Workshop 4 Biochemical Events in Lymphocyte Activation

HERB COOPER and JEAN-CLAUDE MANI

Dept. Microbiol., Boston Univ. Sch. Medicine, Boston, MA. 02118, USA

Factors affecting enhancement of Con A-mitogenesis by vitamin E

R. BELLAS and L. M. CORWIN

Dietary or in vitro vitamin E (E) stimulates the response to T-cell mitogens. It has also been found that E enhances mitogenesis of an Ia - , nylon wool purified T-cell preparation (Scand. J. Immunol. 14: 565, 1981). It was found in addition that E also enhanced mitogenesis when added subsequent to removal of Con A at 18 hr, i.e. before DNA synthesis began. Several groups have shown that when exposure of lymphocyte to a mitogen is terminated before the first cell division, they divide only once. Several of our observations have led to the hypothesis that vitamin E may maintain IL2 receptors on the lymphocyte surface after removal of mitogen, thereby increasing the number of cells entering S phase. First, 48 hr after con A addition and 30 hr after mitogen removal, DNA synthesis reaches a maximum and there is only about a 30 % enhancement of 3H_ TdR uptake by E. At 72 hr after mitogen addition, 3H_ TdR uptake is dramatically reduced by - E T cells, but much less reduction occurs in + E cells. The resulting stimulation of mitogenesis by E at 72 hr is 4-10-fold. KUMAGAI et al. (J. Immunol. 126: 1249, 1981) have shown that insulin, though not mitogenic itself, prevents lymphocytes from entering the resting state and permits responsiveness to IL2. We have shown that addition of insulin stimulates DNA synthesis of - E cells, but not + E cells, thereby abrogating the E effect. Moreover, it was shown that E addition to cells exposed to Con A for only 6 hr to generate IL2 receptors, greatly enhanced the response to IL2 preparations. We suggest that vitamin E, like insulin may enhance T-cell mitogenesis by maintaining IL2 receptors.

Laboratoire de Biochimie des Membranes, ER CNRS 228, ENSCM, 8 rue de l'Ecole Normale, F-34075 Montpellier, France

Role of adenosine in lymphocyte maturation: possible involvement of adenosine receptors coupled to adenylate cyclase

J. C. BONNAFOUS, J. DORNAND, J. FAVERO, and J. C. MANI

Adenosine was recently postulated to induce maturation (1) and pharmacological modifica­tion of immunoregulatory T lymphocytes (2). These effects might be related to increases in cyclic AMP levels. We demonstrate in the present study that immature mouse thymocytes possess adenosine membrane receptors coupled to adenylate cyclase, like many other lympho-

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cyte populations (3): the cyclic AMP level of immature thymocytes (separated by selective agglutination with peanut agglutinin) is strongly increased by adenosine itself or by adenosine deaminase-resistant analogues, such as NECA (5'-N-ethylcarboxamide adenosine); experi­ments on thymocyte homogenates show that this effect is due to direct stimulation of adenylate cyclase through Ra (or A2) type adenosine receptor sites (3). We also present comparison of adenosine receptors in mature and immature thymocytes separated by peanut agglutinin agglutination. Any mechanism which controls the adenosine level might be impor­tant for the possible nucleoside-induced maturation. We compared the activities of several enzymes involved in adenosine metabolism (ecto-ATPases, ecto-5' -nucleotidase, adenosine deaminase, adenosine kinase, S-adenosylhomocysteine hydrolase) in mature and immature thymocytes.

1. ASTALDI, A., C. J. M. LEUPERS, and P. T. A. SCHELLEKENS. 1981. Adenosine induces immunological maturation of thymocytes. Immunobiol. 159: 93.

2. BIRCH, R. E., and S. H. POLMAR. 1982. Pharmacological modification of immunoregulatory T lymphocytes.!. Effects of adenosine, H j and H2 histamine agonists upon T lymphocyte regulation of B lymphocyte differentiation in vitro. Clin. Exp. Immunol. 48: 218.

3. BONNAFOUS, J. c., J. DORNAND, J. FAVERO, and J. C. MAN!. 1982. Lymphocyte adenosine receptors coupled to adenylate cyclase. J. Recept. Res. 2: 347.

University of Alberta, Edmonton, Alberta, Canada

Lymphocytes pulsed with purine and pyrimidine analogues are blocked at the G 1/S boundary

CHRISTINE E. BOUMAH, WENDA L. GREER, and J. G. KAPLAN

Analogues of nucleic acid bases can be useful in the study of RNA metabolism and function. We have studied the effects of the base analogues 8-azaguanine (8-AG) and 5-fluorouracil(5-FU) on the proliferation of mouse lymphocytes stimulated with Con A. The analogues were added for 6 h at different stages of activation and the proliferative response was measured at 48 h after the cultures had been washed and reincubated with an excess of the natural base. A severe inhibition (up to 95 %) of 3H -thymidine incorporation, with very little, if any, decrease in the incorporation of 3H-leucine and 3H-uridine, was observed in cultures pulsed in the first hours of activation, 30 h before the onset of S phase. The near 2-fold stimulation of K+ transport, increase in nuclear and cytoplasmic volume (blast formation) with the progressive disaggregation of the condensed chromatin (i.e. all the preliminary events of transformation) could be demonstrated in such cultures. Moreover, pretreatments with either analogue, at any time of activation, be it before or after the increase in transcriptional activity, all irreversibly prevented the entry of the cultures in the S phase of DNA synthesis and the subsequent mitosis. However, when 5-FU was added to proliferating cultures from 42-48 h, no inhibition was observed. This and other evidence suggested that inactivation of thymidylate synthetase was not responsible for the effects of 5-FU here. There was no difference in the degree of inhibition caused by the presence of 8-AG before or after the entry in S phase. The effects of 8-AG were expressed, provided guanine was not added simultaneously, with a short lag (90 min) and in the presence or absence of protein synthesis. Therefore, the inhibition of proliferation observed was not due to synthesis of a toxic protein but to a block in the formation of the DNA synthesizing system, at the GlIS boundary.

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Abteilung Molekularpharmakologie, Zentrum Pharmakologie und Toxikologie, Medizinische Hochschule Hannover, Konstanty-Gutschow-Stralle, Hannover, FRG

Phosphatidylcholine and phosphatidylinositol show a different activation pattern of their turnover rates of unsaturated fatty acid moieties in plasma membranes of concanavalin A-stimulated thymocytes

M. BRENNECKE and K. RESCH

We examined the course of incorporation of 3H-arachidonic acid, 14C-oleic acid and 3H_ palmitic acid into individual phospholipids of the cytoplasmic membrane from rabbit thymo­cytes (1, 2). After 2 h of prestimulation with ConA cells were pulsed for variable lengths of time from 5 min up to 1 h and compared for their incorporation rates with unstimulated control samples. Phosphatidylinositol showed an increase of 3H-arachidonic acid incorpora­tion of more than tenfold the degree of unstimulated control cells for pulse times up to 15 min and was the most significantly affected phospholipid. Palmitic acid incorporation was not stimulated for anyone of the investigated phospholipid species. To elucidate the time course of the turnover for unsaturated fatty acid moieties, thymocytes were pulsed for 5 min with 3H_ arachidonic acid or 14C-oleic acid after different times of ConA stimulation. Stimulation periods extended from zero min (controls) up to 2 h. Incorporation of 3H-arachidonate was increased detectably after 15 min of ConA-stimulation reaching a peak reincorporation rate after 30 min, and levelling off to an increased steady state incorporation rate thereafter. In contrast, phosphatidylinositol showed a continuous increase of incorporation rates until reaching a plateau level at 60 min stimulation time, at this point exceeding the respective value for phosphatidylcholine. HC-oleic acid revealed a similar pattern than 3H-arachidonic acid.

1. RESCH, K. 1976. In: Receptors and Recognitions (Cuatrecasas, P. and Greaves, M. F., eds.) Series A, Vol. 1, pp. 61-117, Chapman and Hall, London, Halsted Press, New York.

2. RODE, H., M. SZAMEL, S. SCHNEIDER, and K. RESCH. 1982. BBA 688, 66-74.

Departments of Biology and Biochemistry, University of Ottawa, Ottawa, Canada, K1N 6N5

Changes in tubulin content, MTOC activity, and microtubule assembly in mitogen stimulated mouse splenic lymphocytes

D. L. BROWN, I. SCHWEITZER, P. D. WATERHOUSE, P. J. ANDERSON, and J. LITTLE

We have examined changes in the microtubule (mt) component of the lymphocyte cyto­skeleton in mouse splenic T lymphocytes stimulated by concanavalin A. Immunofluorescence and electron microscopy reveal a large increase in numbers of mts assembled from a single microtubule organizing center (MTOC) following stimulation. Counts of mts in the MTOC regions of cells show a doubling of mt number in 48 hr stimulated populations and up to a 5-fold increase in the fully stimulated blast cells. The total tubulin contents of resting and stimulated populations were determined by the measurement of peptides specifically derived

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from tubulin. These determinations show a 2.5-fold increase in tubulin content of 48 hr stimulated populations, which represents about a 5-fold increase in the amount of tubulin in blast cells. Most of the increase in tubulin content and in numbers of mts assembled occurs between 24 and 48 hrs after addition of the mitogen. To determine if there were morphological changes in the lymphocyte MTOC (the centrosome) which could be correlated with the increase in mt assembly observed we have used serial sections through the MTOC regions of cells to produce computer generated, three-dimensional reconstructions. This analysis shows that in both resting and stimulating cell MTOCs the mts are assembled from dense aggregates, termed satellite bodies, which are closely associated with the centriole pair. During stimula­tion, changes are detected in the number, size, and distribution of the satellite bodies. In vitro assembly assays, using detergent permeabilized cells supplied with purified bovine brain tubulin, confirm that the satellite bodies are sites for mt assembly and show an increase in the mt initiation capacity of stimulated cell MTOCs. We conclude that the increase in mt assembly accompanying lymphocyte stimulation is dependent on both an increase in tubulin content due to de novo synthesis and to an increase in the capacity of the MTOC to initiate microtubules.

Immunohematology Research Laboratory, Research Svc., Veterans Administration Medical Center, Sepulveda, CA; Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA, USA

Kinetics of formation of cell-cell linkage in response to diverse T cell mitogens

W. BRUSZEWSKI, H. TONNu, S. FELBERBAUM, J. MEYER, and N. COSTEA

Intercellular linkage is apparently necessary in the activation of lymphocytes that precedes proliferation. Diverse T cell mitogens induce agglutination of lymphocytes. Proliferative response to mitogenic lectins can be ablated by inhibiting intercellular contact and linkage. The structural and chemical nature of the actual linkage mechanism is, however, yet to be demonstrated. We have compared kinetics of establishment of intercellular linkage induced by concanavalin A (Con A) and neuraminidase plus galactose oxidase (NGO) in mitogenesis of human lymphocytes. These mitogens are thought to have widely divergent modes of initial action on the cell surface. They are also postulated to induce dissimilar mechanisms of linkage: Con A may «bridge" receptor sites because it is a polyvalent ligand; NGO is presumed to produce surface aldehyde groups which then form Schiff bases with amino groups on adjacent cell surfaces. We have devised a new method to measure extent of linkage. The proportion of stably-linked aggregates and discrete lymphocytes. Rate and maximum extent of linkage formation during the first 2 h of exposure to mitogen varied directly with cell density. Maximum linkage persisted for about 2 h, and the links then spontaneously decayed. Most significantly, the kinetic pattern for Con A and NGO was always the same, under identical conditions of incubation and cell density. One might expect different kinetics with these functionally different mitogens. We found that inhibiting linkage by agitation of cell suspen­sions during initial exposure to either mitogen ablated proliferation, indicating that linkage formation is essential to mitogenesis. We also studied kinetics in the presence of different mitogen concentrations. The results of our experiments indicate the possibility that the presumed different mechanisms of linkage formation via Con A and NGO are incorrect. An as yet undescribed mechanism common to both mitogens may exist instead.

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Department of Medicine (Rheumatology), University of Iowa College of Medicine, Iowa City, IA 52242, USA

Early activation events in pokeweed mitogen-stimulated human peripheral blood cells

M. M. CHIEN, R. T. MEEHAN, and R. F. ASHMAN

Whereas many biochemical consequences of lymphocyte stimulation with T-cell mitogens have been defined, early events in the response of non-T cells to mitogens have been much less thoroughly studied. DNA synthesis and Ig production induced by pokeweed mitogen (PWM) require the presence of T cells and monocytes or their products, but whether this requirement also applies to earlier activation events is unknown. We have chosen to study PWM-induced choline incorporation into phospholipid, phospholipid methylation, and early protein syn­thesis as examples of early activation events. When unfractionated cells were stimulated by PWM, peak HC-choline incorporation into phospholipid by B cells occurred at 16-20 hr, 2 days before peak incorporation by T cells. Removing either T cells (by E rosetting) or monocytes (by G 10 Sephadex) before PWM addition prevented the increased incorporation by B cells. Synergy for PWM-induced choline incorporation was maximal with equal numbers of T and non-T cells. Monocytes showed a high initial choline incorporation rate which did not change after PWM exposure. Protein synthesis by unfractionated cells, measured as 35S_ methionine incorporation, was significantly increased by about 1.6-fold at 12 hr and 2.0-fold at 21 hr of exposure to PWM, but the increase was not significant earlier than 12 hr. This increase was greater in the monocytes (4-fold) and T cells (2-fold) than in the B cells (only 1.2-fold) . In unfractionated cells, PWM also induced a transient 2-fold increase in 3H-methyl transfer from methionine into phosphatidyl choline at 5 min. By 25 min, lysophosphatidyl choline had replaced phosphatidyl choline as the main 3H-methylated lipid. The cell interac­tion requirements for changes in methyl transfer and protein synthesis can now be analyzed, but the choline incorporation data has already shown us that regulatory cells are required for at least one B cell activation event in the first day after PWM exposure.

Department of Biochemistry, University of Washington, Seattle, WA, USA

Relative contributions of transcriptional and translational control in regulating protein synthesis in mitogen-activated small lymphocytes

J. L. DEGEN, M. G. NEUBAUER, S. J. FRIEZNER-DEGEN, C. E. SEYFRIED, and D. R. MORRIS

In response to a mitogen stimulus, the rate of protein synthesis in T lymphocytes increases ca. 3-fold in the first 10 hours. Of 700 proteins resolved by 2-D gel electrophoresis, 95 % behave in this manner, including the cytoskeletal protein actin. The cellular level of actin mRNA, determined by hybridization using a recombinant plasmid, increases 1.7 to 2.0-fold within the 10 hours after activation, indicating that a major influence on the rate of actin synthesis is new mRNA production. However, substantial modulation at the level of mRNA utilization is also indicated, since the rate of actin synthesis per actin mRNA increases 1.5 to loS-fold. Studies of total translatable mRNA and poly(A)+ sequences indicate that protein synthesis in general is apparently regulated similarly to actin. A detailed examination of actin mRNA-containing polysomes and total polysomes isolated before or after cell activation indicated little or no change in size distribution. These data suggest a model for general protein synthesis in which idle ribosomes are recruited as a function of increased accessible mRNA

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after cell activation. The source of this additional mRNA is apparently both: 1) a pool of unutilized mRNA present in unstimulated cells (ca. 30~5 % of total mRNA) and, 2) new mRNA production. A second class of proteins increases noncoordinately with total protein synthesis. The synthesis of S-adenosylmethionine decarboxylase (SDC), a representative of this class, increases 10-fold by 6 hours after activation. SDC mRNA has been translated in a cell-free system and is being used to study the regulation of this group of proteins.

Departments of Pathology and Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033, USA

Amiloride blocks an essential early event in lymphocyte mitogenesis

J. F. P. DIXON, R. A. FARLEY, and K. UYEMURA

We exposed human peripheral blood lymphocytes to various concentrations of amiloride either at 0-6 hours or 20-26 hours following oxidation with neuraminidase-galactose oxidase. At concentrations of 80 ~ and higher there was significant cytotoxicity; therefore, prior to 3H-Tdr labelling, cultures were adjusted to 106 viable cells per ml. At all concentrations tested (20 to 320~) exposure from 0-6 hours produced significantly greater inhibition of mitogenesis (as measured by 3H-Tdr incorporation at 72 hours) than exposure at 20-26 hours. In all cases sham-treated controls were also run. We then treated lymphocytes with amiloride for different 6-hour intervals during the entire incubation period. For this experiment we chose 40 ~ amiloride since this concentration did not significantly affect viability (as compared to sham-treated controls). When exposure occurred during the first 6 hours, there was about 50 % inhibition. At 6-12 hours this decreased to 35 % inhibition, and at later intervals there was essentially no inhibition. It has previously been shown that immediately following mitogen treatment there is an increase in lymphocyte membrane permeability which results in an increase in the rate of entry of Na+ into the cell. SEGEL and LICHTMAN (1) have produced evidence that it is this increase in intracellular Na+ concentra­tion which activates Na + IK + ATPase and is thus responsible for sustaining the increased Na + and K + active transport in mitogen-treated lymphocytes. Since amiloride is known to be an inhibitor of passive N a + transport, it may be that the inhibition of mitogenesis caused by early amiloride treatment is due to inhibition of this initial increase in intracellular Na + concentra­tion and the consequent chain of events. However, amiloride may also inhibit Ca++ fluxes either by direct or indirect mechanisms (2) and this could also lead to inhibition of mitogenesis. We are currently conducting transport studies that may clarify this problem.

1. SEGEL, G. B., and M. A. LICHTMAN. 1979. Proc. 13th Leuc. Cult. Conf. 399-401. 2. LEVENSON, R., D. HOUSMAN, and 1. CANTLEY. 1980. Proc. Nat!' Acad. Sci. 77: 5948-52.

VAMC and DERC, Dept. of Medicine, University of Iowa, Iowa City, Iowa, USA

Insulin potentiates human T lymphocyte proliferation induced by lectins and mitogenic oxidizing agents in serum-free media

1. ERCOLANI, T. J. BROWN, ]. NOLAN, and B. H. GINSBERG

Freshly isolated human T lymphocytes do not bear insulin receptors. However, upon activation by mitogens, insulin receptors appear on these cells after 12-18 hours culture, well in advance of cell division. Since this data suggests a role for insulin in the proliferation of these

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stimulated lymphocytes, we directly tested this by addition of various concentrations of insulin to activated matched purified T lymphocytes. This was performed 18 hours after culture in serum-free media. As an additional control, non-mitogenic fetal bovine serum (FBS) was added to matched T cell cultures. T cells were incubated with Phytohemagglutinin (PHA) or concanavalin A (Con A), or were neuraminidase treated and then incubated with Soybean agglutinin (SBA), or oxidized with sodium m-periodate (104). A range of lectin or oxidizing condition concentrations were employed. [3-H]-thymidine incorporation by cultured T cells was used as an index of T cell proliferation. Insulin receptors were assayed by [125-I]-Insulin binding. Insulin receptors (5000 sites/cell) with curvilinear scatchard plots characteristic of insulin binding to other insulin sensitive tissues were found on PHA activated T cells after 72 hours culture in serum-free medium without insulin. Similar results were seen in T cell cultures supplemented with 10 % FBS. Addition of 1.0 !-\g/ml insulin significantly increased T cell proliferation to PHA, Con A, SBA, and 104 at optimal and non-optimal mitogenic concentrations, but did not match proliferation of cultures supplemented with FBS. However, increased T cell proliferation to mitogenic agens was not consistently seen at physiologic concentrations of insulin. These findings suggest that insulin in supraphysiologic concentra­tions potentiates human T lymphocyte activation. However, its mechanism of potentiation may not be directly attributable to binding its receptor, but instead may be a consequence of binding to other receptors responsible for cell growth such as the somatomedin receptor for which insulin has weak agonist activity.

Faculty of Pharmacy, U. of Toronto, Toronto, Canada

Early biochemical protein events of Con A-activated murine T splenocytes

M. H. FREEDMA1\, F. CHEUNG, and H. LYONS

Modified 2D-P AGE of radiolabelled cellular proteins of murine T splenocytes demons­trated qualitative and quantitative changes after 4 h following Con A activation. Biosynthetic labelling with l5S-Met or lH-Leu before or during activation and subsequent fractionation of the cells by differential centrifugation indicate that as early as 4 h, Con A stimulation invokes a sequence of intracellular events that results in specific alterations in the plasma membrane (PM) proteins. We have reported that, in the presence of FCS, a mitogenic dose of Con A produced specific changes in the fluorograph of the PM profile in the regions 25-35 Kdal, pI 5-6 and 70-90 Kdal, pI 6-7,with overall increased intensity. We have now demonstrated that Con A activation in the absence of FCS results in the dramatic increase in the production of several additional l5S-Met-labelled PM proteins, notably 80-90 Kdal, pI ,,;; 6 and 15 Kdal, pI ,,;; 5. These proteins appear to be absent, or only minimally present, on quiescent T cells. The total TCA -precipitable dpm in the PM fraction increased on activation by as much as 70 %. Concommitantly, there was an increase in the labelled protein in the nuclear fraction to a maximum of 25 % and a decrease in that of the mitochondrial fraction to a maximum of 33 %. 2D-PAGE and fluorography of the mitochondrial proteins indicate a significant decrease on activation of a 35-37 Kdal protein with pI > 8. The TCA-precipitable dpm of the cytosolic fractions also increased to maximum of 50 % on activation. Furthermore, when T cells were pre-labelled with lH-Leu and activated in the absence of additional isotope the fluorograph of the PM proteins showed increased intensity in the Con A-stimulated samples. Clearly, within 4 h and in specific response to the lectin, labelled proteins have translocated from the cytoplasm to the plasma membrane. The detection of Con A-specific l5S-Met-labelled proteins further suggests that protein modification of the PM occurs rather than simply a general

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overall increase in synthesis. Consequently, it appears that at 4 h not only has there been binding of Con A and probable rearrangement of the PM, but also a definite intracellular response involving the nucleus and mitochondria which results in translocation and modifica­tion of the PM proteins.

Genex Corporation, 16020 Industrial Drive, Gaithersburg, MD. 20877, USA

TCGF and the early intracellular alkalinization permissive for T cell activation

D. F. GERSON

There is a pronounced intracellular alkalinization 6 hours after the mitogenic stimulation of murine T lymphocytes by concanavalin A (ConA). A similar alkalinization occurs in lipopoly­saccharide (LPS) stimulated murine B lymphocytes. ConA stimulated murine spleen lympho­cytes produce T-cell growth factor (TCGF) in detectable quantities approximately 8-12 hours after stimulation, and TCGF is required for subsequent DNA synthesis (at 48 hrs) and mitotic activity. It is possible to partially prevent the early intracellular alkalinization either by acidification of the growth medium or by pre-incubation of the lymphocytes with cyclosporin A. In both cases, subsequent DNA synthesis (at 48 hrs) is inhibited. Since cyclosporin A has been shown to prevent TCGF production, since TCGF production is a prerequisite for DNA synthesis at 48 hrs, and since intracellular alkalinization precedes protein synthesis in other comparable systems, it is reasonable to suggest that intracellular alkalinization is permissive for TCGF production and thus the subsequent mitotic activation of ConA stimulated T lympho­cyte populations.

University of Alberta, Edmonton, Alberta, Canada

Induction by purine and pyrimidine analogs of DNA strand breaks in mouse lymphocytes

WENDA L. GREER, CHRISTINE E. BOUMAH, KATHY M. SEMPLE, and J. G. KAPLAN

Work presented elsewhere in this volume (BOUMAH et aI., 1982) showed that 6 h pulses of mouse lymphocytes with 5-fluorouracil (5-FU) or 8-azaguanine (8-AG) permitted them to pass through blast formation, but blocked them at the GlIS boundary. There was no inhibition observed however, if 5-FU was added from 42-48 h, i.e. after cells have entered S phase. It was surprising to find that such a brief treatment during the early stages of stimulation would have a dramatic effect on a process which does not begin until some 30 h later. In studies using eH] 5-FU to trace the fate of the analog in the cells, about 50 % of the label was recovered from the acid soluble pool in the form ~ ribo and deoxyribonucleoside mono, di and triphosphates, as well as UDP membrane sugars, while most of the remaining label was recovered as RNA. Less than 0.5 % however was in the DNA fraction. There was no obvious change in the level or relative proportion of nucleotides in the pools. It seems therefore that 5-FU in lymphocytes is readily metabolized into nucleotides and RNA in the place of uracil. Since all other aspects of blast transformation appear to proceed normally (BOUMAH et aI., 1982) after treatment with the analogs, it was suspected that they may cause

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damage directly to the chromosomes, thus inhibiting DNA synthesis. A 6 h pulse with either 5-FU or 8-AG was found to cause DNA strand breaks, often to an even greater extent than 500 Rads of cobalt 60 gamma irradiation. Moreover, the breaks were still unrepaired at 48 h. It is also of interest to note that 6-mercaptopurine, an analog which did not inhibit DNA synthesis if added as a 6 h pulse, also did not produce strand breaks. We believe that this damage may at least partially account for the effect on DNA synthesis. It may be that many or all agents that cause irreversible reduction in subsequent DNA synthesis when administered early, during blastogenesis, act by inducing strand breaks or alkali labile bonds.

Basel Institute for Immunology, Basel, Switzerland

Changes of membrane potential and internal pH during mitogenic stimulation of lymphocytes

H. KIEFER

We have recently shown that depolarization of the lymphocyte membrane constitutes an early event in mitogenic stimulation. Evidence has now been obtained that indicates that depolarization is a consequence of specific interactions of mitogens with receptors on the cell surface. Spleen cells from Lipopolysaccharide (LPS) low responder mice (C3HlHeJ) are not depolarized by LPS but only by concanavalin A (Con A), while those of the LPS high responder (C3HfTif Born) depolarize upon addition of either LPS or Con A. Moreover, for T cell populations depleted of I A-bearing cells, the depolarization produced by Con A is only reversed by the addition of growth factors (e.g., TCGF); this indicates that the cell does not recover from this depolarization without the influence of growth factors, which probably constitutes a second, essential signal for the cell to undergo mitosis. Our recent experiments using 3HTPP+ show that only the first signal (mitogen) leads to depolarization. We have tested the effect of purified T cell growth factor (TCGF, interleukin 2, obtained from M. SCHREIER) on whole spleen and purified T cell populations, with and without removal of I A-bearing cells, and found no changes in membrane potential. The interaction of BRMF (B cell replication and maturation factor, obtained from F. MELCHERS) with purified (by nonnal gravity sedimentation techniques) small B cells and lipopolysaccharide (LPS)-induced large blast cells did not result in changes of membrane potential. We conclude that our earlier assumption, that the depolarization is the consequence of a specific triggering signal is correct and that this first signal is followed by a second one, the growth factor(s) . Depolarization seems to be essential but not sufficient to initiate proliferation or differentiation.

Institute for Animal Breeding and Genetics, Veterinary University Vienna, Austria

The kinetics of NOR activation in polydonally stimulated PBL of man, rabbit and dog

B. MAYR and W. SCHLEGER

The ribosomal RNA producing regions of PBL establish characteristic differential patterns of changes induced by different lectins (PHA, PWM, ConA, LPS). These synthesis patterns provide information about activation states and activation kinetics of lymphocyte subpopula­tions. Several tested cytostatic and immunosuppressive drugs cause partly selective modifica­tions of these kinetic patterns of NOR gene expression.

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Hospital for Joint Diseases Orthopaedic Institute, Mount Sinai School of Medicine, New York, N.Y., USA

Regulation of Ia antigen synthesis in T cells and monocytes

P. MERRYMAN, A. DIMITRIU-BoNA, and R. J. WINCHESTER

T cells and monocytes offer two contrasting models to examine the regulation of Ia antigen synthesis in man. Normal T cells have very low levels of Ia antigens detectable on a minor (0-2 %) percentage of cells, and no synthesis detectable by 3H leucine incorporation by immunoprecipitation with monoclonal anti Ia antibody (22c6) and analysis by SDS-PAGE. Following PHA stimulation Ia synthesis was observed. The kinetics of this synthesis showed a maximum at 48 hours with a substantial decline by 72 hours. The expression of Ia antigens on the cell surface detected by FA CS analysis was maximal at 72 hours, at or just after the peak of DNA synthesis and persisted 2-3 days. In certain disease states Ia antigens are found on a considerable proportion of T cells. Evidence was obtained that these Ia + T cells differed from normal T cells in several properties: following PHA stimulation peak DNA synthesis was slightly lower, however, it frequently occurred as much as one day earlier than in normal T cells. Ia antigen synthesis and expression maxima were similarly advanced in these individuals by approximately 24 hours, and were quantitatively considerably greater than that observed in normal T cells. Blood monocytes had a low basal level of Ia antigen synthesis, the quantity of which is increased two to threefold by stimulation with MLC supernates or cultures containing serum. Stimulation by lipopolysaccharides (LPS) results not only in the absence of the increase over basal Ia synthesis but a rapid cessation of all Ia antigen synthesis. The LPS inhibitory effect was also demonstrable in cultures where Ia synthesis was previously stimulated. In contrast, the addition of comparable amounts of LPS to stimulated T and B cells did not result in similarly diminished synthesis of Ia antigens. The accumulated evidence indicates that the synthesis of Ia molecules is characterized by a high degree of regulatory control involving both induction and repression.

Duke University Medical Center, Durham, NC 27710, USA

Plasma membrane depolarization is an early event in anti­immunoglobulin and antigen mediated B cell stimulation

J. G. MONROE and J. C. CAMBIER

In support of surface immunoglobulin (mIg) receptor crosslinking as an active mediator in B lymphocyte activation, a rapid depolarization of murine B cell plasma membranes is demons­trated upon interaction with stimulatory doses of anti-Fab. This response is detectable by 2 min and maximal at 2 hrs following addition of anti-Fab in vitro. The depolarization event is demonstrated to be independent of accessory cell function. Antibody reagents against other surface molecules such as Qa2 and H-2D were ineffective in facilitating the depolarization event. By quantitation of the frequency of B cells stimulated to undergo Go-G\ transition in response to anti-Fab and mitogens and the frequency depolarized by these agents, a correlation is established between depolarization and activation as defined by Go-G\ transition. Further, anti-Fab and mitogen stimulation is demonstrated to result in an increase in surface Ia antigen (mIa) expression indicative of commitment to Go-G\ transition for the mitogen-stimulated cells. Depolarization, induced by 50 mM K +, also stimulates an increase in mIa expression. Further, a correlation exists between the frequency of cells depolarized by K+ (48 %) and the frequency exhibiting elevated levels of mIa (40 %). Thus, depolarization appears causally

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related to increased mla expre~sion. Results using isolated TNP binding B cells with specific antigen, demonstrate depolarization induced by thymus-independent antigen TNP-Ba at frequencies equivalent to that stimulated to undergo Go-G! transition. Interestingly, the thymus-dependent antigen, TNP-ova also stimulates membrane depolarization in these cells, while monovalent hapten-carrier conjugates such as TNP-lysine, were ineffective in inducing depolarization. These results will be discussed in view of current ideas concerning thymus dependent and independent B cell activation.

Supported in part by NIH Grants AI 16128 and CA 09058-07.

Children's Medical Research Foundation, University of Sydney, Australia

Quantitative and qualitative changes in lymphocyte cytoplasmic poly(A)+ RNA during transformation

E. MCCAIRNS, G. E. O. MUSCAT, D. FAHEY, and P. B. ROWE

Cytoplasmic poly(A) +RNA in resting and phytohemagglutinin (PHA) transformed human lymphocytes has been examined. It was found that the translational activity of the cytoplasmic poly(A) +RNA from resting cells was 20 % of that from transformed cells in a rabbit reticulocyte lysate assay. Co-translation of each poly(A)+RNA preparation with rabbit globin mRNA showed that both fractions were essentially free of inhibitors of protein synthesis. Both cytoplasmic poly(A)+RNA fractions were capable of directing the synthesis of peptides in the molecular weight range 15,000-90,000 as determined on SDS gels. Two-dimensional electrophoresis of total proteins from resting and PHA-transformed lymphocytes were qualitatively similar and indicated predominantly quantitative changes. This implied changes in the relative abundance of mRNA species in lymphocyte cytoplasm during transformation. Characterisation of cytoplasmic poly(A)+RNA from resting and transformed lymphocytes on sucrose gradients and agarose gels showed the number average size of mRNA from resting and transformed lymphocytes to be 800 and 950 nucleotides respectively and the average length of poly(A) tails to be 45 and 50 residues respectively. Hybridisation with [3H]poly(U) showed each poly(A)+RNA fraction to have a poly(A) content of 5 %. Complementary DNA synthesised from each species of cytoplasmic poly(A)+RNA when back hybridised to an excess of mRNA from both resting and transformed cells indicated that there exists a large degree of homology between the message sequences found in resting and transformed lymphocytes. However, cross hybridisation after the common sequences have been removed by hydroxylapatite chromatography, show that about 4 % of the cytoplasmic poly(A)+RNA from transformed lymphocytes is not present in the resting cell. This difference may result from PH A-transformation specific gene expression.

Depts. of Internal Medicine and Biochemistry, University of Iowa College of Medicine, Iowa City, IA 52242, USA

Activation of a rearranged immunoglobulin kappa gene: evidence for a control region inside the gene

T. G. PARSLOW and D. K. GRANNER

Cells of the clonal murine line 702/3 constitutively express cytoplasmic immunoglobulin !A. heavy chains without associated light chain synthesis, a phenotype characteristic of the early

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stages of B lymphocyte ontogeny. In response to stimulation with bacterial lipopolysaccharide (LPS), 70Z/3 cells initiate synthesis of kappa light chain protein, and begin to express both heavy and light chains on their surfaces. This induction of kappa protein expression is accompanied by the appearance of kappa mRNA (1.2 kb) in the cytoplasm and of its precursor forms (5.1, 4.4, and 2.8 kb) in the nucleus. The cells harbor a single constitutively-rearranged kappa gene from which these transcripts are derived; the excluded kappa allele retains the germline configuration and is not detectably transcribed. When kappa gene expression is induced by LPS treatment, a discrete segment of DNA inside the gene undergoes a change in chromatin structure which renders it hypersensitive to digestion of nuclei with either pancrea­tic deoxyribonuclease I or certain restriction endonucleases. This hypersensitive site is located 0.5-0.7 kb upstream of the kappa constant region coding sequence, in the region which fortns the large intervening sequence of a rearranged kappa gene. Enhanced accessibility to nuclease digestion arises at this location in both the rearranged and gerrnline kappa alleles after LPS treatment; no additional hypersensitive sites occur in or near either allele. The location of this site does not coincide with known DNA or RNA splice junctions, and serves no other known function. Nevertheless, its sequence has been conserved during mammalian evolution while adjacent intronic sequences have not. Our data indicate that this region undergoes a local change in chromatin confortnation in response to LPS treatment; we propose that this altered structure may playa role in transcriptional activation of rearranged kappa genes.

Soroka Medical Center and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel

Effect of the calmodulin inhibitor, trifluoperazine, on lymphocyte activation

M. R. QUASTEL, F. CHATTAH, and A. SKIBIN

The action of trifluoperazine (TFP) on lymphocyte activation has been studied, and marked time-dependent inhibitory effects on nucleic acid synthesis were observed. TFP, a selective binder of calmodulin (1), was added at various concentrations to cultured normal human lymphocytes, which had been prepared on a ficoll-hypaque density gradient, and stimulated by PHA, Con-A and PWM. The incorporation of tritiated thymidine and uridine into RNA and DNA was measured. Exposure of the cells to TFP led to marked inhibition of nucleic acid synthesis at concentrations greater than 8 X 10-6M, the IDso being 1-2 X 10-sM. At these concentrations, trypan blue staining was not greater than that observed for unexposed control cells. The sensitivity of the activated lymphocytes was independent of the lectin concentration, but highly dependent on the time of administration of the drug. The cells were highly inhibited if the drug was given during the first 18 hours after adding the stimulatory lectin to the cultures. After this time, there was a pronounced decrease in the sensitivity of the cells to the compound. Pulse exposure (from 5 min-3 hrs) of the lymphocytes to TFP, with subsequent washing of the cells prior to the administration of PHA led to inhibition of DNA synthesis to an extent varying with the culmulative time of exposure to the drug. These results indicate a role for calmodulin particularly during the first 18 hours of lectin administration, in parallel to the 20 hour period of Ca + + dependence reported by BARD et al. (2).

1. WEISS, B., and R. M. LEVIN. 1978. Adv. Cyclic Nucl. res. 9: 285. 2. BARD, E., R. COLWILL, R. L'ANGLAIS, and J. G. KAPLAN. 1978. Can. J. Biochem. 56: 900.

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The Neurochemical Institute, Copenhagen, Denmark

Impaired thymidine uptake by T and B cells in presence of supernatants of syngenic polymorphonuclear leucocytes incubated with multiple sclerosis brain antigens

S. C. RASTOGI and J. CLAUSEN

The effect of proteinases released from polymorphonuclear (PMN) leucocytes incubated with multiple sclerosis (MS) and control brain antigens (MSG2) and KG2 respectively) was investigated with regard to proliferation of syngenic T and B cells of normal individuals and MS patients. The supernatants of PMN leucocytes incubated with or without antigens stimulated T and B cells. The proliferation of these cells was determined by (6-3H)thymidine uptake of T + B cell cultures in the presence and absence of PMN leucocyte supernatants. However, the mean value of stimulation of T + B cells by MSG2-PMN leucocyte supernatant was found to be significantly lower than those without antigen- or KG2-PMN leucocyte supernatant. It is concluded that a regulator of T and B cell proliferation may exist in PMN leucocytes and that it is released during incubation of PMN leucocytes with brain antigens. A significantly higher release of neutral proteinase from PMN leucocytes by the action of MSG2 compared to that by KG2 may also correspond to release of higher amount of regulator. The character of the regulator has not yet been studied. Lymphocytes of none of the patients in stable phase revealed a loss of suppressor function when MSG2 was employed as stimulant.

University of Arizona College of Medicine, Tucson, Arizona, 85724, USA

Zn + + - and Hg + + -mediated induction of mitogenesis, cell-mediated cytotoxicity, and interferon production in murine lymphocytes

CH. 1. REARDON and D. O. LUCAS

Zn + + and Hg + + are the chemically simplest nonspecific mitogens for splenic lymphocytes and thymocytes in Balb/C mice. The optimal concentrations of Zn + + (200 1JoM) and Hg + + (10 1JoM) induced several fold increases in 3H-thymidine incorporation in spleen cells at 96 to 144 hours in culture. 2-mercaptoethanol (2-ME) enhanced the spleen cell response to Zn++ but completely inhibited the response to Hg + +. Thymocytes were mitogenically activated by Zn++ only in the presence of both 2-ME and E. coli-derived lipopolysaccharide (LPS). LPS concentrations from 10 Ilg/ml to 1.0 ng/ml promoted a thymocyte response to Zn + +. In contrast, thymocytes were not activated by Hg++ in the presence or absence of 2-ME and LPS. Both heavy metals stimulated production of interferon (pH2 labile) by spleen cells at 96 hours in culture. Zn + + also stimulated low levels of natural killer activity in 168 hour spleen cell cultures with 6 % lysis of Yac-l target cells. In addition, Zn + + (200 1JoM) and Hg+ + (10 1JoM) induced lectin-dependent cellular cytotoxicity (LDCC) activity in 168 hour spleen cell cultures. The presence of PHA (10 Ilg/ml) in the cytotoxicity assay permitted the expression of Zn + + - and Hg + + -induced LDCC with about 50 % and 30 % lysis, respectively, of BW5147 target cells. Zn++ (200~) could not replace PHA in the assay as the LDCC­promoting mitogen. The readdition of Zn + + (200 1JoM) to the washed lymphocytes in the assay containing PHA was found to completely inhibit control cell killing and to inhibit Zn + + -induced killing by 75 % . A theoretical model is proposed for the multiple parametric activation of lymphocytes by Zn + + and Hg + + in which the metals alter «self» la and K or D structures on cell surfaces. Since Zn + + and Hg + + have high affinities for sulfhydryl groups, they may

15th International Leucocyte Culture Conference, Asilomar· 185

directly alter self structures by interacting with intramolecular thiols in the structure or indirectly alter self structures through their interactions with intermolecular sulfhydryl groups in which self structures are cross-linked with other surface components. Therefore, Zn + + - and Hg + + -induced activation of lymphocytes might reflect T cell recognition of surrounding cells expressing heavy metal-induced altered-self structures.

VA Medical Center, Yale University School of Medicine, West Haven, CT. 06516, USA

Acetylated diamines modulate at lymphocyte activation

]. L. RYAN, PH. K. BONDY, L. GOBRAN, and Z. N. CANELLAKIS

Hexamethylene bisacetamide (HMBA) and diaceryl putrescine (DP) inhibit lipopolysac­charide (LPS) stimulated mitogenesis of murine spleen cells in a concentration dependent manner. Maximal inhibition is obtained with 3mM HMBA. Higher doses of the acerylated diamines are toxic to the cultured cells after three days of incubation. 3 mM HMBA or DP reduces the LPS-stimulated (25 ~g/ml) uptake of 3HTdR from 9,092 ± 545 cpm to 1,881 ± 219 cpm in serum free cultures of Balb/c spleen cells. Mitogenesis induced by protein-free LPS is inhibited more efficiently than mitogenesis induced by LPS preparations which contain lipid A protein. The HMBA may be added up to 24 hours after LPS exposure with nearly maximal inhibition. If spleen cells are preincubated with 3 mM HMBA for 6 hours and subsequently cultured with LPS, normal stimulation takes place. If the cells are exposed to HMBA for 24 hours and washed they remain unresponsive to LPS. Inhibition occurs both in the presence and absence of fetal calf serum (FCS) suggesting that serum enzymes play no significant role in mediating this inhibition. Similar doses of HMBA also inhibit LPS induced immunoglobulin secretion in primary spleen cell cultures. In the absence of serum, the polyclonal IgM response (PFC) elicited by LPS is profoundly reduced from 43 ± 15 PFC/106 recovered cells to 2 ± 0 PFC/106 recovered cells in the presence of 2 mM HMBA. In the presence of 5 % FCS the same (2 mM) amount of HMBA inhibits the LPS induced PFC response from 205 ± 40 to 45 ± 18. Addition of nonacerylated diamines, putrescine, spermidine, and spermine over the range of 0.5 mM to 5.0 mM failed to inhibit lymphocyte activation. This level of diacerylated diamine is similar to that which causes Friend erythroleukemia cells to differentiate and which inhibits proliferation of these cells. These data suggest that uncharged acetylated polyamine derivatives penetrate the cell membrane more readily than the positively charged natural molecules where they may be deacerylated to biologically active molecules and regulate cell proliferation.

Depts. of Neoplastic Diseases, Biochemistry and Medicine, Mt. Sinai Sch. of Med., N.Y. and Immunex, Seattle, Wa., USA

Normal and leukemic ~ T ~ cell lymphocyte response to Interleukins (IL-l)

K.]. SCANLON, L. LACHMAN, R. GROSS, and S. WAXMAN

The stimulation of lymphocytes by growth promoting proteins offers a model system for studying the regulation of cell growth. Understanding the difference in nutrient transport systems in the cell cycle of normal and leukemic lymphocytes may reveal important targets for

186 . 15th International Leucocyte Culture Conference, Asilomar

chemotherapeutic agents. Fresh human lymphocytes were cultured in RPMI-1640 sup­plemented with 5 % human serum and variable concentrations of the monocyte derived growth promoting protein IL-l and/or phytohemagglutinin (PHA). IL-l was purified to apparent homogeneity from human monocytes by ultrafiltration and isoelectricfocusing. In the presence of PHA, lymphocytes elicit a dose response curve for optimal normal lymphocyte growth, maximum DNA synthesis and a 2-10-fold increased Na dependent «A» and «ASC» amino acid transport at 72 hrs after stimulation. Amino acid competition experiments for methionine uptake suggest unusual transport systems. Methionine uptake was also sensitive to inhibition by the cancer chemotherapeutic agent, methotrexate. In the presence of suboptimal doses of PHA, IL-l can elicit a dose response curve in normal lymphocytes for the stimulation of methionine and AlB transport by 72 hrs. In contrast, one patient with «T» cell chronic lymphocytic leukemic cells (CLL) did not exhibit quantitative or qualitative changes in growth or membrane transport systems when stimulated with PHA or PHA/Il-l. This preliminary data suggests that the receptor sites on these leukemic cells are either masked and/or other growth factors may be required for stimulating leukemic cell growth. This is currently under investigation. The apparent lack of response to mitogens and interleukins by «T» cell Cll suggests important differences in membrane structure and function between these and normal lymphocytes. Understanding this lack of response to cell growth factors in leukemic cells may improve the efficacy of cancer chemotherapeutic agents.

KJS is a Leukemia Society of America Fellow.

Ruhr-Universitat Bochum, Abteilung Chemie, Lehrstuhl Biochemie, D-4630 Bochum, FRG

40S hnRNP particles in resting and concanavalin A-stimulated lymphocytes and their association with snRNP particles

1. GABALDON DE KOCH, H. E. WILK, and K. P. SCHAFER

Changes in the post-transcriptional apparatus concerned with the processing of heterogene­ous nuclear RNA (hnRNA) have been studied in lectin-stimulated lymphocytes. 40S ribonu­cleoprotein particles (hnRNP) are a basic unit in which hnRNA is organized during matura­tion to mRNA. 40S hnRNP have been isolated from bovine lymphocytes. They contain hnRNA fragments (6-10S) and a typical set of mostly basic core proteins (31-41 kd: 80 % of particle protein mass) also found on other mammalian cells. Associated are other proteins (45-110 kd) and small nuclear RNP complexes (snRNP) consisting of small nuclear RNAs (snRNA: mostly species Ul) and a set of distinct proteins. The snRNP dissociate from the hnRNP above 100 mM NaC!. The amount of 40S hnRNP extractable from lymphocyte nuclei is correlated to the rate of hnRNA synthesis and increases accordingly after lectin stimulation. HnRNP from resting cells, however, lack the smallest of the core proteins (AI: 31 kd) which appears gradually in the 40S complex during Gl phase (up to 30 h). The core proteins contain NG, NG-dimethylarginine. Methylation occurs in vivo and in isolated nuclei using SAM. In contrast to other cells, the core proteins in lymphocytes are repetitively phosphorylated. The synthesis of snRNAs is coupled to hnRNA synthesis. All snRNA species (UI-U6) respond simultaneously showing a lag phase of 6 h after stimulation before their rate of synthesis increases together with other RNA classes. Using highly purified antibodies (anti-RNP), we have identified at least 6 proteins (13-50 kd) and snRNA U 1 as constituents of lymphocyte snRNP.

15th International Leucocyte Culture Conference, Asilomar' 187

Ruhr-Universitat Bochum, Abteilung Chemie, Lehrstuhl Biochemie, D-4630 Bochum, FRG

Lectin-induced changes in mRNAs of lymphocytes: mRNAs for actin, tubulin and calmodulin respond differently

N. KECSKEMETHY and K. P. SCHAFER

The differentiation of T cell-enriched concanavalin A-stimulated bovine lymphocytes was studied in vitro. mRNA was isolated from resting and stimulated cells. The amount of polyadenylated RNA increases from 3.6 to 12 X lQ-9 !!g per cell during 40 h Con A stimula­tion. The maximum length of the 3' poly(A) tract in this RNA is reduced from-240 to 220 residues in stimulated cells. During in vitro translation in the rabbit reticulocyte lysate mRNA from stimulated cells consistently incorporates about 1.6 to 3-fold more radioactivity per !!g

RNA into proteins than mRNA from resting cells. Three translation products have been identified on two-dimensional gels as actin, tubulin, and calmodulin. Large quantitative shifts are seen between proteins translated from mRNAs isolated from resting cells and stimulated cells, respectively. Actin and calmodulin are already major products from resting cell mRNA. Actin, however, increases about 5-fold after stimulation, while calmodulin does not change. Tubulin appears in substantial amounts only among stimulated cell mRNA products. Tubulin and calmodulin on the other hand remain mainly in the polyadenylated RNA fraction after stimulation, while 50 % of the actin together with a group of about 6 other major products is found among proteins translated from nonpolyadenylated RNA. We conclude that in lectin­stimulated lymphocytes, besides a general increase in the amount of mRNA, alterations in post-transcriptional processing reactions are active in determining the fate of individual mRNAs.

Med. Universitatsklinik, 7800 Freiburg, FRG

Human T-Iymphocyte activation depends on the presence of a comito genic serum glycoprotein

R. STAHN, H. A. FABRICIUS. W. BRUMMER, E. KOTTGEN

The induction of mitogenesis with phytohemagglutinin (PHA) in human peripherallym­phocytes (PBL) is a complex process involving several cell types and different mediators which requires the presence of serum. Incubatio(l of PBL with PHA in the absence of serum results in the production of Interleukin 1, which is synthesized by the adhering cell fraction. Mitogen­free IL 1 is able to induce the synthesis of Interleukin 2 (IL 2) in PBL only in the presence of serum. We could identify a 90 KD serum glycoprotein which can be distinguished from other known serum proteins in the same molecular weight range and which can replace whole serum in the process of PHA induced mitogenesis. Its biological activity can be abolished by removal of its sialic acid residues. Media conditioned for IL 2 using the 90 KD serum glycoprotein are (mitogen-free) able to induce mitogenesis in PBL and to support the growth of cytotoxic T­cell lines.

A model for T-cell activation involving a comitogenic serum glycoprotein will be proposed, and the biology of the glycoprotein will be discussed.

188 . 15th International Leucocyte Culture Conference, Asilomar

Northeastern University, Boston, MA 02115, USA

Cytoplasmic DNA from murine splenocytes

P. R. STRAUSS\ A. T. ANDRUTIS, and S. LEONG

We are concerned with the characterization of cytoplasmic DNA (cytoDNA) from spleno­cytes obtained from concanavalin A-stimulated mice. Previously we described the incorpora­tion of thymidineeHTdR) into a cytoplasmic-plasma membrane product (1). The same high molecular weight material was obtained in greater yield in the supernatant from cells lysed with Nonidet NO (NP40sup) and comprised 16-40 % of total cellular (cytoplasmic + nuclear) high molecular weight material after a 2-hr pulse with 3HT dR. CytoDNA from NP40sup was purified by proteinase K followed by phenol extraction, ethanol precipitation, RNase and cesium chloride gradient centrifugation. The OD260/0d28o ratio ranged from 1.7 to 1.9. Using a conversion factor of 50 ~g/OD26o and assuming 3.25 pg DNA/animal cell, the amount of DNA recovered as cytoDNA was 2-4 % of the total initial cell DNA. The low percentage of total cell DNA in the fraction should be compared with the high percentage of incorporated 3HTdR. This is characteristic of metabolically active DNA as defined by PELC (2). Base ratios of nuclear and cytoDNA were analyzed directly by means of HPLC after digestion with venom phosphodiesterase. The G+C content of cytoDNA isolated during 9 different experiments was 43.5 ± 4.4 (SD,9). Unexpectedly, wide deviations were observed in the ratios of G:C and A:T in cytoDNA which were not observed in similar ratios of nuclear DNA. For example, G:C varied between 0.31 and 1.21, while A:Tvaried between 0.58 and 2.2 CytoDNA had a heterogeneous size distribution, ad shown by gel electrophoresis employing 1.2 % agarose. The size varied between 0.3 and 5 kilobase pairs (assuming double stranded markers were appropriate). Nuclear DNA was too large to enter the gel under these conditions. When cyto DNA was treated with nuclease S-I, it could be reduced quantitatively to a single fragment with 74-140 base pairs in length. In conclusion, we have purified cytoDNA from splenocytes from concanavalin A stimulated mice and shown that it is a) metabolically active b) size heterogeneous, and c) at least partly single-stranded.

1. STRAUSS, P. et al. 1979. Molecular Basis of Immune Cell Function. J. G. Kaplan ed., Elsevier Amsterdam.

2. PELC, S. 1968. Nature 219: 162.

Supported by NIH CA24283 and ONR N001482K

Centre de Recherche, Hotel-Dieu de Quebec, Quebec, Canada

Histone synthesis and modification in resting and stimulated lymphocytes

W. I. WAITHE, J. RENAUD, and L. CARON

In most eukaryotic cells, histone synthesis is that there is a low «basal~ level of synthesis in G1• The mode of control of histone gene expression is not yet clear. Transcriptional, post­transcriptional and translational control mechanisms have all been implicated in various cell types. Since peripheral blood lymphocytes are in Go and enter G 1 and S upon activation, we decided to investigate histone synthesis as a preliminary to studies on the control of histone gene expression in activated lymphocytes. We have shown that newly synthesized histones are

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incorporated into chromatin of unstimulated Go and Gj-PHA-stimulated lymphocytes in spite of the absence of DNA synthesis. The Go and G j basal level of synthesis is resistant to 2 mM hydroxyurea. In spite of the overall increase in protein synthesis following PHA activation, histone synthesis does not increase in G j , however, during the first 6-10 hours of PHA activation there is an increase in the labeling of ubiquinated H2A and evidence of other histone modifications. In S phase cultures (40 hrs after PHA), when overall protein synthesis has increased about 7-fold, there is a 20-fold increase in histone synthesis.

Supported by MRC (Canaca) grant # MA4608

Lab. of Immunology, NIAID, and ':"Lab. of Clinical Science, NIMH, Bethesda, MD 20205, USA

An early biochemical pathway of transmembrane signaling in the stimulation of lymphocytes

M. J. WAXDAL, S. TOYOSHIMA, M. IWATA, F. HIRATA", and J. AXELROD"

The binding of mitogenic lectins to lymphocyte surface acceptors induces a series of biochemical events leading to the activation of the cell. Within five minutes of lectin binding and acceptor crosslinkage, the activation signal has passed through the cell membrane. This transmembrane signaling includes the activation of two membrane-associated enzymes responsible for increased phospholipid methylation, followed by an increase of calcium ion flux through the membrane, and results in the activation of phospholipase A2 to release arachidonic acid and lysolecithin (1-3). The specific inhibition of any of these steps also inhibited activation. Similar or identical pathways were followed by the activation of B cells with anti-immunoglobulin or LPS. The calcium ionophore, A23187, had no effect upon phospholipid methylation, but activated phospholipase A2. Two major pathways of arachidonic acid metabolism are known. One, the cyclooxygenase pathway leading to the synthesis of prostaglandins and other compounds, can be specifically blocked with no influence on cell activation. The second, or lipoxygenase pathway, leads to the hydroxy and hydroperoxy ecosatetraeonic acids. No specific inhibitor is available for this pathway, but the use of an inhibitor which blocks both pathways also blocked lymphocyte activation in a dose­dependent manner. These results suggest the existence of a common biochemical pathway for the activation of both T and B lymphocytes.

1. HIRATA, F., S. TOYOSHIMA, J. AXELROD, and M. J. WAXDAL. 1980. PNAS 77: 862-865. 2. TOYOSHIMA, S., F. HIRATA, J. AXELROD, M. BEPPU, T. aSAWA, and J. M. WAXDAL. 1982.

Mol. Immunol. 19: 229-234. 3. TOYOSHIMA, S., F. HIRATA, M. IWATA, J. AxELROD, T. aSAWA, and M. J. WAXDAL. 1982.

Mol. Immunol. 19: 467-475.

LMI, LCI, NIAID, National Institutes of Health, Bethesda, MD, USA

T and B cell responses to membrane potential sensitive cyanine dyes

H. A. WILSON, B. E. SELIGMAN, W. M. LEISERSON, and T. M. CHUSED

Cyanine dyes are lipophilic, fluorescent cations which distribute across the plasma mem­brane according to the Nernst relation, and hence may permit indirect measurement of

190 . 15th International Leucocyte Culture Conference, Asilomar

membrane potential. We have utilized flow cytometry to examine the responses of murine splenic T and B cells to two such probes, dipentylindocarbocyanine [di-I-Cs(3)] and dipentyl­oxacarbocyaine [di-O-Cs(3)]. At dye concentrations lower than 50 nM cells exhibit a unim­odal fluorescence distribution. In the presence of potassium (K +) and the K + ionophore valinomycin, fluorescence decreases (depolarization) as a function of the log K+ over a range from 1.5 to 80 mM K + in isosmolar medium. Dye concentrations above 100 nM are selectively toxic for B cells (identified in two-color studies by FITC-conjugated goat anti-mouse Fab). Under these conditions, a bimodal fluorescence distribution is observed. The lack of a further shift in di-I-Cs(3) fluorescence of B cells on the addition of K + and valinomycin established that the B cells were depolarized. Ionic substitutions and the use of ionophores indicated that these cells were near the sodium equilibrium potential. Chloride, however, was required for T cells to maintain their potential. Addition of valinomycin to dye-depolarized B cells repolarized these cells, suggesting that toxicity results from a selective decrease in K + permeability. A functional correlate to these observations was found in mitogenic responses in the presence of dye. B lymphocyte responses induced by lipopolysaccharide were more susceptible to dye addition than were T lymphocyte responses induced by Concanavalin A. Measurement of lymphocyte membrane potential with cyanine dyes requires recognition of complex dye-cell interactions. These are in some respects cell-type-specific and may help characterize mechanisms for ion fluxes. Understanding these interactions is a prerequisite for applying this technique to the study of lymphocyte activation.