Itaru Moro

Dietary fructooligosaccharides up‐regulate immunoglobulin A response and polymeric immunoglobulin receptor expression in intestines of infant mice

We examined whether or not dietary fructooligosaccharides (FOS) in infancy can have a beneficial effect on the mucosal immune system. Newborn BALB/c mice, accompanied by their dams until 21 days of age, were fed either a control diet based on casein [FOS(–) diet group] or a FOS(–) diet supplemented with 5% (w/w) FOS [FOS(+) diet group]. Total IgA levels in tissue extracts from the intestines of mice in the FOS(+) diet group at 38 days of age were about twofold higher (P < 0·05) than those in the FOS(–) diet group in the jejunum, ileum and colon. Ileal and colonic polymeric immunoglobulin receptor (pIgR) expression in the FOS(+) diet group at 36 days of age was 1·5‐fold higher than in the FOS(–) diet group (P < 0·05). Consistent with these results, the ileal IgA secretion rate of the FOS(+) diet group at 37 days of age was twofold higher than that of the FOS(–) diet group (P < 0·05). Moreover, the percentage of B220+IgA+ cells in Peyers patches (PP) was significantly higher in the FOS(+) diet group than in the FOS(–) diet group (6·2%versus 4·3%, P < 0·05), suggesting that isotype switching from IgM to IgA in PP B cells might be enhanced in vivo. Taken together, our findings suggest that dietary FOS increases the intestinal IgA response and pIgR expression in the small intestine as well as the colon in infant mice.

CD14 IS EXPRESSED AND RELEASED AS SOLUBLE CD14 BY HUMAN INTESTINAL EPITHELIAL CELLS IN VITRO: LIPOPOLYSACCHARIDE ACTIVATION OF EPITHELIAL CELLS REVISITED

ABSTRACT Human endothelial as well as epithelial cells were shown to respond to lipopolysaccharides (LPSs). However, the expression and release of CD14 by these so-called CD14-negative cells have not been studied in detail. We investigated three human intestinal epithelial cell lines (ECLs), SW-480, HT-29, and Caco-2, for their expression of CD14 and CD11c/CD18 as well as their responsiveness to endotoxins. Fluorescence-activated cell sorter analysis revealed no expression of CD11c/CD18, but there was low expression of membrane-bound CD14 on HT-29, Caco-2, and SW-480 ECLs. Both Western blotting and reverse transcription-PCR confirmed the CD14 positivity of all three intestinal ECLs. No substantial modulation of CD14 expression was achieved after 6, 8, 18, 24, and 48 h of cultivation with 10-fold serial dilutions of LPS ranging from 0.01 ng/ml to 100 μg/ml. Interestingly, soluble CD14 was found in the tissue culture supernatants of all three ECLs. Finally, only HT-29 and SW-480, and not Caco-2, cells responded to LPS exposure (range, 0.01 ng/ml to 100 μg/ml) by interleukin 8 release. Thus, we show that HT-29, SW-480, and Caco-2 human intestinal ECLs express membrane-bound CD14. As Caco-2 cells did not respond to LPS, these cell lines might be an interesting model for studying the receptor complex for LPS. The fact that human intestinal epithelial cells are capable not only of expression but also of release of soluble CD14 may have important implications in vivo, e.g., in shaping the interaction between the mucosal immune system and bacteria in the gut and/or in the pathogenesis of endotoxin shock.

Expression of inflammatory cytokine genes in vivo by human alveolar bone-derived polymorphonuclear leukocytes isolated from chronically inflamed sites of bone resorption.

Alveolar bone-derived polymorphonuclear leukocytes (PMNs) were characterized for their ability to produce inflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, tumor necrosis factor α(TNFα), and IL-6in vivo. Periapical exudates (PE) were collected from periapical lesions with chronic periapical periodontitis through root canals. Cells and noncellular supernatants were then isolated by centrifugation. The concentration of cytokines present in the noncellular supernatants were determined by ELISA. High concentrations of IL-1α, IL-1β, and IL-6 were detected in PE, however, TNFα was not. PE contains predominantly PMNs (>95% of residing cells) with a few percent of lymphocytes and/or macrophages. These alveolar bone-derived PMNs were purified by the Ficoll-Hypaque gradient method and were analyzed for cytokine mRNA expression using the cytokine-specific reverse-transcription polymerase chain reaction. Highly purified PMNs (>99.5%) isolated from PE expressed significant levels of mRNA for IL-α, IL-1β, and TNFα. IL-6 mRNA was not detected, although a high concentration of IL-6 was detected in supernatants of PE by ELISA. The IL-6 secretion in PE could be derived from macrophages, T lymphocytes, osteoblasts, or fibroblasts around periapical lesions. These data strongly suggest that human PMNs derived from alveolar bone can spontaneously produce IL-1α, IL-1β, and TNFα at sites of inflammation, and probably initiate inflammation and regulate augmentation of bone resorptionin vivo.

SITE OF CATABOLISM OF AUTOLOGOUS AND HETEROLOGOUS IGA IN NON-HUMAN PRIMATES

Because of similarities between the human and monkey immune systems, we considered the monkey a suitable model for studies on the catabolism of various molecular forms of IgA, for which little information is available. The residualizing label dilactilol‐[125I]tyramine was coupled to monkey (Macaca fuscata) IgA and IgG, as well as to human monomeric and polymeric myeloma IgA1 and IgA2 proteins. When labelled proteins were injected intravenously into monkeys, the non‐metabolizable radioiodinated tracer accumulated at the cellular site of protein degradation, allowing identification of the catabolic sites. To determine the uptake or injected proteins by various tissues, monkeys were sacrificed 6‐7 days after injection of labelled proteins, when blood‐associated radioactivity was ≥ 10% of the injected dose, as measured by plasma clearance. When monkey or human monomeric IgA. as well as human polymeric IgA, irrespective of subclass, was administered to monkeys, the liver showed the greatest tissue uptake relative to total dose injected and to organ weight, and the highest acid soluble radioactivity (degraded protein). Although both hepatocytes and non‐parenchymal liver cells were involved in IgA uptake, the hepatocytes were more active. Therefore, it appears that the liver is the major site of uptake and catabolism of IgA in monkeys and possibly in humans.

p53 gene alterations and p53 protein in oral epithelial dysplasia and squamous cell carcinoma.

To examine the expression of p53 protein and gene alterations in oral epithelial lesions including epithelial dysplasias and primary squamous cell carcinomas, immunohistochemical and temperature gradient gel electrophoresis (TGGE) methods were applied to formalin‐fixed and paraffin‐embedded tissues. Morphologically normal mucosal epithelium stained negatively for p53 protein. Three out of 11 (27·3 per cent) epithelial dysplasias and 19 out of 57 (33·3 per cent) primary squamous cell carcinomas stained positively for p53 protein. Although more than half of the cases were positive for p53 protein in stage I, the positive cancer cases were found at other stages with variable frequency. Immunoreactive products were localized in the nucleus, especially in the basal and suprabasal layers. The analysis by TGGE revealed gene alterations in exons 5–8 in 3 out of 3 epithelial dysplasias and 17 out of 19 (89·5 per cent) primary squamous cell carcinomas which were immunohistochemically positive for p53 protein. These results suggest that p53 gene mutation may be involved in carcinogenesis in the oral squamous epithelium even in the early stage of the dysplasia–carcinoma sequence.

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Detection of Epstein-Barr virus and human herpes virus type 6 in saliva from patients with lymphoproliferative diseases by the polymerase chain reaction

Direct detection of these viruses was made by using the PCR for amplifying viral DNA. In virtually all adults low levels of the herpesvirus can be detected. Therefore it is necessary to quantitate the amount of viral DNA, and a method to compare the plasmid containing the cloned target gene was used here. After 35 cycles of amplification, 10 copies of the viral DNA per 100 microliters saliva were detected. The PCR was used to detect increased levels of EBV and HHV-6 in saliva from patients with lymphoproliferative diseases, suggesting that these viruses may play a part in their pathogenesis. Viral detection by such highly sensitive methods as PCR may allow better monitoring of medication, as well as early detection of EBV- and HHV-6 related diseases that may arise in these patients. The great sensitivity of PCR and its ability to analyse very small samples make this technique most suitable for clinical diagnosis.

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

The polymeric immunoglobulin receptor (secretory component) in a human intestinal epithelial cell line is up‐regulated by interleukin‐1

Secretory component (SC or polymeric immunoglobulin receptor) on mucosal epithelial cells mediates transcytosis of polymeric immunoglobulin into external fluids and functions as a receptor for polymeric immunoglobulin. SC expression in a human colonic adenocarcinoma cell line, HT‐29 has been reported to be up‐regulated by various cytokines, such as interferon‐&ggr;, tumour necrosis factor‐&agr; and interleukin‐4 (IL‐4). However, up‐regulation of SC by IL‐1 is controversial. In this study, we investigated the effect of human recombinant IL‐1 alone on SC expression in HT‐29 cells in detail. Immunocytochemistry and Northern blot analysis revealed that IL‐1&bgr; increased both the number of SC‐positive cells and SC mRNA expression. Enzyme‐linked immunosorbent assay revealed that IL‐1&bgr; enhanced secretion by HT‐29 cells in both time‐ and dose‐dependent manners. IL‐1&agr; had the same effects on HT‐29 cells. Northern blot analysis demonstrated that cycloheximide and actinomycin D abolished the effect of IL‐1. Moreover, we detected IL‐1 receptor (IL‐1R) type I mRNA in HT‐29 cells by polymerase chain reaction (PCR) and sequenced the PCR‐amplified product. We think that it reflects the possibility of the presence of IL‐1R in HT‐29 cells. From these data, we concluded that IL‐1&bgr; and IL‐1&agr; play regulatory roles in SC expression, and their effects depend on de novo protein synthesis and transcription.

Role of nuclear factor-κ B in the expression by tumor necrosis factor-α of the human polymeric immunoglobulin receptor (pIgR ) gene

Abstract We analyzed the mechanism of human polymeric immunoglobulin receptor (pIgR) gene upregulation by tumor necrosis factor (TNF)-α. Northern blot analysis showed that the expression of pIgR mRNA was enhanced by TNF-α stimulation. This activation was completely inhibited by RNA polymerase or protein synthesis inhibitors, suggesting that the regulation of pIgR gene expression depends on de novo RNA and protein synthesis. Furthermore, the stimulation of pIgR mRNA by TNF-α was decreased by pyrrolidinedithiocarbamate and l-1-4′-tosylamino-phenylethyl-chloromethyl ketone, which are known nuclear factor (NF)-κB inhibitors. For further analysis of gene regulation, we cloned and sequenced the 1.5-kb 5′-flanking region of the pIgR gene. In the upstream region, we found two NF-κB-binding motifs (named κB1 and κB2 from the 5′ region). An electrophoretic mobility shift assay indicated that two components of the NF-κB/Rel family, p50 and p65, bound with higher affinity to the κB2 element than to the κB1 element. We also analyzed pIgR gene expression using reporter plasmids expressing the firefly luciferase gene. Stimulation by TNF-α significantly activated the pIgR gene promoter, as a 775-bp upstream region of the pIgR gene increased luciferase gene expression in cells treated with TNF-α. The activation of promoter activity by TNF-α was abolished when a mutation was inserted into κB1 or κB2. These data indicated that pIgR gene expression induced by TNF-α is transcriptionally regulated via activation of NF-κB. In addition, there is a possibility that another factor may act in concert with NF-κB.