Comparison of commercially available target enrichment methods for next-generation sequencing.

Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing (NGS) by allowing researchers to focus on their regions of interest. For the 2010-11 DNA Sequencing Research Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies (Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomic enrichment methods. Both companies were provided with the same genomic sample and challenged to capture identical genomic locations for DNA NGS. The target region totaled 3.5 Mb and included 31 individual genes and a 2-Mb contiguous interval. Each company was asked to design its best assay, perform the capture in replicates, and return the captured material to the DSRG-participating laboratories. Sequencing was performed in two different laboratories on Genome Analyzer IIx systems (Illumina, San Diego, CA, USA). Sequencing data were analyzed for sensitivity, specificity, and coverage of the desired regions. The success of the enrichment was highly dependent on the design of the capture probes. Overall, coverage variability was higher for the Agilent samples. As variant discovery is the ultimate goal for a typical targeted sequencing project, we compared samples for their ability to sequence single-nucleotide polymorphisms (SNPs) as a test of the ability to capture both chromosomes from the sample. In the targeted regions, we detected 2546 SNPs with the NimbleGen samples and 2071 with Agilent's. When limited to the regions that both companies included as baits, the number of SNPs was ∼1000 for each, with Agilent and NimbleGen finding a small number of unique SNPs not found by the other.

Strength, workload, anaerobic intensity and the immune response to resistance exercise in women.

AIM: The mechanism linking exercise intensity to the magnitude of the immune response is not completely understood. The purpose of this investigation was to determine whether the immune response to resistance exercise was associated with (1) changes in workload or (2) anaerobic exercise intensity. METHODS: Previously untrained women underwent 6 months of resistance training for lower and upper body (TOTAL, n = 34) or for upper body alone (UPPER, n = 30). Lymphocyte subsets [T (CD3+), CD4+, CD8+, NK and B], functional markers (CD45RA+ and CD45RO+), and mitogen (phytohemagglutinin-M, concanavalin A and pokeweed mitogen) and superantigen (staphylococcus a. cowans)-stimulated proliferation were measured from blood samples collected pre- and post-exercise for a squat resistance exercise consisting of six sets of 10 repetitions at 75% of one repetition maximum. This protocol was performed before (T0) and after 3 (T3) and 6 months (T6) of training. RESULTS: Lymphocyte recruitment to the circulation and proliferation following resistance exercise did not differ between training groups at any time, although the TOTAL group performed at a higher workload as training progressed. With respect to anaerobic intensity, exercise-induced increases in NK, CD4+, CD8+ and B lymphocyte concentrations were 42 (P = 0.07), 76 (P < 0.05), 72 (P < 0.05) and 242% (P < 0.01) greater in women in the highest compared with the lowest post-exercise lactate quartiles. Lymphocyte proliferation did not differ between lactate quartiles. CONCLUSIONS: Anaerobic intensity, rather than increased strength and workload, is associated with the number of lymphocytes recruited to the circulation, but not T and B cell proliferation responses.

The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans.

The aim of this study was to further investigate the mechanism of suppression of natural killer (NK) cell cytotoxic activity in peripheral blood following strenuous exercise. Blood was collected for analysis of NK cell concentration, cytotoxic activity, CD2 surface expression and perforin gene expression from runners (RUN, n=6) and resting controls (CONTROL, n=4) pre-exercise, 0, 1.5, 5, and 24 h following a 60-min treadmill run at 80% of VO2 peak. Natural killer cytotoxic activity, measured using a whole blood chromium release assay, fluctuated minimally in the CONTROL group and increased by 63% and decreased by 43% 0 and 1.5 h post-exercise, respectively, in the RUN group (group x time, P < 0.001). Lytic index (cytotoxic activity per cell) did not change. Perforin mRNA, measured using quantitative real-time polymerase chain reaction (QRT-PCR) decreased from pre- to post-exercise and remained decreased through 24 h. The decrease from pre- to 0 h post-exercise was seen predominately in the RUN group and was inversely correlated (r=- 0.95) to pre-exercise perforin mRNA. The NK cell surface expression of CD2 (lymphocyte function-associated antigen-2) was determined using fluorescent antibodies and flow cytometry. There was no change in the proportion of NK cells expressing CD2 or CD2 density. We conclude that (1) numerical redistribution accounted for most of the change in NK cytotoxic activity following a strenuous run, (2) decrease in perforin gene expression during the run was inversely related to pre-exercise levels but did not parallel changes in cytotoxic activity, and (3) CD2 surface expression was not affected by exercise.

Lymphocyte proliferation in response to acute heavy resistance exercise in women: influence of muscle strength and total work.

Little is understood about the immune responses to heavy resistance exercise. The purpose of this investigation was to determine the influence of physical strength and the ability to do more total work on lymphocyte proliferation after an acute bout of heavy resistance exercise. A group of 50 healthy but nonstrength trained women were recruited for the study and tested for their one repetition maximum (i.e. 1 RM or maximal mass lifted once). From the normal distribution of strength the top and bottom 8 women [mean age 22.5 (SD 3.1) years] were asked to volunteer to define our two groups (i.e. high strength and low strength). The two groups were significantly different (P < 0.05) in 1 RM squat strength [low strength 39.9 (SD 4.6) kg, 0.65 (SD 0.08) kg.kg body mass-1 and high strength 72.2 (SD 10.7) kg, 1.1 (SD 0.12) kg.kg body mass-1] but were not significantly different in body mass, age, activity levels, and menstrual status (all in same phase). Each performed a resistance exercise protocol consisting of six sets of 10 RM squats with 2 min rest between the sets. The 10 RM loads and total work were significantly greater in the high strength group than in the low strength group. Blood samples were obtained pre-exercise and immediately post-exercise for test for lactate (significant increase with exercise) and cortisol (no changes) concentrations with no differences noted between groups. Immunological assays on the blood samples determined the incorporation of tritiated thymidine by lymphocytes in responses to concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM). Following the squat exercise, there was a significant decrease in lymphocyte responsiveness to PWM in the high strength but not in the low strength group for both total proliferation and proliferation adjusted per B or T cell. On the other hand, lymphocytes from the low strength group proliferated to a significantly greater extent (adjusted per T cell) in response to ConA and PHA. These data indicate that the heavy resistance exercise protocol reduced the lymphocyte proliferative responses only in the stronger group of subjects. This effect may have been due to the high absolute total work and the greater exercise stress created by the resistance exercise protocol in the high strength group. Therefore, individuals performing at the same relative exercise intensity (i.e. 10 RM) in a resistance exercise protocol may have different immune responses stemming from differences in absolute total work performance.

Lymphocyte subpopulations in lymphoid organs of rats after acute resistance exercise.

PURPOSE: The ability of aerobic exercise to change lymphocyte subpopulation distributions is well documented; much less is known about resistance exercise. The purpose of this experiment was to determine the effects of an acute bout of resistance exercise on lymphocyte subpopulations in primary and secondary lymphoid compartments. METHODS: Male rats were operantly conditioned to climb a ladder while carrying weights that were progressively increased to equal body weight. During the acute session, rats performed repetitive climbs until exhaustion. Thymus, spleen, blood, and axial and inguinal lymph nodes were removed; leukocytes were isolated and incubated with monoclonal antibodies against differentiation markers, activation antigens, and adhesion molecules. RESULTS: Exercised versus control rats had greater numbers of leukocytes in the thymus, axial, and inguinal nodes but not in the blood or spleen. The percentage of CD4+ cells increased after exercise in the thymus, spleen, and blood. The percentages of cells expressing the integrin LFA-1beta were elevated in all the tissues except inguinal lymph nodes. In addition, more leukocytes from exercised than nonexercised rats expressed detectable numbers of activation markers, IL-2 receptor-alpha and MHC class II molecules; however, as indicated by proliferating cell nuclear antigen analysis, the cells were not actively dividing at the time of assay. CONCLUSIONS: Based on these and published data, it appears that a single bout of resistance exercise can affect lymphoid cell subpopulations probably by inducing changes in leukocyte trafficking.

Quantitative real-time polymerase chain reaction for the core facility using TaqMan and the Perkin-Elmer/Applied Biosystems Division 7700 Sequence Detector.

Quantitative real-time polymerase chain reaction (PCR) using the Perkin Elmer/Applied Biosystems Division 7700 Sequence Detector provides an accurate method for determination of levels of specific DNA and RNA sequences in tissue samples. It is based on detection of a fluorescent signal produced proportionally during amplification of a PCR product. Turn-around time for data acquisition and analysis by real-time PCR with the 7700 model is short, and results are more reliable than by traditional PCR methods. This technology can be successfully offered as a service in a core faculty setting.

Modulation of levels of a negative transcription factor for IL-2 by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid.

A negatively acting transcription factor, negative regulatory element-A (NREA) that suppresses the transcription of interleukin 2 (IL-2) mRNA, has been described previously. We found that treatment of primary bovine lymphocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, for at least 18 h both increased the levels of the factor and blocked concanavalin A (Con A)-induced proliferation. In contrast, treatments of less than 18 h with TPA decreased NREA levels and increased Con A-induced proliferation. NREA binding activity also increased over basal levels during the first 4 h of stimulation of lymphocytes with Con A in the absence of TPA; after 4 h, NREA levels fell. Phosphorylation of the NREA protein was required for binding to its DNA consensus sequence. Furthermore, incubation of lymphocytes with okadaic acid (OKA), a phosphatase inhibitor, led to increased levels of NREA binding activity and to decreased cell proliferation. Because exposure of lymphocytes to either OKA or TPA should lead to an increase in the phosphorylation and binding of the NREA protein, and a decrease in IL-2 production, proliferation should be decreased. Incubation of lymphocytes with either TPA or OKA inhibited proliferation. However, the mechanisms of action of OKA and TPA appeared to be different because exogenous IL-2 reversed the inhibition of proliferation caused by TPA but not by OKA.

The S-oxalin, N-acetyl-S-oxalylcysteamine, inhibits lymphocyte proliferation, IL-2 production and utilization.

S-Oxalins are a recently described class of cell metabolites that appear to function as negative regulators of proliferation. Previously we have shown that exogenous S-oxalylglutathione (GS-Ox) inhibits the proliferation of lymphocytes by inhibiting the production and utilization of IL-2. In the present study the synthetic S-oxalin, N-acetyl-S-oxalylcysteamine (ACS-Ox), was utilized in similar experiments to determine whether GS-Ox itself, or possibly some metabolite formed following initial conversion of GS-Ox by gamma-glutamyltransferase (GGT), is responsible for the effects (ACS-Ox is not metabolized by GGT). ACS-Ox inhibited DNA synthesis in lymphocytes stimulated by concanavalin A similarly to GS-Ox. IL-2 production and utilization and IL-2R expression were inhibited as well. ACS-Ox also inhibited the proliferation of IL-2 dependent cells at the same concentration as GS-Ox. Because the effects of GS-Ox and ACS-Ox are so similar, presumably the S-oxalin itself, rather than some metabolite, is responsible for the observed effects. Transfer of oxalyl groups from S-oxalins to various proteins thiols is the most likely mechanism involved.

Inhibition of proliferation and of IL-2 production and utilization in lymphocytes by S-oxalylglutathione.

Previously we have shown that S-oxalins (monothiolesters of oxalic acid) are ubiquitous mammalian metabolites whose concentrations decrease when lymphocytes are stimulated to proliferate. The present study was undertaken to further examine the role of S-oxalins in the proliferation process. When added to lymphocytes stimulated with concanavalin A, the S-oxalin, S-oxalylglutathione (GS-Ox), inhibited DNA synthesis by 50% when present at ca. 0.15 mM and virtually 100% at 0.5 mM. The inhibition was reversible. The presence of GS-Ox blocked IL-2 production, but addition of IL-2 did not permit DNA synthesis to proceed. GS-Ox also inhibited proliferation of an IL-2-dependent cell line, BT2. In primary lymphocytes GS-Ox reduced IL-2 receptor expression, but not in an IL-2-dependent blast cell line. Overall RNA synthesis and protein synthesis were not significantly altered by GS-Ox. Levels of the positive transcription factor, NF-kappaB, were decreased after incubation of lymphocytes with GS-Ox, but the amount of a negative transcription factor, NREA, was largely unchanged. The results not only provide further evidence that S-oxalins are small-molecule cell proliferation inhibitors, they also clarify to some extent the specific steps in the activation response modulated by S-oxalins.

The effect of a 10-day space flight on the function, phenotype, and adhesion molecule expression of splenocytes and lymph node lymphocytes.

Previous studies have indicated that space flight affects the activation of lymphocytes from humans, monkeys, and rodents. In rats, where lymphocytes from blood, spleen, and lymph nodes have been tested, the accumulated data suggest that the effects of flight on various cells are lymphoid organ-specific. Thus, cells may be affected by variations in trafficking brought about by fluid shifts in microgravity (< 10(-3) g). In this study we examined lymphocyte activation (IL-2 production) as well as the expression of surface differentiation antigens and of adhesion molecules by splenocytes and lymph node lymphocytes (LNL) after a 10-day flight (Space Shuttle Mission STS-57). For splenocytes and LNL from flight (FLT) animals, IL-2 production decreased in response to the T cell receptor-independent mitogen 12-O-tetradecanoylphorbol-13-acetate plus ionomycin, but was not affected by stimulation with the T cell receptor-dependent mitogens Concanavalin A or phytohemagglutinin. In addition, the percentage, as well as fluorescent intensity, of splenocytes which expressed CD8, CD4, or kappa increased after flight. The percentage of LNL expressing CD2 also increased but those expressing CD5 decreased. The percentage of cells expressing the integrins LFA-1 alpha and beta increased with splenocytes from FLT animals but decreased for LNL. In contrast, FLT animals showed a decrease in the percentage of selectin-positive splenocytes. ICAM-1 expression did not change. In summary, these data are consistent with a model in which microgravity affects lymphocyte redistribution among organs, which in turn influences the activation potential of the cells.

Mechanisms of activation and suppression in rat Nb 2 lymphoma cells: a model for interactions between prolactin and the immune system.

Rat Nb 2 lymphoma cells have been widely used to bioassay human growth hormone and many species of prolactin. Because their morphologic characterization suggests a T-cell lineage, Nb 2 cells were examined for their response to the T-cell mitogens concanavalin A, pokeweed mitogen, and phytohemagglutinin P. As expected, a dose-response to rat prolactin was observed; however, attempts to induce proliferation using the conventional T-cell mitogens failed at concentrations normally stimulatory for rat primary lymphocytes. Moreover, when Nb 2 cells were simultaneously incubated with lectin plus a suboptimal concentration of prolactin, a dose-dependent suppression of the stimulatory effects of prolactin was observed with phytohemagglutinin P and pokeweed mitogen, although not with concanavalin A. Culture medium of prolactin-stimulated Nb 2 cells also contained a factor which inhibited normal rat lymphocyte activation by concanavalin A. The factor did not block induction of the IL-2 receptor and proliferation of IL-2-dependent CTLL-2 cells could be restored by exogenous IL-2. Because Nb 2 cells evolved from a lactogen-dependent lymph node tumor, these results may have implications for further understanding the role of pituitary hormones, particularly prolactin, in the immune response to hormone-dependent tumor progression.

IL-2 mRNA levels and degradation rates change with mode of stimulation and phorbol ester treatment of lymphocytes.

Transient expression of interleukin 2 (IL-2) in activated T lymphocytes may be due to transcriptional and post-transcriptional regulation. As incubation of lymphocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to mitogenic stimulation results in decreased levels of IL-2 mRNA, we asked if IL-2 mRNA stability was affected. We found that in TPA-treated cells, IL-2 mRNA was degraded more rapidly than in untreated ones whether the mitogenic stimulus was Concanavalin A (Con A), Con A plus TPA, or TPA plus ionomycin. The degradation was blocked if the TPA pre-incubation included cycloheximide. In contrast, when TPA was included as a co-mitogen, i.e. added at the same time as the mitogen, the IL-2 mRNA levels and stability significantly increased. Compared to the levels found in Con A stimulated cells, TPA plus Con A increased IL-2 mRNA levels by as much as 20-fold and the half-life by 5-fold. TPA plus ionomycin increased the message levels at least 100-fold and half-life by nearly 10-fold. These effects on IL-2 mRNA were not general because IL-2 receptor mRNA stability was not changed even though it also is transiently expressed during the course of lymphocyte activation.

The expression of forms of prolactin receptors in splenocytes and thymocytes of neonatal rats: the effect of milk ingestion.

Because prolactin (PRL) plays a role in neonatal immune development, we examined the expression of prolactin receptors (PRL-R) in neonatal lymphoid tissues. We had shown previously that deprivation of milk-borne PRL, days 2-5 in the neonatal rat, leads to enhanced in vitro mitogenesis of thymocytes and splenocytes as well as a change in lymphoid-specific, cell surface antigens (GROVE et al. 1991). In this present study, we asked if neonatal lymphocytes express PRL-R; which forms of PRL-R are expressed (long vs. short form); when these forms are expressed during development; and if milk ingestion plays a role in receptor expression. Two approaches were taken using neonatal rat thymocytes and splenocytes: RNA was analyzed by polymerase chain reaction (PCR) and cells were stained with antibody to PRL-R and analyzed by flow cytometry. In regard to cell surface expression, the percentage of PRL-R positive splenocytes was greater than thymocytes at all ages tested. In the spleen, the percentage of PRL-R positive cells gradually increased to adult levels by day 10; in the thymus the percentage fell to adult levels by the first day after birth. Finally, milk ingestion in the first 7 h decreased the percentage of cells expressing cell surface PRL-R. Tissues from animals deprived of milk during this time expressed PRL-R at the same level as the newborn.

Cytochalasans and PMA induce IL-2 receptors on CD8+ lymphocytes.

The cytochalasans, fungal metabolites that interact with actin, can affect lymphocyte proliferation; high concentrations inhibit lectin-induced proliferation and low concentrations augment it. The phorbol ester tumor promoter, PMA, alone is not mitogenic for primary lymphocytes but enhances the activity of mitogenic lectins. Because the cytochalasans have been reported to increase intracellular Ca2+ and because PMA activates protein kinase C, lymphocytes were treated with PMA and cytochalasin B (CyB) to determine if this combination would induce DNA synthesis. While this treatment by itself did not cause proliferation, lymphocytes cultured with PMA and CyB overnight, washed, and recultured with IL-2 proliferated to the same degree as lymphocytes stimulated with Con A. Three different cytochalasans, cytochalasin B, cytochalasin D, and chaetoglobosin C, all of which bind to cellular actin with different affinities and only one of which affects glucose transport, induced IL-2 receptors in combination with PMA. Flow cytometric analysis with an antibody to the IL-2 receptor alpha subunit confirmed the induction of receptors on CD8+ cells. However, no IL-2 was produced after the exposure of lymphocytes to the combination of cytochalasans and PMA. Therefore, there was sufficient signal to induce IL-2 receptor expression but not to induce IL-2.

Effect of neonatal milk-prolactin deprivation on the ontogeny of the immune system of the rat.

A growing body of evidence suggests that prolactin (PRL) is involved in regulation of the immune system in the adult. PRL provided to the neonate in mother's milk also has been shown to be important in development of the neonatal neuroendocrine regulation of PRL secretion. Therefore, in this study we asked if deprivation of the neonate of milk-PRL on days 2-5 postpartum affected the ontogeny of the immune system. Two aspects, DNA synthesis (3H-thymidine incorporation) of neonatal lymphocytes in response to polyclonal mitogens in vitro and expression of lymphoid cell surface antigens, were examined. Splenocytes and thymocytes from neonates ranging in age from 5 to 28 days were taken from mothers treated with bromocriptine or saline on days 2-5 of lactation. Splenocytes from pups of vehicle-treated mothers showed a gradual increase in surface antigen expression by day 5 to 28. Thymocyte patterns and percentages of these surface proteins were at adult levels at the earliest times tested. Thymocytes from day 5 and 10 neonates were more responsive to Con A than were splenocytes, but both thymocytes and splenocytes showed an increase in mitogenic responsiveness until day 18, a sharp decline at day 21, and an increase again at day 28. The fact that day 21 is the time of intestinal closure (cessation of absorption of macromolecules from the gut) suggested that milk-borne material plays a role in immune cell maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Negative regulation of interleukin-2 production in primary lymphocytes by 12-O-tetradecanoylphorbol-13-acetate.

Stimulation of quiescent T lymphocytes to proliferate involves a complex series of events both between and within cells. At least 70 genes are known to be induced or activated from the time of the initial stimulation until DNA synthesis. While some of these gene products, e.g., interleukin-2 (IL-2) and IL-2 receptors, are required for proliferation, others, e.g., gamma-interferon and colony-stimulating factor, are ancillary to activated T cell function. Several biochemical signal transductions are among the early events. One of the earliest is phospholipase C-mediated hydrolysis of phosphatidylinositol leading to release of diacylglycerols and inositol phosphates, which in turn activate protein kinase C and elevate intracellular free calcium levels. The discovery that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) both enhances proliferation and activates protein kinase C strengthens the evidence for a general role of protein kinase C in proliferation. Yet, the exact consequences of stimulation of protein kinase C in regard to specific proliferation proteins is still not clear. In this study, we present evidence that protein kinase C activation is directed to production of IL-2 but not to IL-2 receptors. Under conditions of TPA treatment in which protein kinase C was chronically reduced in T lymphocytes, IL-2 production was greatly depressed as were the level of IL-2 mRNA and [3H]thymidine incorporation. In contrast, these cells still expressed high affinity IL-2 receptors and proliferated when endogenous IL-2 was added. Because neither phosphatidylinositol metabolism nor Ca2+ flux was affected, the block appeared to be mediated directly or indirectly through protein kinase C.

Differential activation and inhibition of lymphocyte proliferation by modulators of protein kinase C: diacylglycerols, "rationally designed" activators and inhibitors of protein kinase C.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) can enhance or inhibit lymphocyte proliferation. Enhancement correlated with increased interleukin 2 (IL-2) production and activation of protein kinase C while inhibition correlated with decreased IL-2 and downregulation of protein kinase C activity (D.S. Grove and A.M. Mastro, Cancer Res. 51, 82-88). In this study, various activators and inhibitors of protein kinase C were used in order to try to separate the effects of TPA on this enzyme from its effects on IL-2 production and determine if protein kinase C activity was directly or indirectly related to IL-2 production. 1,2-Dioctanoylglycerol, 1-oleoyl-2-acetyl-glycerol, phospholipase C, and two "rationally designed" activators, 6-(N-decylamino)-4-hydroxy-methylindole and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, were tested. Some activators enhanced proliferation in the presence of a Ca2+ ionophore, ionomycin, but not concanavalin A. Some activators suppressed proliferation and downregulated protein kinase C. Others neither downregulated protein kinase C nor inhibited IL-2 production and proliferation. However, inhibition or downregulation of protein kinase C activity always correlated with decreased IL-2 and depressed proliferation. Thus, the evidence in this and the previous study suggests that activation of protein kinase C is directly related to IL-2 production in activated T cells.

Differential activation and inhibition of lymphocyte proliferation by phorbol esters, mezerein, teleocidin, and okadaic acid.

Lymphocytes can be stimulated to proliferate in vitro by mitogens such as concanavalin A. The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) can enhance this proliferation, partly because of an increase in interleukin 2 (IL-2) production. However, if lymphocytes are treated with TPA for 24 h before concanavalin A exposure, IL-2 production and proliferation are depressed. The target of the action of TPA is protein kinase C, which is activated after a short exposure but down-regulated after a longer one. This study was designed to determine if the modulation of IL-2 was separable from the modulation of protein kinase C. When phorbol esters phorbol 12-retinoate-13-acetate, phorbol 12,13-dibutyrate, 12-deoxyphorbol 13-phenylacetate, and 12-deoxyphorbol 13-phenylacetate-20-acetate, as well as nonphorbol tumor promoters mezerein, telocidin, and okadaic acid, were tested, all but okadaic acid reproduced the effects of TPA. However, 12-deoxyphorbol 13-phenylacetate and 12-deoxyphorbol 13-phenylacetate-20-acetate were required at nearly 100-fold higher concentrations than TPA to suppress IL-2 production, suppress mitogenesis, and cause down-regulation of protein kinase C. A comparison of structures indicated that an R group at the 12-position was less important for IL-2 production and mitogenesis than for down-regulation of protein kinase C and the suppression of mitogenesis. In no case was the modulation of protein kinase C separated from the effects on IL-2 production and proliferation.

Effect of macrophages on the translocation of protein kinase C in concanavalin A-stimulated lymphocytes.

The concanavalin A (Con A)-induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12-0-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activating PKC, while IL1 appears to act at a different step in proliferation.

Prevention of the TPA-mediated down-regulation of protein kinase C.

Protein kinase C activity in lymphocytes was down-regulated upon exposure of the cells to 12-0-tetradecanoylphorbol-13 acetate. This down-regulation was prevented by preincubating the cells with sphingosine, a reported protein kinase C inhibitor. Two other protein kinase C inhibitors, palmitoylcarnitine and phloretin, were ineffective in preventing down-regulation by the phorbol ester.