G. Sandberg

Inhibition by xanthine derivatives of adenosine receptor‐stimulated cyclic adenosine 3′, 5′‐monophosphate accumulation in rat and guinea‐pig thymocytes

1 The effect of stable adenosine analogues, including adenosine 5′‐N‐ethylcarboxamide (NECA) and N6‐l‐phenylisopropyl‐adenosine (l‐PIA), were studied on cyclic adenosine 3′, 5′‐monophosphate (cyclic AMP) accumulation in rat and guinea‐pig thymocytes. 2 NECA was approximately 10 times more potent than l‐PIA, in thymocytes from both species. d‐PIA was more potent in guinea‐pig than in rat thymocytes. The effect of a number of adenosine analogues followed the order: NECA > 2‐chloro‐adenosine > l‐PIA > N6‐cyclohexyl‐adenosine (CHA), an order of potency characteristic for adenosine receptors of the A2‐subtype. Thymocytes may be used as a model system to study the pharmacology of such receptors. 3 Several xanthines were studied as antagonists of the NECA (1 μm)‐induced cyclic AMP accumulation. The order of potency was: 1,3‐diethyl‐8‐phenylxanthine > 8‐phenyl‐theophylline > IBMX = 8‐p‐sulphophenyltheophylline = verrophylline > theophylline > caffeine > enprofylline > theobromine > pentoxiphylline. The pA2 value for 8‐phenyltheophylline was 0.35 μm, and the antagonism was shown to be competitive. The order of potency of the xanthine is virtually identical to that found earlier in several other systems in which the receptors are of the A1‐subtype. None of the xanthine derivatives tested thus seem to discriminate between A1 and A2‐receptor‐mediated adenosine actions.

Cyclic amp in freshly prepared thymocyte suspensions. evidence for stimulation by endogenous adenosine

Abstract Thymocyte suspensions were prepared from guinea pig thymus by gentle mincing and repeated washings. The mincing resulted in a ten-fold increase of the cyclic AMP level, most of the cyclic AMP being intracellular. The level remained high during the washing procedures. During incubation at 37° the level of intracellular cyclic AMP gradually fell by a temperature dependent process. The cyclic AMP content was increased by non-methylxanthine phosphodiesterase inhibitors (papaverine, Ro 20-1724, ZK 62.711) but decreased in the presence of theophylline or isobutylmethylxanthine. Adenosine and phenylisopropyl adenosine acutely increased cyclic AMP by a theophylline inhibited mechanism. An increase was also obtained by inhibitors of adenosine uptake (dipyridamol, dilazep) and of adenosine deaminase (EHNA). The results were compatible with a release of adenosine during the isolation of thymocytes, and with an adenosine-induced increase of intracellular cyclic AMP. This interpretation was supported by in vivo labelling of purine nucleotides with [3H]adenine. A marked rapid fall of purine nucleotides was found during the preparation of the thymocytes and at the same time adenosine metabolites appeared in the medium.

Regulation of thymocyte proliferation by endogenous adenosine and adenosine deaminase

Spontaneous proliferation of thymocytes after 20-25 h of culture was significantly increased by the presence of adenosine deaminase (ADA) or theophylline. The effect of ADA was counteracted by the ADA inhibitor EHNA. When given alone, EHNA inhibited proliferation. This effect was not blocked by inhibition of adenosine uptake with dipyridamol. These results suggest that proliferation in culture is regulated by a balance between endogenous adenosine and ADA, controlling the influence of adenosine on the intracellular cyclic AMP level via an adenosine receptor on the surface of thymocytes. According to the hypothesis, ADA would stimulate proliferation by decreasing extracellular adenosine levels and theophylline by blocking adenosine receptors on thymocytes. EHNA would inhibit proliferation by increasing extracellular adenosine levels. In accordance with this interpretation, the adenosine analogue phenylisopropyl adenosine (PIA) inhibited proliferation and the effect could be inhibited by theophylline. The postulated effect of endogenous adenosine could not be mimicked by a single administration of exogenous adenosine. Whereas most doses of adenosine were without effect, a high dose of adenosine (0.1 mM) in combination with EHNA unexpectedly stimulated proliferation. Since the effect was blocked by dipyridamol, an intracellular site of action for adenosine is suggested in this case.

Quantitation of B and T Lymphocytes in Guinea Pigs with Evidence for a Release of both Cell Types from the Spleen into the Blood

B and T lymphocytes were quantitated in lymphoid organs and in blood of young normal guinea pigs by the use of EAC rosettes and rabbit erythrocyte (RE) rosettes as markers. Special attention was focused on the release of B and T cells from the spleen, estimated from the difference between the content of B and T cells in splenic efferent and afferent blood. The following frequencies of resette-forming cells (RFC) were found. Thymus: 0.3% EAC- and 88% RE-RFC; spleen: 44% EAC- and 46% RE-RFC; lymph nodes: 13% EAC- and 39% RE-RFC; arterial blood: 13% EAC- and 42% RE-RFC, and bone marrow: 2% EAC- and 7% RE-RFC. A fairly large number of cells in the lymph nodes and blood could not be identified by any of the two markers. Some possible explanations for this are discussed. The content of both EAC- and RE-RFC in splenic efferent blood significantly exceeded that in the afferent blood, indicating a release of both B and T cells from the spleen into the blood. The possibility of a release of a third type of mononuclear cell cannot be excluded from the present results.

Leukocytes from Patients Allergic to Chromium and Nickel Examined by the Sealed Capillary Migration Technique

Leukocytes from patients allergic to chromium and nickel were studied by a sealed capillary migration test. The migration indices were determined at 2, 5 and 24 h. The chromium-allergic group could be differentiated from the control group at all investigated times, and especially at 2 and 5 h, by using a potassium dichromate concentration of 1.7 x 10(-5) M in the capillaries. The migration indices in the nickel-allergic group could not be used for discrimination from the controls. The highest chromate (1.1 x 10(-4) M) and nickel sulfate (3.8 x 10(-4) M0 concentrations inhibited the migration of leukocytes from both patients and controls, indicating toxic effects.

Mitotic Activity of Thymocytes in a Synthetic Tissue Culture Medium. Effect of L-AIanine

DNA synthesis in guinea pig thymocytes suspended in RPMI 1640 medium increased to a peak after 4-5 h in culture and was followed by increased mitotic activity, indicating that many thymocytes in S phase proceeded through G2 into mitosis. Addition of L-alanine to the medium markedly increased the DNA synthesis within 1 h and the mitotic frequency from 6 h. The increase in DNA synthesis when L-alanine was present in the medium was thus caused by an increased number of cells in S phase. Human thymocytes cultured in RPMI 1640 for 18 h had a low mitotic frequency. Addition of L-alanine immediately started DNA synthesis in the arrested thymocytes resulting in increased mitotic activity from 6 h later. The results show that L-alanine is a growth factor for guinea pig and human thymocytes and should be included in tissue culture media used for such cells. Growth of thymocytes in vitro was partly synchronized, and the mitotic studies indicated that many cells had entered S phase near the start of incubation.

The sealed capillary migration technique and thymocyte migration in vitro.

The in vitro migration of thymocytes in sealed capillary tubes was influenced by cell number, temperature, cell viability and oxygen supply. Migration was successively slower over a 24 h period. There was no major influence of gravity or the degree of initial packing of the cells by centrifugation. Migration was inhibited by cytochalasin B, although the cells escaped from this effect after 8 h. A transient stimulatory effect on migration was seen after addition of serum or supernatants from cultured thymocytes. There was no effect of isoproterenol, theophylline or carbacholine. A paradoxic effect was obtained with high doses of some metabolic inhibitors, which produced increased migration in spite of cell death. The technique may be used for studies on lymphocyte migration in vitro, but care must be taken to exclude a toxic influence of added substances, and expected effects should preferably be looked for early after onset of migration, since cell viability is gradually decreased.

Lymphatic vessels and Kurloff cells in the thymus of estradiol-treated guinea pigs

SummaryMale guinea pigs were given a single subcutaneous injection of estradiol, which induces formation of Kurloff cells, and serial sections of thymus were examined after 10, 12, 15 and 21 days. Kurloff cells were found in large numbers in lymphatic vessels, both outside the thymus, in the interlobular tissue, at the cortical surface and inside the cortex, suggesting migration via such structures. Large extrathymic or interlobular lymphatics communicated with a previously undescribed thymic structure-the ‘lymphatic centre’-surrounded by a marginal sinus. The orientation of lymphatic valves, and the concentration of Kurloff cells within this ‘lymphatic centre’ at an early time after the administration of estradiol, indicate the existence of an afferent migratory pathway. The different morphology at different times after estradiol suggest that the treatment caused a dynamic remodeling of thymic lymphatic structures.

Effect of L-alanine and some other amino acids on thymocyte proliferation in vivo

L-alanine was shown earlier to play a significant role for the proliferation of lymphocytes in vitro. In the present work the effect of L-alanine and some other amino acids on thymocyte proliferation was studied in vivo by local administration into one thymus lobe of guinea pigs. Proliferating cells were pulse labelled with bromodeoxyuridine (BrdUrd). The labelling index of the treated lobe significantly exceeded that of the contralateral, control lobe at 48 h after treatment with 10 or 100 micrograms L-alanine, indicating stimulated proliferation. The higher dose, which was also tested after other time intervals, stimulated also at 24 h. The difference in proliferative activity between the lobes was verified by mitotic studies. The effect of L-alanine was mainly on the large, low density, highly proliferating precursor cells. No other amino acids tested (D-alanine, cysteine, hydroxyproline, serine, tryptophan), or pyruvate produced significant differences between the treated and control lobes.

Kinetic study of Kurloff cells in guinea pig thymus.

One single injection of estradiol to male guinea pigs resulted in the appearance of Kurloff cells in the thymus and spleen in a maximal number after 2-3 weeks. In both organs, Kurloff cells of three categories were observed with different size and number of the inclusion(s). In the thymus, cells with small and medium-sized inclusions were present almost exclusively among low-density thymocytes. Cells with one large inclusion were initially present among low-density cells, but with time an increasing proportion were found among high-density thymocytes. This indicates that the different categories of Kurloff cells represent maturational stages and follow the normal differentiation step of thymocytes from low to high density. According to electronic cell volume distribution analysis, estradiol treatment was associated with a shift in cell volume towards larger cells, and this shift was correlated in time with the appearance of Kurloff cells with large inclusions. In thymus sections there was evidence for a massive and selective migration of Kurloff cells via interlobular lymph vessels. No difference in localization of Kurloff cells was noted at various times after estradiol treatment. Attempts to induce Kurloff cell formation from bone marrow, spleen or thymus cells during 2 weeks in vitro were unsuccessful, also when culturing the cells in serum from estradiol-treated animals. This negative result, the long lag phase of estradiol-induced Kurloff cell formation in vivo, and the reported lack of estradiol receptor in Kurloff cells indicate that the Kurloff cell induction by estradiol may be indirect.