Gall bladder: the predominant source of bile IgA in man?

The sedimentation profiles of IgA and Secretory Component (SC) and the concentrations of IgA, IgG, IgM, SC and albumin were evaluated after an overnight fast in gall bladder bile of six adult subjects without hepatobiliary disease. The sedimentation profiles differed from those previously obtained in hepatic bile in three ways: gall bladder bile contained a greater percentage of free-SC, a greater percentage of polymeric-IgA (p-IgA), and a major peak of 14 to 19 S p-IgA associated to SC. In contrast to hepatic bile in which IgG is the predominant Ig, IgA clearly was the predominant Ig in gall-bladder bile, its concentration averaging 92 micrograms/ml. Relative-to-albumin coefficients of excretion of proteins in gall bladder bile averaged 0.99 for IgG, 8.6 for monomeric IgA, 196 for p-IgA and 31 for IgM, indicating that there was a selective excretion of IgA and IgM into gall bladder bile. As compared to hepatic bile, the enrichment of gall bladder bile with IgA and IgM was respectively 6.5 and 11.5 times greater than with IgG. These results suggest that quite a significant amount of p-IgA could have been added to bile during its storage in the gall bladder which should therefore be regarded as the predominant source of bile IgA in humans.     

Rat bile as a convenient source of secretory IgA and free secretory component

Rat hepatic bile contains three proteins as major constituents: secretory IgA (SIgA), free secretory component (FSC) and albumin. Traces of α-macroglo-bulin, transferrin, IgG and IgM are also detectable. The bile duct daily pours between 5–12 mg each of SIgA and FSC into the rat duodenum. The origin and function of these proteins in bile may represent important clues in the understanding. of the SIgA system.     

Synthesis of IgA by human gastric mucosa in vitro

Human gastric mucosa was removed at gastrectomy for duodenal ulcers. Intact mucosal layer and homogenized mucosal scrapings were incubated in vitro with L-[U-14C]leucine and with D-[U-14C]glucose and incorporation of radioactive label into IgA was investigated. 14C of both the precursors was incorporated into tissue IgA when intact gastric mucosa was incubated indicating synthesis of IgA by the tissue in vitro. IgA isolated from the media which bathed the luminal sides of the mucosae during incubation was also radioactive implying secretion of the newly-synthesized immunoglobulin into the media. When mucosal scrapings were incubated as homogenized suspensions, radioactivity of IgA was below the level of sensitivity of our methods. For analysis, IgA was separated from the bulk of radioactive mucosal proteins of the samples by isopycnic CsCl gradient centrifugation followed by gel filtration on Sephacryl S-200 or Sephadex G200 and then Sepharose 4B. The IgA-enriched Sepharose 4B fractions were subjected (1) to immunoprecipitation in agar gels by double diffusion against specific anti alpha-chain serum and (2) to SDS-polyacrylamide gel electrophoresis after reduction. Radioactivity of precipitated IgA, and of the protein band which corresponds to the alpha-chain of IgA on SDS-PAGE gels, was demonstrated autoradiographically. It is suggested that in vivo the mucosa is involved in local synthesis of the constituent IgA of gastric mucus.     

IgA antibodies in the bile of rats. V. Primacy of the GALT as a source of IgA.

In each of a series of rats the common bile duct and the thoracic duct (cisterna chyli) were cannulated so that both bile and thoracic duct lymph could be collected quantitatively for several hours. The concentrations of IgA in samples of lymph and bile were measured by radioimmunoassay so that the output of IgA per unit time could be calculated. Although the output of IgA in the lymph did not decline significantly, the output in the bile fell so that by 2 hr it had been reduced to less than 20% of the peak value. Similar experiments in rats which had been immunized actively by injecting antigens into the GALT showed a corresponding rapid decline in titres of specific biliary antibodies after fistulation of the thoracic duct. The low levels of IgA in the bile of rats that had been drained of thoracic duct lymph were restored quickly to normal values by the intravenous infusion of a volume of thoracic duct lymph equal to that which had been lost; this restoration was transient, and the concentration of IgA in the bile soon declined again after the infusion ceased.     

Distribution of IgA 1 and IgA 2 plasma cells in various normal human tissues and in the jejunum of plasma IgA-deficient patients.

The distribution of IgA 1 and IgA 2 plasma cells was studied in normal human tissues. IgA 2 is a minor constituent in peripheral lymph nodes as well as in serum and in bone marrow plasma cells. An increased proportion of IgA 2 plasma cells was observed in gastric and intestinal mucosa, as well as in bronchial mucosa and salivary and mammary glands. Tonsils and mesenteric lymph nodes exhibit values intermediate between those of central and peripheral lymphoid systems. In patients with plasma IgA-deficiency, IgA 2 is the predominant intestinal IgA plasma cells. This may explain the frequent association of an asymptomatic condition and plasma IgA deficiency.     

Distribution of plasma cells in normal rectal mucosa

In a survey of 36 histologically normal rectal biopsies, plasma cell counts were recorded at different depths of the lamina propria. The necessity of surveying the lamina propria at every level from the muscularis mucosae to the epithelium, in order to obtain an accurate estimate of plasma cell frequency is demonstrated. The relative accuracy of counting smaller areas of lamina propria is tested and the ratio of epithelium to lamina propria area established.     

A novel cell-surface marker found on human embryonic hepatoblasts and a subpopulation of hepatic biliary epithelial cells

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM-5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM-5 is found on a 50-kDa protein present on the surface of these cells. In tissue sections of first-trimester human embryos, GCTM-5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM-5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM-5-positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM-5-positive cells that did not express cytokeratin 19 or N-CAM, classical makers of ductular reactive cells. Live GCTM-5-positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM-5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.     

Hydrophobic bile acids suppress expression of AE2 in biliary epithelial cells and induce bile duct inflammation in primary biliary cholangitis

Understanding the mechanisms of chronic inflammation in primary biliary cholangitis (PBC) is essential for successful treatment. Earlier work has demonstrated that patients with PBC have reduced expression of the anion exchanger 2 (AE2) on biliary epithelial cells (BEC) and deletion of AE2 gene has led to a PBC-like disorder in mice. To directly address the role of AE2 in preventing PBC pathogenesis, we took advantage of our ability to isolate human BEC and autologous splenic mononuclear cells (SMC). We studied the influence of hydrophobic bile acids, in particular, glycochenodeoxycholic acid (GCDC), on AE2 expression in BEC and the subsequent impact on the phenotypes of BEC and local inflammatory responses. We demonstrate herein that GCDC reduces AE2 expression in BEC through induction of reactive oxygen species (ROS), which enhances senescence of BEC. In addition, a reduction of AE2 levels by either GCDC or another AE2 inhibitor upregulates expression of CD40 and HLA-DR as well as production of IL-6, IL-8 and CXCL10 from BEC in response to toll like receptor ligands, an effect suppressed by inhibition of ROS. Importantly, reduced AE2 expression enhances the migration of autologous splenic mononuclear cells (SMC) towards BEC. In conclusion, our data highlight a key functional role of AE2 in the maintenance of the normal physiology of BEC and the pathogenic consequences of reduced AE2 expression, including abnormal intrinsic characteristics of BEC and their production of signal molecules that lead to the chronic inflammatory responses in small bile ducts.     

Variously-formed inclusions in the rough endoplasmic reticulum of plasma cells in the human oviduct mucosa

Human oviducts, obtained from 60 women having undergone total hysterectomy due to diseases of the uterus or ovary, were studied for cyclic changes by electron microscopy. In the studies, plasma cells were frequently observed in the lamina propria of the oviductal mucosa. Russell bodies were occasionally encountered in the plasma cells. In the oviduct mucosa obtained from a patient with cervical carcinoma, a plasma cell contained C-shaped or worm-like structures in an extensively-distended cisterna of the rER. These structures were morphologically quite similar to type A curvilinear bodies in lysosomes of certain lysosomal diseases. Additionally, intracisternal tubules were noted in slightly dilated cisternae of other rER. The origins and functional significance of curvilinear inclusions and intracisternal tubules in the plasma cell remain obscure.     

Oxidative stress-induced apoptosis of bile duct cells in primary biliary cirrhosis

There has been a relative paucity of effort at defining effector mechanisms of biliary damage in PBC. We hypothesize that biliary cells are destroyed secondary to the immunologic relationships of inflammation and biliary epithelial apoptosis and, in particular, that biliary damage is a result of reduced levels of glutathione-S-transferase (GST), the production of hypochlorous acid (HOCl) and its association with eosinophil peroxidase (EPO). To address this issue, we examined the expression of EPO and GST in PBC and control livers and demonstrated an increase of EPO within the portal areas of PBC. We also demonstrated that macrophages have evidence of phagocytosed EPO. Furthermore, we studied the influence of HOCl on apoptosis in cultured human biliary epithelial cells (BEC) as well as the associated activity of Bcl-2, Bax, p-JNK, JNK, p53, Fas and caspase-3. HOC1-induced apoptosis in BEC in a dose-dependent fashion increased the activity of caspase-3 and the expression of p53 and p-JNK. Pretreatment with l-buthionine-(S,R)-sulfoximine, a glutathione (GSH) inhibitor, potentiated the sensitivity of BEC to HOCl-induced apoptosis. We conclude that intracellular GSH reduction leads directly to BEC apoptosis. Modulation of these events will be critical to reduce immune-mediated destruction.     

Quantitation of immunoglobulin-producing cells in small intestinal mucosa of patients with IgA nephropathy

The numbers and proportions of immunocytes producing IgA, IgM, or IgG were found to be normal in the proximal small intestinal mucosa of patients with IgA nephropathy. There was no indication of any quantitative aberration in the mucosal humoral immune system at this secretory tissue site.     

Apoptosis of bile duct epithelial cells in hepatic allograft rejection

Liver biopsy remains the 'gold standard' for monitoring rejection in liver transplant patients. Portal inflammation, bile duct damage and endothelialitis are recognized features of hepatic allograft rejection. The pathogenesis of the bile duct injury during rejection, however, remains unclear. To define the mechanism of bile duct damage, we studied the light- and electronmicroscopic appearance of hepatic tissue from selected patients in whom allograft failure was solely due to rejection. Of the 25 orthotopic liver transplant rejection cases examined, 17 were mild, seven were moderate and one was severe rejection. Light microscopy examination of the damaged bile duct epithelium revealed evidence of apoptosis which was confirmed by electronmicroscopy. Furthermore, there appeared to be a positive correlation between the grade of rejection and the number of apoptotic cells. Also included in the study were 13 cases of chronic active hepatitis and 10 normal livers which showed the least apoptotic cells. We conclude that the identification of apoptotic cells in damaged bile ducts in allograft biopsies might be helpful in the diagnosis of rejection and in assessment of the severity of rejection.     

IgA deficiency in children. Immunoglobulin-containing cells in the intestinal mucosa, immunoglobulins in secretions and serum IgA levels

Twenty-eight children with IgA deficiency were investigated by studying immunoglobulin-containing cells in the jejunal and rectal mucosa by direct immunofluorescence and by measuring immunoglobulins in the intestinal juice and saliva. Serum IgA was measured by radioactive single radial immunodiffusion.     

Appearance of IgG and IgA antibodies in human bile after tetanus toxoid immunization

Humans immunized intramuscularly with one dose of tetanus toxoid exhibited IgG, and in some cases IgA antibody, in their bile as well as serum. Both isotypes appeared in bile transiently with titres declining after about day 10 for both classes. These kinetics resembled those of the serum IgA response but were markedly different to those for IgG antibody in serum. Measured IgG titres in bile were between 0.07 and 4.2% of those in paired sera, and IgA titres were between 6.8 and 124% of sera. Peak responses in bile, while generally of smaller size, exceeded those of paired sera when expressed as antibody/mg of IgG or IgA present. This calculation showed that during the peak response bile was up to nineteen-fold more abundant in IgG antibody than was serum taken at the same time, and up to forty-five-fold more for IgA. Enrichment of antibody in bile is not consistent with the Ig of bile being solely conferred by plasma, and may mean the involvement of local synthesis too. This study indicates that tetanus toxoid immunization of humans results in biliary antibody and raises the possibility of intra-hepatic antibody production for export to the intestinal tract in man.     

The human IgA system: a reassessment

In healthy adults the total daily production of secretory and serum IgA exceeds that of other immunoglobulin classes. Secretory and serum IgA display features of mutual independence: they are represented by molecules with different physiochemical and immunochemical properties and antibody activities and are produced by cells with different organ distributions. Secretory and serum IgA also exhibit different effector functions: interaction of secretory IgA with environmental antigens results in prevention of the penetration of such antigens by a variety of mechanisms. Although the function of polymeric serum IgA antibodies in certain animal species involves elimination of antigenic substances by noninflammatory means, the primary function of serum IgA remains unknown. It is proposed that in humans monomeric serum IgA (which prevents activation of the complement systems, inhibits phagocytosis and antibody-dependent cellular cytotoxicity) may protect endogenous antigens expressed on various cells and tissues by preventing their interaction with humoral and cellular immune mechanisms that may lead to tissue damage.     

Small mucus-granule-containing ciliated cells in the human gastric mucosa: a transitional form to metaplastic ciliated cells

Summary Ciliated cells were found in the gastric mucosa in close association with intestinal metaplasia, mainly in the pyloric mucosa, of Japanese patients. The occurrence of ciliated cells is believed to be an acquired phenomenon and is considered to be a type of metaplasia; the term ciliated metaplasia is used to describe this phenomenon. Ciliated cells are found in the basal part of the glands among normal-looking mucous cells, mucous neck cells and neuroendocrine cells, but never on the surface or in foveolar epithelium. In ciliated cellcontaining glands, mitoses were noted in the neck region and the ultrastructural features of these cells were identical to those of undifferentiated neck cells. However, cell metaplasia from undifferentiated cells to metaplastic ciliated cells has never been demonstrated previously. The small mucus-granule-containing ciliated cells found in our present study may arise subsequent to division of undifferentiated neck cells into mucous cells with some daughter cells then exhibiting differentiation characteristics specific to ciliated cells. Thus they contain a mixture of both small mucus granules and numerous basal bodies and cilia, at the same time as a transitional form.