Antigen modulation of the immune response. VI. Rate of large memory cell appearance in lymph nodes and thoracic duct lymph

We have studied the rate of appearance of memory B-cell subpopulations in the antigen draining lymph nodes and thoracic duct lymph of rats using 1g velocity sedimentation and adoptive transfer. Five days after immunization 100% of the memory response was attributable to large cells. By Days 7, 14, 28, and 77 after priming the large cells contribution to the memory population dropped to 86, 35, 15, and 10% respectively. At the same time the small cell contribution rose from 20% on Day 14 to 46% on Days 28 and 77. The same results were obtained with thoracic duct lymphocytes with the large cells contributing 53% of the response on Day 7 and 20% on Day 150. Appropriate controls were included to show that differential suppression was not responsible for these results. Furthermore, when purified large memory cells were passaged through intermediate hosts for 7 to 11 days, between 76 and 81% of the large cells matured into medium or small lymphocytes. These data show that the earliest memory cells formed after antigen encounter are the blast-like large lymphocytes and that these evolve, through a series of antigen-independent events, first into medium and then small lymphocytes. A model of memory cell development incorporating these results and the results of others is presented.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.     

Antigen-inhibited B-lymphocyte differentiation. X. Sedimentation velocity characterization of antigen-binding cells and antibody-forming-cell progenitors in the in vitro microculture immune response of unprimed CBA mice to NIP--POL antigen.

The B lymphocytes serving as antibody-forming-cell (AFC) progenitors have been investigated using two different types of separation methods. Sedimentation velocity fractionation was used to separate subsets of B lymphocytes differing primarily in size. Fractionation on a 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-gelatin matrix was used to separate NIP-binding cells, a population highly enriched for cells with surface Ig receptors specific for the NIP hapten. Assessment of the functional capacity of the separated B cells was by culture at limiting dilution in the presence of thymus “filler” cells, using the T-independent antigen NIP-POL (polymerized bacterial flagellin) to induce antibody formation. Splenic AFC-progenitors from both adult conventional and neonatal germ-free mice were a physically heterogeneous population, with activity in small, medium, and large lymphocytes. The cells enriched by NIP-gelatin binding, whether isolated and counted directly or isolated and assayed as AFC-progenitors, were no less heterogeneous. These NIP-binding cells resembled in sedimentation characteristics the overall B-cell and overall NIP-specific AFC-progenitor populations, except for some relative enrichment of medium-sized cells (S value, 5.5 mm/hr). The small (S value, 3.4 mm/hr) B-cell region of adult mouse spleen contained both NIP-binding cells and cells responsive as AFC-progenitors in the microculture assay. This contrasts with the results of the in vivo adoptive immune assay, where the smaller B-cell region is unresponsive in unprimed adult animals.     

Antigen-bearing Langerhans cells in skin,dermal lymphatics and in lymph nodes

Ferritin-challenged skin sites and draining lymph nodes were studied in normal guinea pigs and in guinea pigs which had been passively sensitized to ferritin or peroxidase by lymphoid cell transfer to ascertain whether Langerhans cells can bind antigen in skin and carry it to lymph nodes. After intradermal challenge with amounts of ferritin as small at 5 μg, ferritin-containing Langerhans cells were seen by electron microscopy in the marginal sinus and cortex of draining lymph nodes in ferritinscnsitized animals and, to an apparently lesser degree, in control animals. Lymph nodes from unchallenged normal guinea pigs contained rare Langerhans cells, none of which had ferritin. The findings indicate that Langerhans cells may pick up antigen in skin and from there circulate to draining lymph nodes, thus carrying out a function analogous to macrophages. In this way they may exhibit antigen to lymphocytes both in skin and in lymph nodes.     

A proposed role for prostaglandins in the modulation of the relaxation response to urotensin I in isolated rat arteries

Urotensin I (UI) elicits dose-dependent relaxation responses in isolated helical strips of rat tail and mesenteric arteries contracted by 10⁻⁵M norepinephrine (NE). The rat mesenteric artery demonstrated a 40 fold lower threshold sensitivity to UI (0.25 mU/M1 versus maximal relaxation at 0.25 mU/m1). Complete relaxation of the rat tail artery with UI could not be achieved, even at doses exceeding 10 mU/m1. Pretreatment of the arterial strips with cyclooxygenase inhibitors had no effect on the contractile response to NE in the tail artery, but reduced NE responsiveness in the mesenteric artery. Significant enhancement of UI relaxation responses in both types of arterial strips was achieved by pre-treatment with the cyclooxygenase inhibiters, suggesting a modulatory role for prostaglandins (PGs) in the expression of the UI relaxation response in NE contracted arterial strips. The major enzymatically formed PG (as assessed by [1-¹⁴C] PGH₂ metabolism in broken cell preparations) in both the rat tail and mesenteric arteries was 6-keto PGF_1α, the stable hydrolysis product of PGI₂. Using a specific RIA to quantify 6-keto PGF_1α release, it was found that UI elicited nearly a two-fold increase in the release of this PG compared to the NE control in both rat tail and mesenteric arteries. These data suggest that PGI₂ may modulate the relaxation response to UI either by direct physiological opposition (PGI₂ elicited contractile response in NE contracted tail and mesenteric arteries at doses exceeding 10−8M) and/or by some as yet undefined mechanism (eg. effects on Ca²⁺, cAMP).     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Membrane dynamics of lymphocyte interaction with antigen and tolerogen.

The principle of hapten-specific carrier-dependent immunologic tolerance was used to study the in vivo and in vitro interaction of lymphocyte membrane receptors with antigen (DNP-KLH) and tolerogen (DNP-MGG). Direct fluorescent techniques were employed to illustrate the binding of tolerogeu and antigen to the same population of lymphoid cells and the subsequent in vivo and in vitro events related to capping and regeneration of membrane receptors.     

Control mechanism of lymphocyte traffic. A study of the action of two sulfated polysaccharides on the distribution of 51Cr- and [3H]adenosine-labeled mouse lymph node cells

The effects of dextrans of varying molecular weights and of pentosan sulfate on the distribution of ⁵¹Cr-labeled mouse lymph node cells were studied in vivo, i.e., in recipients treated with the sulfated polysaccharides, and in vitro, i.e., by following the fate of cells treated in vitro, in intact syngeneic recipients. Both types of experiments demonstrate that dextrans, especially dextran sulfate (DXS) and pentosan sulfate (PS), considerably reduce lymph node entry of lymphocytes, with concomitant increases in the blood and, in the case of DXS, in both the blood and lungs. A parallel quantitative autoradiographic analysis of the distribution of [³H]adenosine-labeled cells confirmed the data with the ⁵¹Cr-labeled cells and, in adidtion, indicated that DXS and PS slow down circulation of lymphocytes through the marginal zone and red pulp of the spleen and, in the case of DXS, in the pulmonary capillary bed. Unusually large numbers of unlabeled lymphocytes were found in the endothelial wall of the post-capillary venules in lymph nodes of PS-treated mice.     

A non-linear model for the synaptic basis of neuronal memory.

We propose a mathematical model for the synaptic basis of neuronal memory. The model incorporates non-linear effects in analogy with population growth problems of human beings, animals, biological species, crystal growth, etc., and provides a mechanism whereby the excitatory and inhibitory inputs produce alterations in a neurone which result in a long-lasting increase in transmitter release at a synapse.     

Stimulation and modulation of adenylate cyclase by Sendai virus.

Stimulation of adenylate cyclase activity occurs in membranes prepared from toad erythrocytes preincubated briefly (at 37° or 4°) with ultraviolet light-inactivated Sendai virus. Stimulation occurs with as few as five virions per cell, and it is blocked by pretreating the virus with the membrane glycolipid, ganglioside GM₁. Virus treatment also alters modulation of adenylate cyclase by hormones, nucleotides and sodium fluoride. Interactions of viral envelope antigens with plasma membrane components may thus elicit functional changes possibly important in the pathogenesis of viral infections.     

Regulation of immune responses. II. The cellular basis of cyclic AMP effects on humoral immunity.

DbcAMP, when added at 10^?3M for the first 12 hr, can increase the number of AFC to SRBC (a TD antigen) and POL (a TI antigen) in antigen-stimulated CBA/ J spleen cell cultures. The cellular basis of dbcAMP action was therefore investigated. It was found that dbcAMP does not act by a direct B cell effect. It also does not stimulate the activity of T helper cells, and it inhibits the function of macrophages. The stimulatory activity of dbcAMP to anti-SRBC and anti-POL responses is through inhibition of a θ-bearing regulator (or suppressor) cell. Removal of T cells by anti-θ treatment has the same effect on the anti-POL response as treatment with dbcAMP. Furthermore, in the absence of T cells, the enhanced anti-POL response was insensitive of dbcAMP treatment. The data also support the hypothesis that the number of anti-SRBC AFC formed is regulated by the ratio of T helper to T regulator cells.     

Time-dependent loss of prostaglandin E release by adherent cells in response to stimulation by thyroid cells: Prevention of this loss by phytohemagglutinin

Preplating human adherent peripheral blood mononuclear cells (PBMC) for up to 24 hr results in a progressive decrease in their basal PGE release, and in the loss of their ability to increase PGE release during a subsequent 72-hr coculture period with allogeneic human thyroid cells. Phytohemagglutinin (PHA) present during a 24-hr adherent-cell preplating period prevents, in part, the loss of this PGE response to thyroid cells. These data indicate that adherent cells require continual stimulation by the thyroid cells or by PHA in order to maintain their ability to increase PGE secretion in response to thyroid cells.     

Role of silicon in diatom metabolism. VII. Silicic acid-stimulated DNA synthesis in toluene-permeabilized cells of Cylindrotheca fusiformis.

Human diploid cell populations were fractionated on the basis of cell size by gravity sedimentation. This cell separation procedure yielded fractionated cell populations that were enriched for both large cell volumes and for slow and/or non-replicating cells (cells which did not have labeled nuclei after a 48 h incubation with [³H]TdR). It also yielded fractionated cell populations that were enriched for small cell volumes and for rapidly replicating cells (cells with labeled nuclei). However, upon reintroduction to tissue culture conditions, cell populations lost their fractionated properties and soon resembled unfractionated cell cultures at similar levels of in vitro passage.     

Studies on the role of lymphocyte-activating factor (Interleukin 1) in antigen-induced lymph node lymphocyte proliferation

The in vitro proliferation of primed lymph node lymphocytes (LNL) in response to the soluble antigen ovalbumin (OVA) was dependent upon the presence of adherent cells. Restoration of OVA-induced LNL proliferation could be achieved by addition of highly purified lymphocyte-activating factor (LAF; Interleukin 1, IL 1): LAF (IL 1) did not stimulate LNL proliferation in the absence of the priming antigen or T lymphocytes. Furthermore, treatment of the LNL with antimacrophage serum completely blocked the ability of the LNL to respond to OVA and LAF (IL 1), suggesting that the residual macrophages in the LNL population were necessary to provide an additional function or signal, possibly antigen presentation, in conjunction with LAF (IL 1). These data therefore support the two signal hypothesis of macrophage-mediated lymphocyte activation and demonstrate the ability of LAF (IL 1) to provide one of these signals.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Modulation of a neural antigen during metamorphosis in Drosophila melanogaster

Transplantation of the acousticolateral placode to the evacuated eye position in embryos of the frog Rana pipiens has been used to force axons of the VIIIth cranial nerve to penetrate the diencephalon. These ectopic axons establish a growth trajectory that is strikingly similar to their normal growth trajectory within the medulla oblongata despite the fact that no other axons within the diencephalon normally follow this route. The result is discussed in terms of the "blueprint" and substrates pathway hypotheses which have been advanced to explain the initial development of axon tracts within the central nervous system.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

Biosynthesis of transcobalamin II by adult rat liver parenchymal cells in culture.

The biosynthesis of transcobalamin II was investigated in primary cultures of adult rat liver parenchymal cells maintained in serum-free media. The data indicate that these hepatocytes secrete a vitamin B₁₂-binding substance into the culture medium which is identical to rat serum transcobalamin II as judged by the following criteria: (i) gel filtration on columns of Sephadex G-200; (ii) ion-exchange chromatography on columns of diethyl aminoethyl cellulose and carboxymethyl cellulose; (iii) polyacrylamide-gel electrophoresis at pH 9.5; and (iv) the ability to facilitate cellular vitamin B₁₂ uptake by HeLa cells and mouse L-929 fibroblasts in culture. The secretion of transcobalamin II by the liver parenchymal cells was blocked by cycloheximide, puromycin, and p-fluorophenylalanine. The inhibition by cycloheximide, but not that of the other inhibitors, was partially reversed upon removal of the drug. The liver parenchymal cells incorporated radioactive amino acids into transcobalamin II which was absorbed from the growth medium using affinity chromatography on Sepharose containing covalently linked B₁₂. Collectively, these data indicate that rat liver parenchymal cells, in culture, are capable of the biosynthesis de novo of transcobalamin II and the subsequent secretion of this protein into the culture media.