Rapid active transport of immunoglobulin A from blood to bile.

Immunoglobulins were isolated from the serum or ascitic fluid of Lou/Wsl rats bearing plasmacytomas and labeled with 125I. When labeled IgA was injected i.v. it disappeared from the blood serum much more rapidly than IgG2 so that after 3 h less than 10% remained. This rapid disappearance of the injected IgA was not seen in rats with ligated bile ducts. In rats with cannulated bile ducts, the labeled IgA appeared rapidly in the bile so that 25% of the injected dose was recovered in 3 h; at the peak of this biliary excretion the specific radioactivity of the bile (cpm/milligram protein) was about 200 times greater than that of the blood serum. Thus much of the IgA which finds its way into the blood is rapidly and actively transported across the liver so that it enters the gut lumen via the biliary tract.

Secretory component as the receptor for polymeric IgA on rat hepatocytes.

Rat hepatocytes in short-term monolayer cultures bound radiolabeled polymeric rat IgA but not IgG. The binding of 125I-IgA was inhibited equally well by unlabeled polymeric IgA and by antiserum to rat secretory component (SC). The antibody to SC, after specific purification and radiolabeling, was bound to hepatocytes as effectively as the IgA. These results indicate that SC acts as the receptor for polymeric IgA on rat hepatocytes as it does on human gut epithelia, and that the transport of IgA from blood to bile in rats across the liver is analogous to that of IgA across human enterocytes.

Endocytic vesicles in liver carry polymeric IgA from serum to bile.

The distributions both of endogenous IgA and of injected 125I-labelled IgA were determined amongst the components of a liver homogenate. Rate zonal sedimentation, under conditions where separation was principally determined by particle size, showed that IgA was tightly bound to material which sedimented in the size range of the larger endoplasmic reticulum fragments. Further fractionation of the components within this size range according to their densities, by isopycnic centrifugation, showed that the IgA was associated with small vesicles with a density range of 1.12--1.17 g/ml, quite distinct from endoplasmic reticulum fragments. We therefore conclude that the IgA is present in liver cells in a distinct class of vesicles, which are, presumably, responsible for the transport of IgA from blood to bile.

The use of detergents and immunoperoxidase reagents for the ultrastructural demonstration of internal immunoglobulin in lymph cells.

Lymph-borne immunoblasts were fixed in dilute glutaraldehyde and then treated with saponin. This treatment made most parts of the cells permeable to ferritin, so that anti-immunoglobulin (Ig) antibodies which had been conjugated to horse radish peroxidase (HRP) had no difficulty in gaining access to Ig which thus could be demonstrated at an ultrastructural level. Best results were obtained by fixing the cells in 0.1% glutaraldehyde for 7 min and then treating them with a 1% solution of saponin for 100 min at 55 degrees C before exposing them to the Ig-HRP conjugate. The method yielded reproducible results and although it causes a small amount of ultrastructural damage, it may be of value in detecting a variety of intracellular antigens.

The antigenic interrelations of some mammalian IgG subclasses detected with cross-reacting fowl antisera to human and mouse IgG-Fc.

Two fowl antisera to plyclonal human IgG and one to a mouse IgG1 myeloma protein, after absorption either with homologous F(ab)2 alone, or also with myeloma proteins of known subclasses, cross-reacted in immunoelectrophoresis or immunodiffision withthe IgG of many mammalian species. Often more than one IgG precipitation line was formed in the corss-reacting systems. By testing isolated IgG1 and IgG2 fractions from mouse, rat, guinea-pig, bovine and dog sera, it was shown that the distinct arcs seen with whole serum corresponded to the known IgG subclasses of these species. With rabbit serum as antigen, the two anti-human-Fc sera diffusing from neighboring wells formed a 'spur', showing that they were reacting with determinants on different molecules, and that therefore rabbit IgG contains two antigenically distinct Fcpopulations. With the possible exception of canine IgG1 and human IgG4, no antigenic homologies were found between the subclasses of different species, thus supportingthe view obtained from sequence data, namely that IgG subclasses within species havearisen independently.

The biological half-lives of four rat immunoglobulin isotypes.

Purified preparations of monoclonal rat IgG2a, IgG1, IgM and monomeric IgA, and of polyclonal IgG, were lightly radiolabelled with 125I and injected intravenously into rats. Blood samples were taken so that the half-life of each immunoglobulin isotype could be calculated. Half-life was defined as the time taken for the specific radioactivity present in the blood 24 h after injection to fall by half. The half-life values thus obtained were 106 h for IgG2a, 53 h for IgG1, 63 h for polyclonal IgG, 25 h for IgM and 27 h for monomeric IgA.

Studies on the lymphocytes of sheep. II. Some properties of cells in various compartments of the recirculating lymphocyte pool.

The proportion of cells bearing surface immunoglobulin (Ig) and the normal lymphocyte transfer (NLT) reactivity of mononuclear cells obtained from the blood, afferent lymph, and efferent lymph from both somatic and intestinal nodes of sheep were measured. The proportion of Ig-positive lymphocytes was approximately 30% in blood and efferent lymph, but only about 10% in afferent lymph. The proportion of unlabeled lymphocytes (T cells) was about the same in all the preparations, as afferent lymph contains 15--20% macrophages. Despite this apparent uniformity in the T cell content, the NLT reactivity of efferent lymph was 3 to 5 times higher than that of blood or afferent lymph. No humoral factors could be detected by incubating cells in the various plasmas in vitro before injection. No inhibitory cells could be detected when mixtures of cell populations were used to initiate lesions. We suggest that lymphocytes in efferent lymph are more reactive than others because they are "groomed" by macrophages during their recirculatory passage through the substance of the node.

Levels of adenosine deaminase in some experimental animal tumours and the possible therapeutic effect of the ADA inhibitor 2-deoxy-coformycin.

The intracellular adenosine deaminase activities (ADA) in 12 different experimental animal tumours were measured. Unlike the leukaemic lymphoblasts of man, those of two spontaneous rat leukaemias did not have elevated levels of the enzyme. Very high levels were found in a rat plasma-cell tumour (IR 461) and an attempt was made to treat such tumours with the specific enzyme inhibitor, 2-deoxy-coformycin. The shortage of this drug prevented a systematic study, but a daily dose of 8 mg/kg had a significant inhibitory effect on the growth of tumours.

Studies on the lymphocytes of sheep. III. Destination of lymph-borne immunoblasts in relation to their tissue of origin.

Lymph-borne immunoblasts were labeled in vitro with 125I[]iodo-deoxy-uridine, washed and returned by intravenous injection to the sheep from which they had been collected. Twenty h later the sheep were killed and the distribution of the immunoblasts was determined by assaying the radioactivity in various organs. Immunoblasts from the efferent lymph of peripheral somatic lymph nodes (PSLN) went mainly to the spleen, lungs and other PSLN, while immunoblasts from intestinal lymph went mainly to the small gut. This ability of intestinal immunoblasts to home to the gut was demonstrated also in the sterile environment of fetuses in utero; apparently the migratory behavior of immunoblasts, like that of small lymphocytes, is not primarily "antigen-driven". A technique was devised for the collection of peripheral (i.e. afferent to the mesenteric node) intestinal lymph which was found to contain 10-20 times the numbers of small lymphocytes that occur in the peripheral lymph from other tissues. Immunoblasts from peripheral intestinal lymph also homed to the gut. The immunoglobulin content of immunoblasts was studied by making detergent extracts of lymph cells, by applying immuno-peroxidase techniques to cell films and by investigating the incorporation of 14C-labeled amino acids into immunoglobulins by immunoblasts in vitro. Immunoblasts from both somatic and intestinal lymph contained and made IgG and IgM, but many intestinal immunoblasts contained and made IgA. It is not known whether this immunoglobulin mediates the extravasation of immunoblasts into the gut. Nonetheless, there is compelling evidence that there are two major migratory pathways for lymphoid cells; one through the gut-associated lymphoid tissue and the other through the somatic-splenic lymphoid tissues.

The detection of F(ab')2-related surface antigens on the thymocytes of children.

A Fowl antiserum to human polyclonal F(ab')2 with specificities for variable region antigens of both light and heavy chains and for the constant portion of the Fd fragment bound to about 20 per cent of thymocytes from three children. Binding was completely inhibited by purified human polyclonal IgG and only partly by L chains. The fowl anti-F(ab')2 also bound to a subpopulation of peripheral blood lymphocytes which was somewhat larger than the B-cell population identified with other anti-immunoglobulin sera. Binding was detected by autoradiography and the amount of radiolabel seen on thymocytes was nearly as great as that on peripheral blood lymphocytes. As no binding to thymocytes was seen with other anti-immunoglobulin sera we propose that the fowl anti-F(ab')2 recognizes immunoglobulin or material with structural similarities which is synthesized by thymocytes but of which predominantly V region determinants are exposed on the membrane surface.

The elimination of circulating complexes containing polymeric IgA by excretion in the bile.

Radiolabelled human dimeric IgA injected intravenously into rats behaved like oligomeric rat IgA in that 40% of the injected dose was recovered in the bile within 6 h. Monomeric IgA was not transported into bile. In addition, dimeric IgA was able to carry with it macromolecular material in the form of a complex that also included rat SC. This was demonstrated in rats which had received an intravenous injection of specifically purified radiolabelled rabbit antibody tao the idiotype of a human IgA myeloma: when a later injection of unlabelled IgA dimer with the corresponding idiotype was given up to 18% of the rabbit antibody when the monomeric form of the same IgA was injected. This mechanism for clearing complexes containing polymeric IgA is mediated by hepatocytes and is distinct from the phagocyte-mediated mechanisms which clear conventional complexes.

The origin of IgA in chicken bile: its rapid active transport from blood.

In the chicken, there are two bile ducts, one draining the left lobe of the liver directly into the duodenum and the other draining the right lobe via the gall bladder. Cannulation of these ducts enabled us to collect bile in unanesthetized birds and to compare the IgA concentrations of the hepatic bile, gall bladder bile and blood serum. Bile from the cystic and hepatic ducts of the same bird contained similar amounts of IgA (1.7 mg/ml), roughly 10 times as much as in serum, but considerably less than that found in the concentrated bile collected by aspiration from the gall bladder (8 mg/ml). Ligation of the two bile ducts resulted in a three to fourfold increase in the concentration of IgA in serum suggesting that IgA is normally removed from serum by the biliary route. This was confirmed by a substantial recovery of i.v. injected, radiolabeled monoclonal dimeric human IgA in the bile corresponding to a rapid active clearance from the blood circulation; negligible amounts of monomeric human IgA, similarly injected, were recovered from the bile.

The transport by hepatocytes of immunoglobulin A from blood to bile visualized by autoradiography and electron microscopy.

Polymeric myeloma IgA, labelled with 125I, was injected intravenously into rats that were killed 5, 30, or 60 min later and the livers removed, fixed and sectioned. Autoradiographs of ultra-thin sections examined in the electron microscope showed that the IgA first became bound to the plasma membrane of the hepatocytes but after 30 min much of it was transported across their cytoplasm and became localized around the bile canaliculi. At this time, autoradiographs of 1 micrometer sections examined in the light microscope showed the contents of the bile ducts in the portal tracts to be labelled heavily. These results confirm the previous finding of rapid transport of IgA across the liver and show directly that the hepatocytes are the cells that carry it out. No intracellular organelle or vesicular structure, discernible within the resolving power of the techniques used, could be implicated in the transport mechanism.

Distribution of secretory component in hepatocytes and its mode of transfer into bile.

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound (125)I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [(14)C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.

Elimination into bile of circulating antigen by endogenous IgA antibody in rats.

Rats injected once into their Peyer's patches with insoluble precipitates of chicken antibody and antigen produced antibodies to chicken IgG (CGG) of the IgM and IgA classes which rose steeply between days 3 and 5 after injection; IgG antibody was detected later and rose more slowly. The IgA antibody was almost entirely in the bile whilst IgM and IgG were found predominantly in serum. In immunized rats with cannulated bile ducts injected intravenously with radiolabelled CGG on day 5 up to 22% of the injected dose was recovered in the bile in 24 hr; in control rats the maximum recovery was 0.4%. Although the complexes were unstable, intact 125I-CGG (some of it bound to rat IgA and secretory component) was demonstrated in bile collected from immunized rats between 1.5 and 3 hr after injection.