Jane Peppard

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.

Occurrence of Specific Antibodies of the IgA Class in the Bile of Rats

In pooled bile, obtained by cannulating the common bile duct of normal rats, the immunoglobulins were mainly of the IgA class but the total immunoglobulin concentration was 20 times less than in the b

EFFECT OF COLCHICINE ON THE TRANSFER OF IgA ACROSS HEPATOCYTES INTO BILE IN ISOLATED PERFUSED RAT LIVERS

In hepatocytes a stream of endocytic vesicles moves rapidly from the sinusoidal surface to the cyto- plasm in the region of the bile canaliculus. The vesi- cles carry polymeric IgA or the haptoglobin-haemo- globin complex if these are available in the blood [l-4]. The rapid concentration in a particular region of cytoplasm suggests that cytoskelktal elements may be involved. Accordingly we have examined the effects of the microtubule-disruptive drug, colchicine, on the transport of IgA across hepatocytes. Transportation of IgA across hepatocytes involves at least 4 separate processes which could require participation of microtubules. (1) Before uptake of IgA can take place, the receptor for IgA, the glycoprotein secretory component, which is synthesised in hepatocytes [5], must itself reach the sinusoidal plasma membrane [6]. Such movement of glycoproteins from the Golgi apparatus to the plasma membrane is inhibited by colchicine although glycoprotein synthesis is unaffected [7]. (2) However, if this were the only point of interfer- ence, the secretory component already on the membrane before the addition of colchicine would be expected to continue to transfer any IgA available to it, and also continue to move to the bile on its own [4]. The actual formation of endocytic vesicles is unlikely to be inhibited since in [8] the movement of the plasma mem- brane enzyme 5’-nucleotidase into the interior of the cell was unaffected by colchicine. t

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.

Lymphatic Physiology and Secretory Immunity

The chyle-filled lacteals of the mesentery, and the various components of the gut-associated lymphoid tissue (GALT) were noted by the earliest anatomists, but their role in mucosal immunity became apparent relatively recently and is still a somewhat controversial topic. It is true, unfortunately, that even amongst mammals, differences in reproductive and digestive physiology and age-related changes in the GALT make a general understanding of the immune systems of their various mucosae particularly difficult. Therefore, it may be helpful to review briefly some of the factors which are thought to govern the behaviour of those lymphoid cells and humoral factors that are associated with immune reactions at mucous surfaces.

ELIMINATION OF CIRCULATING ANTIGEN INTO BILE BY ENDOGENOUS IgA ANTIBODY IN RATS

The active transfer of polymeric IgA from blood to bile in several species, by a mechanism that involves secretory component (SC] of rats, is now well documented.’ That an antigen that is combined with polymeric IgA antibody can also be carried across the liver into bile by this mechanism has been demonstrated using several model systems.’-‘ These models showed that antigen-antibody complexes combining polymeric IgA, but not IgM, IgG, or monomeric IgA, injected intravenously (i.v.1 would traverse the liver and appear in bile, using SC as a receptor. We were interested in determining whether endogenously produced polyclonal IgA antibody would mediate in the same way the transhepatic clearance of i.v. injected antigen in vivo. To stimulate an IgA-antibody response to a soluble antigen in rats, insoluble immune complexes of chicken antibody and antigen were injected into Peyer’s patches. This injection elicited an antibody response to chicken IgG (CGG) detected by radioimmunoassay in both blood and bile, beginning at about three days after immunization. Serum antibody was predominantly of the IgM and IgG isotypes, whereas bile antibody was all IgA. Rats immunized in this fashion 5 days before were given bile duct cannulae and [“‘I] CGG was injected i.v.; up to 22% (TCA precipitable) of the injected dose of [lZ5I] CGG was recovered in the bile in 24 hours, whereas in control (unimmunized) rats, a maximum of 0.4% was collected. The [lZ5I] CGG was intact antigenically in that it was precipitated by anti-chicken IgG sera. In contrast, however, to previous experiments of ours using a “model” System,’ it was difficult to show that the radiolabeled protein was combined with IgA or SC after being transported. Only those samples of bile tested immediately after collection contained small amounts of such complexes. These experiments, described completely in Reference 5, show that polyclonal polymeric IgA antibodies would perform the function in vivo of “mopping up” specific antigen from the systemic circulation by clearance through the liver, and would presumably also function at the gut epithelial surface, should antigen manage to penetrate from the gut. It seems likely, however, from these observations that most of the antigen carried by such IgA, which may be of low affinity, would, at least in bile, be present free, and not in combination with IgA and SC. Whether this dissociation is carried out by the action of bile or within the transporting cell itself remains to be seen.