Capping of saccharides on the plasma membrane of lymphocytes as studied by fluorescein-labelled lectins

The capping of saccharides on the plasma membrane of rat splenic lymphocytes was studied by means of fluorescein-labelled lectins. Treatment of unfixed splenic lymphocytes with any one of the three lectins, concanavalin A (Con A), Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) led to the formation of caps of each saccharide receptor on the plasma membrane. Treatment of unfixed lymphocytes with Con A was found to result in the formation of caps of saccharide receptors for RCA, whereas cap formations were never noted in such double treatment of the cells with all other combined uses of two lectins. These results are taken to indicate that the saccharide receptors for Con A are associated with those for RCA in the plasma membrane of rat splenic lymphocytes.     

Characteristics of human IgA and IgM genes used by plasma cells in the salivary gland resemble those used in duodenum but not those used in the spleen

Immunologically, the parotid salivary gland is an effector site that secretes large quantities of polyspecific Abs into the saliva, mainly of the IgA isotype. It is considered to be part of the common mucosal immune system but the inductive site for the Ab-producing cells of the salivary gland has not yet been clearly identified. The origin and diversity of cells of B lineage can be investigated by analyzing their Ig heavy chain genes (IgH). We have obtained sequences of IgM and IgA VH4-34 genes from plasma cells in human salivary gland, duodenal lamina propria, and splenic red pulp. Related sequences were found in different areas sampled within each tissue studied, indicating that the plasma cells carrying these genes are widespread with limited diversity. Examples of related IgH genes that are isotype switched were also seen in the salivary gland. The genes from plasma cells of the salivary gland were highly mutated, as were duodenal plasma cell sequences. The level of mutation was significantly higher than that seen in splenic plasma cell sequences. Analysis of CDR3 regions showed that the sequences from salivary gland had significantly smaller CDR3 regions than sequences from spleen, due to differences in number and type of DH regions used. Sequences from duodenum also had smaller CDR3 regions. Therefore, plasma cells from human duodenum and salivary gland showed characteristics that differed from those of human splenic plasma cells.     

Reversal of immunosuppression induced with plasma of malarious rats by supplemented complement

Suppression of antibody producing splenic lymphocytes by plasma from rats infected with Plasmodium chabaudi malaria was confirmed. Suppressive activity was found in plasma drawn on the sixth, seventh and eighth day of infection. It was temporally associated with anemia, elevated levels of soluble immune complex, reduced titers of lytic complement and elevated titers of immunoconglutinin (IK) in the plasma. Heat inactivation of the plasma to destroy complement and removal of IK by absorption did not reduce the suppressive activity. Incubating the plasma-treated lymphocytes with normal rat complement largely, but not completely, reversed the suppressive action. Soluble immune complexes prepared from bovine serum albumin (BSA) and antiBSA (BSA-antiBSA) alexinated complex (BSA-antiBSA-C') and immunoconglutinated complex (BSA-antiBSA-C'-IK) each suppressed the capacity of splenic lymphocytes from rats immunized with sheep blood cells to produce hemolytic Jerne plaques. Incubating the complex-treated cells with fresh complement largely reversed the suppressive activity. It is suggested that the suppressed responses of lymphocytes from malarious animals to antigens or mitogens, reported by others, may have been in part induced by complexes in blood of the animals, and that antibody producing cells might also have been suppressed. Since suppressive activity was not influenced by complement inactivation, but was reversed when plasma-treated cells were incubated with fresh complement, it is suggested that the hypocomplementemic state of suppressive plasma may have contributed to immunosuppression.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

Splenic storage volume in the unanesthetized resting beagle

Determination of the dog's splenic storage volume without surgical procedures requires measurement of both circulating and total red cell volumes. The estimation of circulating red cell volume by radioisotope techniques is impeded by the rapid uptake of tagged cells in the spleen. The circulating cell volume might be calculated from plasma volume and large vessel hematocrit, provided that the latter is corrected for the unequal distribution of red cells in the circulatory system. However, the correction factor can only be estimated in the splenectomized dog. We describe here a method to determine the factor in the intact dog, "physiologically splenectomized" by the severe exercise. The values obtained by this method slightly exceed those in the resting dog, as shown by studies in splenectomized exercising beagles in which splenic function was simulated by infusion of packed cells. The method was tested in beagles exercised by swimming and treadmill running and it was concluded that in the unanesthetized resting beagle about one-third of all erythrocytes is stored in the spleen. Labeled cells are equilibrated with about one-half of the splenic storage volume within 10 min after their injection. During maximal exertion the mean increase in large vessel hematocrit was 38.6 +/- 3.3%, the mean decrease in plasma volume 13.6 +/- 1.7% and the mean increase in plasma osmolarity 2.8 +/- 0.9% (percentages of control values).     

Enhanced differentiation of splenic plasma cells but diminished long-lived high-affinity bone marrow plasma cells in aged mice

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.     

Constant number of immunogloblin G positive plasma cells around the penicilli in murine spleen with increasing age

The splenic lymphoid tissue of mice reared in a conventional environment regressed with increasing age. Immunogloblin G positive plasma cells gathered along the penicilli in the senile spleen as well as in the adult control spleen. The number of plasma cells around the penicilli showed no difference in the senile spleen as compared to the adult spleen. Since mature plasma cells remain in the lymph node for a short time only and seldom divide there, our results indicate that the transformation from B lymphocyte to plasma cell is not affected by age.     

Nicotinamide adenine dinucleotide splitting enzyme: a plasma membrane protein of murine macrophages.

The subcellular distribution of NADase in splenic and peritoneal macrophages of the mouse has been studied. Conventional procedures for fractionation and isolation of subcellular components demonstrated that the NADase of murine macrophages was localized in the microsomal fraction. By using the diazonium salt of sulfanilic acid, a nonpenetrating reagent known to inactivate ecto-enzymes in intact cells, purified plasma membrane preparations, and marker enzymes, 5′-nucleotidase for plasma membrane and glucose 6-phosphatase for the microsomal fraction, we have shown that: (i) NADase of murine macrophages is a plasma membrane ecto-enzyme and (ii) the microsomal fraction is a mixture of endoplasmic reticulum and plasma membrane elements. At 5 × 10^?4 M concentration, the diazonium salt of sulfanilic acid drastically decreased NADase in intact splenic and peritoneal macrophages of the mouse. 5′-Nucleotidase was similarly inhibited by this reagent, whereas the activity of glucose 6-phosphatase remained unaffected. There was a good recovery of NADase of high specific activity in plasma membrane preparations that were characterized by high 5′-nucleotidase and low glucose 6-phosphatase activity.     

Induction of expression of protein disulphide-isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide

Protein disulphide-isomerase (PDI) activity, and the level of immunodetectable PDI protein, were monitored in splenic lymphocytes and in BCL1 cells during culture in the presence of various activating factors. Bacterial lipopolysaccharide stimulated induction of PDI in splenic B cells and BCL1 cells. The time-course and specificity of induction indicated that the increase in expression of PDI is closely coupled to the final stages of B cell differentiation into antibody-producing plasma cells. The system will prove valuable in studies on the control of expression of PDI.     

Plasmablast and plasma cell production and distribution in trout immune tissues

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.     

Oral administration of freeze-dried kefir reduces intestinal permeation of and oral sensitization to ovalbumin in mice

An increase in plasma ovalbumin concentrations after intragastric administration of ovalbumin was suppressed by concomitant freeze-dried kefir in BALB/c mice. Serum levels of ovalbumin-specific immunoglobulin G and proliferation of splenic mononuclear cells in mice immunized orally with ovalbumin were suppressed by feeding freeze-dried kefir. We propose that kefir reduces intestinal permeation of food antigen, which contributes to suppression of oral sensitization.     

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.     

Biochemical and enzymatic characterization of thymic and splenic lymphocyte plasma membranes from inbred rats.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.     

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.     

Trypanosoma lewisi: electron microscopy of the infected spleen

The spleens of Lewis rats, both normal and infected with Trypanosoma lewisi were examined by electron microscopy. Special attention was directed to clusters of splenic cells which occur in the course of the infection. The reticular cells first showed alterations of their structure by the second day of infection, with considerable surface membrane activity. By the fourth day and thereafter various cells were found gathered around the reticular cells. These cell clusters mainly contained lymphocytes, plasma cells, and erythropoietic elements in many stages of differentiation. It was not unusual that several cell types were found adjacent to the same central reticular cell. These arrays, similar in geometry to the erythropoietic island of the bone marrow, became more predominantly “plasma cell” islands as the infection progressed. Parasites were recognizable within the reticular cells, and were noted to be in regions where the cellular membranes of adjacent cells demonstrated vesiculations resembling rhopheocytosis. A further observation was the pinching off of neighboring plasma cell cytoplasm into the reticular cells.     

Genetic control of the B cell response to LPS: opposing effects in peritoneal versus splenic B cell populations

Lipopolysaccharide (LPS) from gram-negative bacteria activates B cells, enabling them to proliferate and differentiate into plasma cells. This response is critically dependent on the expression of TLR4; but other genes, such as RP105 and MHC class II, have also been shown to contribute to B cell LPS response. Here, we have evaluated the role of genetic control of the B cell response to LPS at the single cell level. We compared the response to LPS of peritoneal cavity (PEC) and splenic B cells on the BALB/c genetic background (LPS-low responder) to those on the C57BL/6J background (LPS-high responder) and their F1 progeny (CB6F1). Both PEC and splenic B cells from B6 exhibited 100% clonal growth in the presence of LPS; whereas, BALB/c PEC and splenic B cells achieved only 50% and 23% clonal growth, respectively. Adding CpG to the LPS stimulus pushed PEC B cell clonal growth in the low responder strain BALB/c up to 90%, showing that the nonresponse to LPS is a specific effect. Surprisingly, PEC B cells on the F1 background behaved as high responders, while splenic B cells behaved as low responders to LPS. The data presented here reveals a previous unsuspected behavior in the genetic control of the B cell response to LPS with an opposing impact in splenic versus peritoneal cavity B cells. These results suggest the existence of an, as yet, unidentified genetic factor exclusively expressed by coelomic B cells that contributes to the control of the LPS signaling pathway in the B lymphocyte.     

T cell recruitment from the thymus to the spleen in tumor-bearing mice

Summary After inoculation of tumor cells (methylcholanthrene-induced sarcoma), the number of Thy 1⁺ cells and PNA (peanut agglutinin) binding cells, which were shown to be different subpopulations were increased in the spleen of thymus-intact mice, in contrast this increase was not observed in adult thymectomized mice. In experiments performed concurrently with splenic cell analysis, we found that the plasma PGE₂ levels declined in parallel with the tumor growth. Prevention of such a decline of plasma PGE₂ level by replenishment with exogenous PGE₂ inhibited the splenic cell increase in tumor bearers. In the tumorbearing mice, cell traffic systems from the thymus to the periphery was ascertained by injecting fluorescein diacetate (FDA) into the thymus and observing fluorescein positive cells in the periphery. We suggest that increased recruitment of thymic cells to the periphery may be mediated by PGE₂ in the presence of a tumor.Abbreviations PNA⁺ cells, peanut agglutinin binding cells - Ig⁺ cells, surface IgM positive cells - Thy 1⁺ Thy 1.2 antigen positive cells - PGE₂ prostaglandin E₂     

The surface-connected canalicular system in the sinus endothelial cells of rat spleen

The existence of a surface-connected canalicular system in the splenic sinus endothelial cells of the rat has been demonstrated by transmission electron microscopy with lanthanum nitrate acting as a tracer for the extracellular space. In addition, the three-dimensional arrangement of the canaliculi has been revealed by computer-aided reconstruction. The surface-connected canalicular system of the sinus endothelial cells consists of slender canaliculi that are branched, anastomosed, and that show continuity with the plasma membrane. They twist in and out among the organelles and are often found in close apposition to the spherical invaginations of the plasma membrane and run alongside them. Canaliculi which are not infiltrated by lanthanum nitrate take the form of electron-lucent tubules and are accompanied by numerous spherical invaginations of the plasma membrane. From a computer-aided reconstruction, the canaliculi, which invaginate from various sites of the plasma membrane, have been found to be continuous with each other and to penetrate to the surface of the sinus endothelial cell; they also branch and anastomose to form a complex network in the cytoplasm. Although the surface-connected canalicular system in blood platelets and thrombocytes is believed to function as the main route for the discharge of granules and the uptake of foreign materials and also to take part in the storage and transport of calcium, it is unclear at present whether the network of the surface-connected canalicular system in splenic sinus endothelial cells has any physiological significance.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.