Uncoupling lymphocyte proliferation from differentiation: Dissimilar dose-response relations for pokeweed mitogen-induced proliferation and differentiation of normal human lymphocytes

Dose-response relations for pokeweed mitogen (PWM)-induced B- and T-cell proliferation and differentiation of human peripheral blood B lymphocytes were derived. For each tested concentration of PWM used in stimulating mononuclear cells, proliferation, assayed by cell population size and distribution of cells with respect to cell cycle phases; and differentiation, assayed by incidence of cytoplasmic immunoglobulin, were determined as a function of time following PWM stimulation. Balanced T- and non-T-cell proliferation occurred without necessarily being associated with B-cell differentiation. Differentiation, in contrast, was not observed without proliferation. The onset of balanced T- and non-T-cell proliferation preceded the differentiation of B lymphocytes into plasmacytoid cells bearing detectable cytoplasmic immunoglobulin. The dose-response relations for PWM-induced proliferation and differentiation were dissimilar. Optimum proliferation occurred at a PWM concentration $\text{1}\text{100}$ of that required to induce differentiation. The results indicate that while B- and T-cell proliferation may be necessary for B-cell differentiation, it is not sufficient. Proliferation can be uncoupled from differentiation. The dissimilarity of the dose-response relations for the two responses makes it improbable that PWM triggers a unique cellular process seminal to proliferation coupled inevitably to subsequent differentiation.     

Distinctive expression of the T4 antigen in normal and stimulated lymphocytes

Correlated light scatter and fluorescence flow cytometric analysis of human peripheral blood lymphocytes showed that the expression of the T4 antigen was higher in the larger lymphocytes than in the smaller lymphocytes. A similar expression pattern was observed for HLA Class I antigens but not for T3 and T8, whose expression was independent of cell size. Results with lymphocytes from spleen, lymph node, and tonsil were comparable to those of peripheral blood. Thymocytes, however, were smaller and expressed less T4 and T8 than peripheral lymphocytes. In studies of lymphocytes stimulated in vitro with allogeneic cells or pokeweed mitogen, two populations of T4-positive cells were observed: one of large cells expressing high amounts of T4 and one of small cells expressing low amounts of T4. Similar patterns were seen with T8, although less consistently. In contrast, the expression of T3 was the same in both large and small cells. The large cells expressing high amounts of T4 were not restricted to cells engaged in DNA synthesis or mitosis. This was established by selectively analyzing cells in the G0G1 phases of the cell cycle and by studying stimulated lymphocytes no longer undergoing proliferation. Taken together, these results suggest that immature T lymphocytes are small and express low amounts of T4 and T8. We postulate that as they differentiate, cell size and T4 expression increase proportionally, both parameters increasing even further after antigenic or mitogenic stimulation. The quantitative expression of T4, and probably of T8 but not of T3, is therefore intimately related to maturation and activation of lymphocytes, a fact that may conceivably be related to a functional role of these surface molecules.     

Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro

The results reported here show some characteristics of adenylate cyclase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F⁻ under conditions where cyclic nucleotide degradative pathways are effectively inhibited.Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 × g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E₁ and E₂, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F_1α and F_2α. Stimulation by prostaglandin E₁ or E₂ is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E₁ is equal to that caused by optimal fluoride concentrations. Stimulation c caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts

When treated with the synthetic glucocorticoid dexamethasone, HT1080 human fibrosarcoma cells show changes in morphology, adhesion, and the extracellular matrix. Dexamethasone treatment results in a tenfold increase in the rate of fibronectin biosynthesis in HT1080 cells and a twofold increase in untransformed, normal human fibroblasts. Maximal induction levels are attained within one cell generation, while decay of the response requires several cell cycles. Pulse-chase studies showed that most of the newly synthesized fibronectin is secreted into the medium. The glucocorticoid antagonist, RU-486, blocks the dexamethasone-induced changes but does not alter the basal rate of fibronectin production. Therefore, fibronectin biosynthesis appears to be controlled by two distinct mechanisms--one, regulating basal rates of fibronectin production, which is transformation-sensitive and glucocorticoid-independent; and another, which is mediated by the glucocorticoid receptor, resulting in elevated rates of fibronectin biosynthesis upon dexamethasone treatment both in normal fibroblasts and in HT1080 cells.     

Bromodeoxyuridine and light treatment enhances responsiveness of pokeweed mitogen-stimulated human lymphocytes to autologous and allogeneic determinants

We have investigated a novel system whereby lymphocytes from normal human subjects can be induced to develop exaggerated reactivity to histocompatibility antigens in vitro. Peripheral blood mononuclear cells (PBMC) stimulated with pokeweed mitogen (PWM) showed increased and accelerated subsequent proliferation to both autologous and allogeneic stimulators. Addition of bromodeoxyuridine (BUdR) during the period of maximal PWM-induced DNA synthesis followed by light exposure caused unexpected, but marked enhancement of this secondary proliferation. While untreated cultures contained a preponderance of T8⁺ cells after PWM activation, BUdR plus light-treated cultures were largely T4⁺ cells. Because removal of suppressor cells in nonsuicided cultures with anti-T8 and complement just before restimulation failed to unmask enhanced autoreactivity, events critical in the induction of the enhanced response must have occurred during priming. Cultures of PBMC with medium alone or concanavalin A, as well as purified T cells cultured with PWM, gave no enhanced autoproliferation after BUdR and light; thus T and non-T cells must be acted on by a T- and B-cell mitogenic stimulus to prime T cells for enhanced responsiveness. The interactions between T cells and activated B cells in this in vitro system may be relevant to regulatory mechanisms important in the induction of pathological autoimmune responses.     

Synthesis of prostaglandins by the ductus arteriosus of the bovine fetus

Previous studies demonstrated that prostaglandins are local or tissue hormones which can be released from blood vessel walls. In the present study, we investigated the capacity of bovine ductus arteriosus to synthetize prostaglandins . After incubation of slices of ductus arteriousus in Krebs' solution with (1-¹⁴C) arachidonic acid for 3 hours, more than 40% of the radiolabeled material recovered from the incubating medium were metabolites of arachidonic acid. The major product was indistinguisable from 6 keto-PGF_1α as determined by its chromatographic mobility and resistance to alkaline conversion to PGB.The PGI₂ synthetic capacity of the ductus arteriosus, as revealed by the predominance of its major metabolite 6 keto-PGF_1α, suggests that this metabolic pathway of arachidonic acid may contribute to the hemodynamic changes occurring during fetal life and at birth.     

Volatile metabolites in sera of normal and diabetic patients

The profiles of volatile metabolites in serum samples from normal individuals and from individuals with diabetes mellitus with varying degrees of polyneuropathy have been studied. The transevaporator procedure was used to obtain sample extracts which were chromatographed on a highly efficient glass column coated with Silar 10C (106 m × 0.25 mm I.D.). Differences in profiles between normal subjects and diabetic subjects on no drug therapy were noticed. However, correlations between the severity of the neuropathy and the concentrations of certain ketones could not be established. Compounds present both in diabetic and normal sera have been identified by mass spectrometry.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Alanine racemase of Pseudomonas. Observations on subtrate and inhibitor specificity

Systematic studies with purified alanine racemase and a number of substrate analogs permit the generalization that effective competitive inhibition is limited to 2- and 3-carbon compounds. A free α-amino group was not necessary for relatively tight binding; compounds lacking an amino group, or with an α-amino group acylated even by a bulky substituent, were bound as tightly as alanine. Substitution at the α-carbon of alanine (i.e., replacement of the α-H) eliminated binding, while substitution at the β-carbon generally reduced binding. Of several inhibitory compounds tested for substrate activity by H exchange with ³H₂O, only glycine appeared active. Covalent binding to the enzyme by halo analogs was not demonstrated.     

Inhibitory factor of phospholipase A2 in normal human serum

Normal human serum and plasma were shown to contain a factor inhibiting phospholipase A2. This factor has been separated from human serum and plasma by chromatography on a Blue-Ultrogel column and was eluted by tris-HCl buffer (pH 7.2); the proteins eluted by 1 M NaCl-tris HCl buffer exhibited phospholipase A2 activity. This activity was abolished when the inhibitory factor was added to proteins possessing such activity. The inhibitory factor was not dialysable, sensitive to both heat and trypsin treatment, suggesting that it is a protein. In vitro, the same factor inhibited phospholipase A2 rat serum.     

Simultaneous extraction and assay of cyclic nucleotides, prostaglandins, and DNA from cat alveolar bone

A method for the simultaneous extraction of cAMP, cGMP, PGE₂, PGF_2α, and DNA from a small sample of mineralized bone and the subsequent assay of these substances is described. Various solvents were tested for efficiency of extraction for the fatty acids, and water or 40% ethanol was found to extract more than 90% of labeled prostaglandin. In order to avoid enzymatic degradation, the substances were extracted at ?5°C requiring a solvent which would not freeze during extraction. Frozen alveolar cat bone samples were homogenized in 40% ethanol in the presence of 5 mm EDTA to inhibit phosphodiesterase. Small aliquots of the homogenate were withdrawn for the spectrofluorophotometric assay of DNA. After centrifugation, the supernatant was extracted first with petroleum ether, in order to take out neutral lipids, followed by ethyl acetate partition. The ethyl acetate layer was dired with N₂ gas, reconstituted with assay buffer, and assayed for PGE₂ and PGF_2α. A portion of the aqueous fraction was used for cAMP binding assay, while the rest was column chromatographed to elute the cGMP for radioassay. On the basis of per microgram of DNA, values for each of the following in cat alveolar bone were: 0.346 ± 0.049 pmol for cAMP, 0.026 ± 0.001 pmol for cGMP, 5.52 ± 1.46 pg for PGE₂, and 1.00 ± 0.29 pg for PGF_2α. Values calculated after the dilution of the sample aliquots or addition of standards to cAMP, cGMP, or PGE₂ showed no significant difference (P < 0.05) to their respective values. Within the limits of the sensitivity for each of the assay systems, it is feasible to measure cAMP, cGMP, PGE₂, and PGF_2α in alveolar bone from the same sample.     

Locomotion of human lymphoid cells. I. Effect of culture and con A on T and non-T lymphocytes.

Human peripheral lymphocytes were separated from whole blood on a Ficoll-Hypaque gradient. They were then depleted of monocytes, separated into T and non-T fractions, and assayed for locomotor responses toward casein and endotoxin-activated serum in Boyden chambers. Non-T cells showed higher random motility than did T cells. Culture prior to assay was necessary in order to demonstrate locomotor activity of T cells, but this requirement, although desirable, was not essential for non-T lymphocytes. It was not necessary for Con A to be present in the culture medium or for either T or non-T lymphocytes to be in blast form to show locomotion.     

Abnormal hemoglobin synthesis in some leukemic patients.

Hemoglobin chain synthesis during leukemic processes has been studied on patients having fetal hemoglobin. All cases showed the following abnormalities : (1) a relatively increased synthesis of the beta chain ; (2) an important increase of the free dimeric precursors pool, with, most of the time, a predominance of alpha chain. If the first point suggests an alpha-thalassemia feature, the presence of free alpha chains shows evidence for a more complex mechanism not only due to a decrease of messenger RNA. The hypothesis of a clonal disorder could neither be demonstrated nor ruled out. The observed abnormalities could be due to a defect in a alpha chain depending regulation mechanism.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E₂(PGE₂) and F_2α (PGF_2α) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE₂ at a dose of 10 μg per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE₂ and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE₂ in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 μg/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE₂ and PGF_2α stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

Effects of systemic and intrafollicular injections of LH,prostaglandins, and indomethacin on the luteinization of rabbit Graafian follicles

The possibility that initiation of luteinization in ovarian follicles by luteinizing hormone (LH) is mediated by prostaglandins (PG's) was investigated in rabbits. Estrous rabbits, given an ovulatory dose of LH (50 μg) intravenously, were administered indomethacin (IM), an inhibitor of PG biosynthesis, by various routes. Progesterone levels in the serum and in the induced corpora lutea (CL) were subsequently measured by radioimmunoassay. Continued daily subcutaneous injections of IM from 2 days before through 2 days after LH treatment reduced the corpus luteal level, measured at 72 hours post-LH, of PGF from 208 ± 43 to 98 ± 20 pg/CL (P < 0.025) and that of PGE from 272 ± 31 to 115 ± 9 pg/CL (P < 0.005). At the same time, progesterone levels were 72 ± 12 and 93 ± 10 ng/CL (P > 0.05) in the oil-treated and IM-treated rabbits, respectively. Serum progesterone continued to rise in a linear fashion during the period from 24 to 72 hours following LH treatment, whether IM was injected or not. Intrafollicular treatment with LH (100 ng/follicle) raised the progesterone content in the treated follicles 72 hours later from 1.1 ± 0.5 to 50.1 ± 13.5 ng. (P < 0.01). This progesterone content reached 21.5 ± 15.8 ng (P < 0.05) in follicles similarly treated with PGE₂ (5 μg/follicle), but remained meagre at lower doses of PGE₂ (100 ng/follicle and 2 ng/follicle). Serum progesterone increased from 0.5 ± 0.1 to 1.2 ± 0.1 ng/ml (P < 0.005) within 72 hours in rabbits treated intrafollicularly with LH, but remained unaltered in those similarly treated with PGE₂ (P > 0.1). Intrafollicular injections with PGF_2α failed to induce changes in either level of progesterone. It is concluded that prostaglandins probably do not mediate the luteinizing action of LH in rabbit Graafian follicles, although some degree of luteinization can be induced by high levels of exogenous PGE₂.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Effects of dietary saturated, n-6 and n-3 polyunsaturated fats on blood pressure and prostaglandins outflow from perfused mesenteric vascular bed in rats

Effects of the dietary administration of saturated fat and of n-6 and n-3 polyunsaturates on blood pressure, prostaglandin metabolism in small vessels, tissue fatty acid distribution and urinary PGE₂ excretion were compared. Rats were divided into three groups. Diets contained 10% hydrogenated cocunut oil (HCO), 10% safflower oil (SFO) or 10% cod liver oil (CLO) added to a basic fat free diet for 10 weeks. Systolic blood pressure was increased in the CLO group animals. Urinary PGE₂ excretion was decreased in the HCO and CLO groups as compared to that in the SFO group animals. PGE₂, 6-keto-PGF₁ and thromboxane (Tx) B₂ outflow from isolated perfused mesenteric arterial beds were extremely decreased in the CLO group animals, and to a lesser extent in the HCO group as compared to the SFO animals. In the tissue phospholipid, 20:3n−9/20:4n−6 ratios were increased in the HCO group indicating essential fatty acid deficiency, and n-6 and n-3 polyunsaturates were elevated in the SFO and the CLO group animals respectively. Arachidonic acid concentration was highest in the SFO group, while there was no significant differences between the HCO and the CLO group. These results suggest that dietary fatty acid manipulation effects urinary PGE₂ excretion and PGI₂, PGE₂ and TxA₂ synthesis in mesenteric arterial beds and also changes the tissue fatty acid distribution. Furthermore, n-3 polyunsaturates caused an extreme reduction of 2-series PGs synthesis in small resistance vessels.