enables mariners to submit hydrographic data directly to the UKHO using their mobile device. The ADMIRALTY H-Note App is a free digital version of the UKHO's Hydrographic Note and will allow important
Intercargo Calls for Bulk Carrier Safety
The International Association of Dry Cargo Shipowners (Intercargo) issued a statement which again reminds stakeholders of the continued dangers associated with the carriage of bulk cargoes that may have a potential for liquefaction.
USN Railgun at Future Force EXPO
The Electromagnetic Railgun-a weapon that will play a significant role in the future of the U.S. Navy-will be on display to the public for the first time on the East Coast Feb. 4-5 at the Naval Future Force Science and Technology (S&T) EXPO in Washington, D.C
IMB: Asia Maritime Piracy Attacks on the Rise
Piracy, armed attacks on ships in Asia at highest since 2006. Asia accounted for three-quarters of global maritime piracy last year after a surge in tanker hijackings helped to fuel a 22 percent jump in armed robbery and pirate attacks on ships in the region.
Hoegh Osaka Salvors Battling High Winds
All precautions are in place to hold the Hoegh Osaka in place as tonight's weather forecast predicts high winds, the U.K. Maritime and Coastguard Agency (MCA) reported. The vessel remains held in position at anchor on the Alpha Anchorage.
US Navy to Display Electromagnetic Railgun
The U.S. Navy will display its revolutionary weapon at Future Force EXPO The Electromagnetic Railgun – a weapon that the U.S. Navy says will play a significant role in its future – will be on display to the public for the first time on the East Coast Feb
AIS Marks Hazardous Reefs Following Oil Spills
Vesper Marine has been awarded the contract to provide Virtual Automatic Identification System (AIS) Beacons to mark seven reefs hazardous to shipping in the Bay of Plenty, as well as provide a coastal monitoring capability. The beacons will alert ships' crews and the local
Towing Vessel Sinks off Oahu
A 95-foot towing vessel, Nalani, sank approximately two and half miles west of Barbers Point Harbor, Oahu, Thursday, the U.S. (USCG) Coast Guard reported. The 11 people on board Nalani were rescued safely. The pilot aboard the sinking towing vessel placed a call at 3:13 p.m
Australia Bans Waste Dumping on Barrier Reef
Australia has ordered a ban on dumping dredge waste on most of the Great Barrier Reef, the environment minister Greg Hunt stated, as part of a push to cease the UN declaring the website in danger. The atmosphere minister has given orders for new regulations to be put in place for the


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Hope Floats(1998)Birdee Pruitt seems to have it all. She's been married for years to her handsome highschool sweetheart, and has a brilliant and sensitive child. But when her picture perfect life comes crashing down around her - when her beloved husband dumps her on national tv - Birdee must start over. With no place else to turn, the former beauty queen, along with daughter Bernice, heads back home... to Smithville, Texas. There her life becomes even more complicated as she tries to deal with a mother she must get to really know for the first time, a daughter who desperately misses her father, and the prospect of a new romance. As Birdee undertakes her emotionally charged journey, she begins to find strength to reclaim her life, rediscover her family and return to something she had almost given up... hope. For more about Hope Floats and the Hope Floats Blu-ray release, see Hope Floats Blu-ray Review published by Michael Reuben on July 30, 2011 where this Blu-ray release scored 3.0 out of 5. Starring: Sandra Bullock, Harry Connick, Jr., Gena Rowlands, Mae Whitman, Michael Paré, Cameron Finley Director: Forest Whitaker » See full cast & crew Hope Floats Blu-ray, Video QualityHope Floats was shot by Caleb Deschanel, one of the most stylish cinematographers working today. Deschanel's trademark look is rich and textured, especially in scenes of nature (his early work was with Carol Ballard), and Hope Floats is no exception. Even in the TV studio scenes, he resists the temptation to light harshly and crassly. Through Deschanel's lens, the people on The Toni Post Show look better than they ever would on TV. And when the film quickly reaches Texas, he's in his element. The 1080p, AVC-encoded Blu-ray offers an excellent image with richly detailed surfaces, a lightly visible grain pattern, saturated colors and excellent detail. Blacks are genuinely black, and Smithville is the kind of setting where the nights are dark enough so that the image would visibly suffer without a true black. Nevertheless, shadow detail is well preserved in the kind of scenes where you need to be able to see what's happening, e.g., when Justin shows Birdie the house he's building and they go out back, where there's almost no light. Fox has used a BD-25, but the film is neither long nor action-packed, and there are no real extras; as a result, there are no compression-related issues (or, at least, none that I noticed). Hope Floats Blu-ray, Audio QualityThe DTS 5.1 lossless track for Hope Floats is restrained but detailed, especially when Birdie returns home to Smithville. Little sounds of the countryside and small town life are layered into the sound mix and often float into the surrounds: birds chirping, wind blowing, trees rustling. Dialogue is clear and centered. The primary beneficiary of the track's fidelity is the musical score consisting of original underscoring by Dave Grusin and a fine assembly of moody tunes, some original and some covers, overseen by soundtrack producers Don Was and director Whitaker. Lyle Lovett's version of "Smile" and Sheryl Crow's "In Need" are particularly noteworthy. Indeed, as far as emotional authenticity is concerned, the soundtrack easily bests the film it accompanies.
Similar titles suggested by membersHope Floats Blu-ray, News and Updates• Romance Blu-ray Wave from Fox in January - October 19, 2010 An early announcement to retailers indicates that 20th Century Fox Home Entertainment will release three romantic movies from its catalog on Blu-ray on January 4, 2011, in time for Valentine's Day: Ever After, starring Drew Barrymore and Dougray Scott; Hope Floats, ...
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Definition of feast day in English:
A day on which a celebration, especially an annual Christian one, is held: on feast days a High Mass was sung the feast day of Saint Mark
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- The Maltese love festivals, and between May and October almost every town and village in Malta and Gozo celebrates the feast day of its patron saint.
- Every city or town also celebrates the feast day of its patron saint with processions, dancing, and bullfights.
- Each village celebrates the feast day of its patron saint.
Definition of feast day in:
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Donkey and Goat The Recluse Broken Leg Vineyard Syrah 2009
Syrah/Shiraz from North Coast, California
Winemaker's Notes
Charcuterie, white and black pepper, tobacco and leather seduce the senses immediately. Cherry, blackberry and blueberry all laced with floral hints and blood orange prepare the palate. An iron ferrous quality runs through with long and chewy tannins. The finish will linger with more notes of cured meats, smoke and mulberry. This Syrah illustrates why we love this varietal and will offer hours of enjoyment as she reveals her secrets (if you can wait that long).
Blend: 97% Syrah and 3% Viognier
Critical Acclaim
International Wine Cellar - "Saturated ruby. Varietally accurate aromas of dark berries, violet and smoke, with higher-pitched mineral and spice nuances building with air. Juicy, incisive and animated in the mouth, offering fresh blackberry and mulberry flavors and a touch of bitter chocolate. The spiciness comes back on the very long finish, which features lingering notes of soy sauce and candied violet. Approachable now but I'd come back to this in a couple of years."
Donkey and Goat Winery
Donkey & Goat is a family owned and operated winery located in Berkeley California. Tracey & Jared Brandt craft their natural wines from Rhône varietals, Chardonnay and Pinot Noir grown in the Anderson Valley, Mendocino Ridge and the undiscovered El Dorado appellation in the Sierra Foothills. Tracey & Jared trained in France, under Éric Texier, an up and coming Rhône winemaker who taught them the art and craft of making wines that speak to the soul about the earth from which they originate. Their maverick winemaking has helped pave the way for the natural wine movement. They make their wines for the table not the cocktail glass. They make Rhône varietals in both colors plus an atypical Chardonnay and a Pinot Noir true to the varietals nature, striving to make wine as naturally as possible and done so since day one. View all Donkey and Goat Wines
About North Coast
View a map of North Coast wineriesBeyond Napa and Sonoma in the north you find a couple of other counties producing great wine. Among these are Mendocino and Lake County. The northernmost California winegrowing regions, these two counties are right above Napa and Sonoma, geographically. Yet, wine-wise they are very different – both from their southern neighbors and from each other.
Notable Facts
Mendocino has a high amount of organic vintners and vines. The first winery to settle here was Fetzer, which practices organic viticulture and holds some of the most vineyard land in the area. Mendocino has many pockets of micro-climates while Lake County, being smaller in size, is less diverse climactically. As for the grapes, Chardonnay is the most popular in both counties, but there are also some excellent Sauvignon Blancs, particularly in the Lake County. In red wine, Zinfandel leads the way, followed by Rhone Blends and Petite Sirah. The reds in both counties are complex and sumptuous. Anderson Valley is a sub-AVA of Mendicino and is quite well known for its excellent cool climate, producing the delicious Roederer Estate sparkling wines and some wonderful cool-climate Syrah.About California
It's not rare to see a wine's country of origin listed as "California." A country into itself in the wine world, California makes enough varieties and styles to match many European wine countries. It produces a diverse range of wines that span the quality spectrum.The most famous of the California wine regions is Napa Valley, and these wines are certainly outstanding – but it's not as broad and diverse as its larger neighbor, Sonoma County. Down south, Santa Barbara's Santa Maria Valley is well-known for its Rhône blends, as well as cool-climate varieties like Pinot and Chardonnay. The Central Coast, the largest California AVA, has many different microclimates that lead to a wide range of wines with many sub-AVAs.
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gwendolyn - Oakland, CA59/27/2011so tempting to drink this now because it has all those delicious floral and spice notes on the nose, but wait, because this gets even better with a few years in the cellar. I would totally buy a case, then drink one every year just to see it evolve. This wine has character, a sense of place and a whole bunch of layers that have you wanting to savor each sip, but also wanting to drink more! LOVE this wine!Related ProductsAlcohol By Volume Guide
Most wine ranges from 10-16% alcohol by volume. Some varietals tend to have higher (for example Zinfandel and Cabernet Sauvignon) or lower alcohol levels (Pinot Noir and many white varietals), but there is always some variation from producer to producer. Some wine falls outside of this range, for instance Port weighs in closer to 20%, while Muscat and Riesling are usually a bit below 10%.Wine Style Guide
Light & Crisp
- Light to medium bodied wines that are high in acid and light to medium fruit. Typically no oak.
Fruity & Smooth
- Light to medium bodied wines with lots of juicy fruit, typically medium acid and medium oak.
Rich & Creamy
- Full bodied wines that have typically undergone malo-lactic fermentation and/or spent time in oak.
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Native Plants
Q. Who is Mr. Smarty Plants?
A: There are those who suspect Wildflower Center volunteers are the culpable and capable culprits. Yet, others think staff members play some, albeit small, role. You can torture us with your plant questions, but we will never reveal the Green Guru's secret identity.
Did you know you can access the Native Plant Information Network with your web-enabled smartphone?
Ask Mr. Smarty Plants is a free service provided by the staff and volunteers at the Lady Bird Johnson Wildflower Center.
Need help with plant identification, visit the plant identification page.
rate this answer
Saturday - October 03, 2009
From: Prairieville, LA
Region: Southeast
Topic: Propagation, Transplants, Cacti and Succulents
Title: Removal of pups from Century Plant after blooming in Prairieville LA
Answered by: Barbara Medford
Will the main part of the century plant always die after it grows a stalk? I have babies coming off the base and need to know if I should separate them to keep them alive.ANSWER:
There are nine members of the agave genus with the common name "Century Plant." Of these, only Agave americana (American century plant) is considered native to Louisiana, although others no doubt have been purchased and grow there. You are correct, once a Century Plant has put on its spectacular bloom, the whole plant then dies. The little offshoots, or "pups," are the plant's attempt to propagate itself. The pups should be separated from the adult plant as soon as possible.
How do you transplant an Agave? Like kissing a porcupine-very carefully! All agaves reproduce by pups in about the same way, so these instructions should apply to your plant. Since the "mother" plant is already dying, you might begin by cutting away some of its menacing leaves from the area where you will be working.
First, and no kidding this time, approach this task with care. The agaves have survived in very hostile environments by being pretty hostile themselves to grazing by livestock or digging up to clear land for farming. You can, indeed, make new plants of the "pups", but first, protect yourself with heavy leather gloves. With clippers, remove the fierce spines on the pup before you separate the plant from its parent. If there is a clump of several new plants, gently break them up by hand or with a knife. One standing alone can probably be popped out of the ground with a trowel or small shovel. Again, careful, you are close to a very forbidding parent plant that won't hesitate to get you in the eye or the arm or the back with those long, sharp-tipped leaves. Get rid of loose roots and then, as if it was an onion, peel away leaves until you get to the best-quality leaves in the center of the rosette. Carefully discard all the removed spines and leaves where someone won't come along and step on them. And the compost pile is out, you don't want to stick your hand into that!
If it was necessary to cut the transplant, you need a clean cut on the base of the wound. You can dab the wound with sulphur before putting the cutting in a pot filled with a "cactus" potting mix. Top off the pot with more sand or "cactus mix", pack it down and put it out of doors in full sun. Don't overwater it and in a couple of months it should be ready to be on its own.
Now you're ready to decide on the permanent location for the new plant. Since it can be anywhere from 8 to 40 years before the plant summons up the energy to bloom and then it dies, you should not worry about where it can bloom best, but where the plant can spread out and the plump leaves be seen best. And, of course, where it's out of foot traffic or where a child or pet might blunder into it. Since the average annual minimum temperatures in Ascension Parish are 15 to 20 deg. F, you should be aware that the agaves should be protected from teen winter temperatures to avoid damage. A short spell of freezing weather shouldn't harm it.
If you choose to keep the new plants indoors in a pot, the agaves are so slow-growing that you shouldn't have to transplant into a larger pot very often. They can do very very well indoors in a good light from a window. Remember, they are succulents, and like most other succulents, they need less water and can tolerate quite a bit of shade. For more information on container gardening, read this article from the Lady Bird Johnson Wildflower Center "How To Articles".
Pictures of Agave americana (American century plant) from Google.
From our Native Plant Image Gallery:
More Transplants Questions
Transplanting butterfly weed in Hamden OH
July 10, 2009 - I have tried transplanting butterfly weed from it's native location to my yard and each time the plant wilts and dies.
Any suggestions?
view the full question and answer
Transplanting and germination of Pride of Barbados in Adkins, TX
April 02, 2012 - What is the root system like of the Pride of Barbados? I have a lot of new plants coming up in my beds from seeds. Can these be transplanted to a new location easily without damaging the plants? If...
view the full question and answer
Removing leaves before transplanting from Miami
August 27, 2009 - What is good idea to remove some leaves before transplanting a plant??
view the full question and answer
Transplanting Tecoma stans in Texas
October 26, 2008 - I have a pair of Tecoma stans planted too near the house. They're in shade most of the day. The branches that can reach a little sun are blooming nicely. Would they survive being transplanted fart...
view the full question and answer
Transplanting Turks Cap, when and how
September 10, 2007 - Mr. Smarty Pants - We have an enormous healthy Turk's Cap - not the lily, but the one with red flowers(Malvaviscus arboreus v. drummondii) It has also produced a new plant nearby. Please tell us how...
view the full question and answer
Support the Wildflower Center by Donating Online or Becoming a Member today. Mr. Smarty Plants wants you to be his Facebook friend. Click the Facebook icon to add yourself to Mr. Smarty Plants list of friends. |


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Annual Houston Port Coffee Symposium.
HOUSTON -- The 4th Annual Houston Port Region Coffee Symposium was held November 17-18. The two-day conference was themed "Houston Port Region: Coffee Gateway to Latin America/" and was sponsored by the Port of Houston Authority, the Greater Houston Coffee Association and the Economic Alliance Port Region.Sessions focused on the Brazilian perspective of the coffee industry and Houston's increasing influence as the 4th designated green coffee port in the U.S. as well as a panel on getting coffee through customs and an advice session for small business entrepreneurs.
"In 2007, Brazil ranked as our largest coffee importer, followed by Columbia and Mexico, demonstrating Latin America's continued significance to the Houston Port Region's economy," said Jim Edmonds, Port of Houston Authority chairman.
In addition, Edmonds served as keynote luncheon speaker. Other speakers included Alan Kaiser (National Coffee Association of the U.S.), Jose Maria Ribeiro Neto (coffee planter and member, Cooxupe), Don Pisano (American Coffee and chair of the Green Coffee Association Traffic & Warehouse Committee), L.B. Booty (U.S. Food and Drug Administration), Vera Braun (Coffee America USA, Inc.), Matt Braunner (Braunner Int.), Tammy Deininger (Volcafe USA LLC), Mike Sinclair (U.S. Customs and Border Protection) and Scott Singleton (Kadena Strategies, Inc.).
Reader Opinion
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Title Annotation: | World News |
Publication: | Tea & Coffee Trade Journal |
Date: | Dec 1, 2008 |
Words: | 203 |
Previous Article: | Coffee authority destroys 60 tons of poor quality coffee. |
Next Article: | Calendar. |
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France refuses EU order to include drugs, prostitution in GDP figures
France is refusing to comply with a European Union demand to include proceeds from the drug trade and prostitution in its gross domestic product (GDP) figures. The national statistical institute (Insee) says that neither can be counted a voluntary transaction.
Britain, Belgium, Italy and Spain have all said they will abide by new norms set by the European System of Accounts, based on a recommendation from the Eurostat statistics authority.
Member-states have been told to calculate wealth created by the majority of illegal activities, including drugs and prostitution, and include them in national statistics.
The Bank of Italy estimates that the "criminal economy" was worth 10.9 per cent of Italian GDP in 2012, while the UK's Office of National Statistics puts income from drugs and prostitution in 2009 at about 9.7 billion pounds (12 billion euros), 0.7 per cent.
But French ministers have slammed the idea that prostitution is a free commercial transation and Insee says it will not include it or narcotics in its GDP figures.
The institute considers that it already accounts for prostitution practised under cover of other activities along with tax fraud and unregistered work, according to economic syntheses and studies director Eric Dubois.
"But there is also street prostitution, which is principally the work of mafia networks and is pretty much a trade in people without legal status," he told Le Monde newspaper. "Basically the criterion of mutual consent is probably not confirmed."
Insee also considers that drug addicts are not exercising free will, he explained, although the trade will be included in calculations of gross national revenue to be sent to EU institutions.
A French government minister has joined a Belgian colleague in protesting to the European Commission about the new methods of calculation.
"Prostitution is not a commercial transaction that has been freely entered into," Women's Rights Minister Najat Vallaud-Belkacem and Belgian Interior Minister Joëlle Milquet wrote in a letter. "Believing that it is is an ideological choice, it is a mirage and an offence to millions of victims of sexual exploitation around the world."
Comments (1)
Legalize and tax prostitution
Legalize and tax prostitution immediately, without any prohibition about the matter.


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In a wonderful profile of master diagnostician Dr. Thomas Bolte, this especially pleased me:
A large fraction of Bolte's patients have been poisoned. They get better when the poison is stopped. The mother of a friend of mine was near death — so near that her children decided to put her in a hospice. By mistake her six or seven medicines were stopped. And she recovered! Her medicines were what had been killing her. |
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Cougar Town
Tuesdays 10:00 PM on TBSFollow Cougar Town
Watch Cougar Town Season 1 Episode 4 online via TV Fanatic with over 2 options to watch the Cougar Town S1E4 full episode. Affiliates with free and paid streaming include Amazon and iTunes.
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Episode Details
Jules accidentally tells Grayson she thinks he's not and tries to get him to admit she's also hot; Ellie feels unattractive after seeing Jules' sex tape
- Show:
- Cougar Town
- Season:
- Cougar Town Season 1
- Episode Number:
- 4
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Cougar Town Season 1 Episode 4 Quotes
Jules: Oh look who got laid last night
Andy: That's right chumps, missionary accomplished!
- Permalink: Oh look who got laid last night That's right chumps, missionar...
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Ellie: Why are you out of breath
Jules: Cause I'm sanding down my disgusting elephant heels. With all the shavings here I could make a second foot
- Permalink: Why are you out of breath Cause I'm sanding down my disgusting...
- Added: