Partial characterization of a prostaglandin-induced suppressor factor

Glass adherent splenic T cells, cultured in the presence of prostaglandin E₂ (10^?5M), were found to elicit a factor capable of nonspecifically suppressing PHA- and LPS-induced mitogenesis. Cells from C57B1/6J, Balb/C, and C3H/He mice were all capable of producing this suppressor factor, although some degree of variability in the response of cells from C3H mice to the factor was observed. The suppressor (designated prostaglandin-induced T-cell derived suppressor, PITS) was characterized biochemically and it was found that the activity was resistant to boiling, and treatment with RNase and DNase, yet was sensitive to treatment with proteinase K, trypsin, and Pronase. Further, PITS supernatants were found to contain at least two suppressors with approximate molecular weights of 35,000 (PITS_α) and 5000 (PITS_β). Results from experiments with cycloheximide-treated glass-adherent T cells indicate that prostaglandin E₂ may function by inducing the release rather than de novo synthesis of the PITS. These results indicate that the reported overall suppressive effect of prostaglandin E₂ on lymphocytes may in part be due to the release by certain T cells of a suppressive factor.     

alpha-Fucose inhibits human mixed-lymphocyte culture reactions and subsequent suppressor cell generation

Carbohydrate moieties serve as important sites of interaction for many lymphocyte activities. The potential role of saccharides in the cellular interactions involved in mitogen-, antigen-, and alloantigen-induced proliferation was investigated. Eight different monosaccharides were tested for their inhibitory potential when added to uni- and bidirectional mixed-lymphocyte culture (MLC) reaction as well as to mitogen (Con A, PHA, PWM)-stimulated cultures. Only alpha-L-fucose blocked the MLC reaction in a dose-dependent fashion while having no effect on mitogen stimulation, although antigen-specific stimulation was also blocked by fucose. Similarly alpha-L-fucose specifically inhibited the MLC-induced generation of suppressor cells. Pretreatment of the MLC responder cells with fucose dehydrogenase abolished the MLC reaction while stimulator cell pretreatment had no effect, suggesting that the recognition site of the former contained alpha-L-fucose. The generation and the effector phase of Con A-induced suppressor cells was not affected by fucose, indicating that different receptors are involved in the latter. Apparent competitive inhibition by exogenous fucose of the cell-cell interaction required for the MLC reaction suggested that this monosaccharide is an essential constituent of allogeneic recognition sites.     

The association of suppressor cells with mononuclear cell aggregates in spleens of tumor-bearing mice

Spleen cell suspensions from mice with progressive B-16 melanoma consistently contained significant numbers of aggregates of mononuclear cells (MN-Agg), when compared to spleen cell suspensions from normal mice or mice in the early stages of tumor growth. Histological, histochemical and immunological characterization of the cells involved in MN-Agg from tumor-bearing mice indicated that aggregates were composed of macrophages and T and B lymphocytes. The formation of MN-Agg was dependent upon the macrophage content of the spleens of tumor-bearing mice since the appearance of MN-Agg correlated temporally with an increase in the number of splenic macrophages demonstrable in tumor-bearing animals. An antigen nonspecific suppressor cell was identified in the spleens of mice 15 days following the appearance of palpable B-16 tumor, and the appearance of the suppressor cell population closely correlated with the appearance of MN-Agg. Additionally, fractionation of MN-Agg-containing cell suspensions demonstrated that fractions highly enriched in MN-Agg were concomitantly enriched for suppressor cells. The suppressor cell associated with MN-Agg was a T lymphocyte since suppressor activity of MN-Agg could be abolished by treatment of MN-Agg with a rabbit anti-mouse brain serum and complement. It is proposed that the generation of suppressor cells in mice with B-16 melanoma may require specific interaction between macrophages and lymphocytes which is manifested in the spleens of tumor-bearing mice by the formation of MN-Agg.     

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). I. Analysis of the PITS beta factor

Within populations of mitogenically (PWM) stimulated normal human lymphocytes, the proliferation of B lymphocytes is terminated by T cells. In contrast, T cells limit their own proliferation. T cells thus apparently measure and terminate the proliferation of B cells as well as themselves, suggesting an important role for them in limiting amplification during immune response. Under the culture conditions employed, PWM-induced B- and T-cell proliferation was uncoupled from B-cell differentiation into plasmacytes. Termination of B-cell proliferation in this in vitro model of humoral immune response is independent of B-cell differentiation.     

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). II. Resolution of multiple PITS beta factors

It has been shown that T cells cultured with prostaglandin E2 are induced to release at least two peptide-containing lymphokines (PITS). Both the high-molecular-weight (35,000; PITS alpha) and the low-molecular-weight (2000 to 5000; PITS beta) factors were shown to be potent inhibitors of both T-cell- and B-cell-dependent mitogen responses. Data are reported here which show that the PITS beta factor may be reproducibly resolved into seven components by high-pressure liquid chromatography. Although each of these components is capable of suppressing the phytohemagglutinin-induced blastogenic response, not all of these factors will suppress the in vitro antibody response to sheep erythrocytes, or a mixed lymphocyte reaction. Nevertheless, the broad-range suppressive effects previously reported for PITS beta now seem to be mediated by multiple low-molecular-weight PITS factors.     

Macrophage activation during experimental murine brucellosis: II. Inhibition of in vitro lymphocyte proliferation by brucella-activated macrophages

During infection of CBA mice with Brucella abortus strain 19, there is a massive accumulation of macrophage-like cells in the spleen with resultant gross splenomegaly. In vitro cultures of cells from these spleens show a reduced proliferative response to brucellin and to other mitogens (phytohemagglutinin, concanavalin A, and lipopolysaccharide). The effect could be overcome by the addition of high concentrations of mitogen. Removal of adherent cells from spleen populations derived from 20-day infected mice abrogated the suppressive effect. Conversely, adherent cells from the spleens of 20-day infected mice inhibited proliferation of normal spleen cell cultures. Inhibition of responsiveness of normal spleen cells by cells from the spleens of infected mice occurred even when the two populations were separated by dialysis membranes. Although proliferation was measured by uptake of tritiated thymidine, inhibition in this system was not due to the release of unlabeled thymidine from macrophages.     

Control of the expression of interleukin-2 activity

Mitogen stimulation of lymphocytes activates phospholipase A₂, which in turn generates arachidonic acid by its action on phospholipids. Cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandins and related cyclic compounds, whereas lipoxygenases direct the formation of straight-chain hydroxylated derivatives such as, for example, the leukotrienes. The studies in this report suggest a correlation between arachidonic acid metabolism and production of the lymphokine, interleukin-2 (IL-2). Inhibitors of phospholipase A₂ activation, mepacrine, tetracaine, glucocorticoids and estradiol, all inhibited the expression of IL-2 activity in concanavalin A-stimulated mouse spleen cells. Inhibition of cyclooxygenase and lipoxygenase activities also resulted in decreased IL-2 production. This was established by the use of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin, and nordihydroguajaretic acid (NDGA). A more direct attempt at influencing the arachidonic acid metabolism by addition of the fatty acid to IL-2 production cultures demonstrated that arachidonic acid bound very tightly to IL-2. Extensive dialysis or partial purification of the lymphokine by reverse-phase high-performance liquid chromatography failed to remove the bound arachidonic acid. It was shown, however, that no covalent interactions were involved. In addition to an active arachidonic acid metabolism, continuous protein synthesis was required for expression of IL-2 activity. Thus incubation with puromycin inhibited IL-2 production.     

Immunoregulation by macrophages: differential secretion of prostaglandin E and interleukin 1 during infection with Salmonella enteritidis

We have investigated the temporal relationship between bacterial clearance in vivo, macrophage bactericidal activity in vitro, and the secretion of immunoregulatory molecules, prostaglandin E (PGE) and Interleukin 1 (IL1) in vitro, during infection with an avirulent strain of Salmonella, Salmonella enteritidis 11RX. The two model systems used were normal mice challenged intraperitoneally with SE11RX (NSE) and previously sensitized mice rechallenged 24 days later with SE11RX (SESE). The increasing nonspecific bactericidal activity of the peritoneal macrophages from NSE and SESE mice after the second day of infection paralleled the clearance of bacteria observed in vivo. Prostaglandin secretion by normal macrophages cultured for 4 hr with LPS correlated inversely with intracellular bacterial numbers but showed a positive correlation when cultured with opsonized SRBC or C3-zymosan complexes. PGE was the major arachidonate metabolite secreted. The cells from sensitized mice secreted tittle prostaglandin with any stimulus, and this secretion showed a positive correlation with bacterial number. The capacity to secrete IL1 in response to LPS increased during infection in both NSE and SESE mice. There was an inverse correlation between IL1 secretion and PGE production by cells from sensitized mice. We propose that changes in the capacity of peritoneal macrophages to secrete IL1 and PGE in response to stimulants in vitro reflect the initiation and regulation of the immune response through the course of infection.     

Anti-clotting activity of endothelial cell cultures and heparan sulfate proteoglycans

A photoaffinity probe for the vitamin D-dependent chick intestinal calcium binding protein (CaBP) has been prepared by conjugation of methyl-4-azidobenzoimidate (MABI) to lactoperoxidase-¹²⁵I-iodinated CaBP to yield ¹²⁵I-CaBP-MABI: [3 moles MABI per mole CaBP]. After incubation $\text{in}\text{vitro}$ of ¹²⁵I-CaBP-MABI (28,000 daltons) in model systems with bovine intestinal alkaline phosphatase (AP) (67,000 daltons), a UV light-dependent crosslinking occurred to yield a conjugate with a molecular weight of 95,000 (by SDS-gel electrophoresis); no crosslinking occurred with $\text{E.}\text{coli}$ alkaline phosphatase. The formation of the ¹²⁵I-CaBP-MABI-AP was found to occur only in the presence of calcium.     

An improved method of reconstitution of adipocyte glucose transport activity

The glucose transport activity of rat epididymal fat cells was reconstituted into egg lecithin liposomes with a high degree of reproducibility. The activity was solubilized with 20 mm sodium cholate in Buffer B (10 mm Tris-HCl, pH 7.5). After elimination of small molecules by gel filtration, the transport activity was incorporated into egg lecithin liposomes (Sigma, Type IX-E, homogeneously dispersed into Buffer B) by sonication (5 s), freezing (?70°C), thawing, and a second sonication (5 s). The sonication was done in a 16.8-mm polystyrene test tube (Sarstedt, 55-468) placed in a cup horn (from Heat Systems Ultrasonics) connected to a Branson's sonicator (W-185) at setting No. 3 (70 W of output). The optimum sample size was 80 μl, and the optimum clearance between the test tube and the sonicator horn was 2–3 mm. The concentration of egg lecithin at the reconstitution step was 25 mg/ml, and that of the microsomal protein was approximately 0.3–0.5 mg/ml. The glucose transport activity of reconstituted liposomes was assayed by incubating the latter with a mixture of d-[³H]glucose and l-[¹⁴C]glucose. The incubation was terminated by the addition of HgCl₂, and the reaction mixture was filtered with a Millipore filter (GSWP). The difference in the rates of uptake of d-glucose and l-glucose was regarded as representing the carrier-mediated glucose transport activity. The results of the assay indicated that the glucose transport activity could be reconstituted in a highly reproducible manner. The reconstituted activity was proportional, within a limit of experimental error, to the amount of protein used for reconstitution and was almost completely blocked by cytochalasin B, phloretin, or HgCl₂. However, a small amount of d-glucose was found to bind with the egg lecithin preparation.     

A fluorometric procedure for measuring the neuraminidase activity: its application to the determination of this activity in influenza and parainfluenza viruses

A fluorometric procedure for quantitating the amount of N-acetylneuraminic acid enzymatically released by the neuraminidase activity from N-acetylneuraminyl-lactose (sialyl-lactose) has been developed. The liberated lactose is hydrolyzed with beta-galactosidase, and the released galactose is oxidized with galactose dehydrogenase and NAD+; finally, the NADH produced is measured by fluorometry (excitation at 340 nm and analysis of emitted light at 465 nm). The fluorometric assay is about 10-fold more sensitive than the spectrophotometric procedure that measures NADH at 340 nm. It readily measures amounts as little as 2 nmol of sialic acid, and does not require the use of radioactive isotopes. Interferences due to sucrose or other substances, which cause errors in some cases with the use of the periodate-thiobarbiturate method for neuraminidase activity determination, are avoided. The procedure reported here provides a sensitive, rapid, and relatively simple method (feasible with commercialized reagents) for measuring the neuraminidase activity not only in purified samples from different sources but also directly in biological materials such as viruses. The technique has been tested with some viruses recently isolated belonging to Orthomyxoviridae or Paramyxoviridae families, known to be rich in neuraminidase. Reciprocally, this method can also be employed for determining the sialic acid concentration in acylneuraminyl-lactose-containing compounds when using purified neuraminidase for hydrolysis.     

Threshold of plasma testosterone required for normal mating activity in male sheep

The finer control of mating activity by testosterone in male sheep was investigated using the castrated ram (wether) as an experimental model. Adult wethers that had been castrated before puberty were injected with graded doses of testosterone propionate (TP) and mating behavior was assessed in standardized libido trials at various times during treatment. Doses of 1 to 2 mg TP/day elicited mounting behavior in wethers but did not result in intromission or ejaculation. On the other hand, TP doses of 4 mg/day or greater stimulated the complete mating response which included intromission and the ejaculatory reflex. The threshold dose of TP required for complete mating activity (4 mg/day) produced plasma testosterone levels which were lower than those normally observed in intact rams. The results of this study indicate that the behavioral aspects (arousal mechanisms) of mating in rams have a lower testosterone threshold than intromission and ejaculation (consummatory mechanisms). Also, since complete mating activity was stimulated in wethers having relatively low plasma testosterone levels, this may explain why there is no apparent relationship between plasma testosterone and mating drive in intact rams.     

Murine lymphocyte cell surface proteolytic activity is strain related

A/J and C57 Br/cdj mice bear the H-2 haplotypes which are generally associated with high responses to bovine gamma globulin and type 3 pneumococcal polysaccharide. A/J has been reported, however, to produce higher levels of antibody than C57 Br/cdj mice against the antigens. The two strains of mice were used as model systems in this study to determine whether the level of lymphoid cell surface proteolytic activity is also genetically controlled. The results of this study illustrate that the level of lymphoid cell surface proteolytic activity is strain related. Since A/J lymphocytes were found to have a significantly higher rate of proteolysis than C57 Br/cdj lymphocytes, a correlation between lymphoid cell surface caseinolytic activity and immune responsiveness is suggested.     

Phosphodiesterase induction in Dictyostelium discoideum by inhibition of extracellular phosphodiesterase activity

Adenosine 3′,5′-monophosphate (cAMP) is a chemoattractant in Dictyostelium discoideum; it also induces phosphodiesterase activity. Recently it was shown (M. H. Juliani, J. Brusca, and C. Klein, (1981)Develop. Biol.83, 114–121) that N⁶-(aminohexyl)adenosine 3′,5′-monophosphate (hexyl-cAMP) effectively induced phosphodiesterase activity, while this compound was chemotactically inactive and did not effectively bind to the cell surface receptor for cAMP. It was suggested that hexyl-cAMP and cAMP induce phosphodiesterase activity via a chemoreceptor-independent mechanism. In another recent report (P. J. M. Van Haastert, R. C. Van der Meer, and T. M. Konijn (1981)J. Bacteriol.147, 170–175) investigation of induction of phosphodiesterase by several cAMP derivatives revealed that phosphodiesterase induction and chemotaxis had similar cyclic nucleotide specificity. Based on this result it was suggested that cAMP induces phosphodiesterase activity via activation of the chemotactic receptor. In this report we show that hexyl-cAMP transiently inhibits extracellular and cell surface phosphodiesterase. This transient inhibition of the inactivating enzyme and the permanent release of small amounts of cAMP by the cells leads to a transient increase of extracellular cAMP levels. Hexyl-cAMP does not inhibit beef heart phosphodiesterase, and is not degraded by this enzyme. Addition of hexyl-cAMP to a cell suspension containing beef heart phosphodiesterase does not result in an accumulation of extracellular cAMP, and phosphodiesterase induction is absent. We conclude that hexyl-cAMP inhibits phosphodiesterase activity which leads to the accumulation of cAMP; consequently cAMP binds to the chemotactic cAMP receptor resulting in the induction of phosphodiesterase activity.     

Desensitization of ornithine decarboxylase activity to norepinephrine in the testis of rat

Injection of norepinephrine (NE) at a dose of 10 micrograms per testis caused the testis refractory in terms of ornithine decarboxylase (ODC) activity at 24 h. This desensitization was found to be both time and dose dependent. Injection with follicle stimulating hormone, luteinizing hormone, prostaglandin F2 alpha, cyclic AMP or epinephrine to norepinephrine desensitized testis caused stimulation of ODC activity. This indicates that the refractoriness caused by norepinephrine is specific to this agent alone.     

Differential response of testicular and ovarian heme oxygenase activity to metal ions

The response of the microsomal heme oxygenase in the testis to metal ions distinctly differed from that of the ovarian source. The activity of the ovarian enzyme in rats treated with Co2+ (250 mumol/kg, 24 h) responded in consonance with that of the liver and the kidney, i.e., heme oxygenase activity was elevated. In contrast, similar treatments did not increase the activity of testicular heme oxygenase. In addition, other metal ions, such as Cu2+, Sn2+, Pb2+, and Hg2+, known for their potency to increase heme oxygenase activity, were ineffective in increasing the enzyme activity in the testis. The unprecedented response of heme oxygenase in the testis to metal ions did not reflect an unusual nature of the enzyme protein insofar as it displayed a similar cofactor requirement and inhibition by known inhibitors of the enzyme activity, such as KCN and NaN3. Moreover, the apparent Km's for oxidation of hematoheme by the testicular and ovarian microsomal fractions were comparable and measured 2.3 and 1.4 microM, respectively. In the testis of Co2+-treated rats, the concentration of cytochrome P-450 in the rough and smooth endoplasmic reticular fractions was significantly decreased. The decrease in the hemoprotein level, however, did not reciprocate the activity of heme oxygenase in the fractions. The inability of metal ions to induce heme oxygenase activity in the testis did not represent the general refractory nature of the enzymes of heme metabolism to metal ions in this organ, since in rats treated with Co2+ the activity of delta-aminolevulinate synthetase was significantly decreased 24 h after treatment. However, the activities of uroporphyrinogen-I synthetase, delta-aminolevulinate dehydratase, and ferrochelatase and the content of porphyrins were not altered in the testis of rats treated with Co2+. The response of delta-aminolevulinate synthetase in the ovarian tissue to Co2+ treatment contrasted that of the testis. In the ovary, the enzyme activity significantly decreased 6 h after treatment. This decrease was followed by a rebound increase at 24 h after administration of Co2+. The presently described inability of metal ions to induce testicular heme oxygenase activity suggests that the activity of the enzyme in the testis is controlled by factor(s) which differ from those regulating the enzyme activity in other organs, including another steroidogenic organ, the ovary.     

Spontaneously active and ATPMg-dependent protein phosphatase activity in vascular smooth muscle

A spontaneously active (Mr greater than 350,000) and an ATPMg-dependent phosphatase (Mr congruent to 140,000) were identified in bovine aortic smooth muscle. The spontaneously active phosphatase was effective in dephosphorylating both phosphorylase a (240nmol32P/min/mg) and phosphorylated myosin light chains (1000nmol32P/min/mg). In contrast, the ATPMg-dependent phosphatase was only effective in dephosphorylating phosphorylase a (400nmol32P/min/mg). Phosphorylase phosphatase activity of the ATPMg-dependent enzyme was suppressed by the well-characterized modulator protein (inhibitor-2), whereas the activity of the spontaneously active enzyme was unaffected. The aortic spontaneously active phosphatase did not convert to an ATPMg-dependent form when it was stored at 4 degrees or incubated at 30 degrees C in either the presence or absence of modulator protein. These findings suggest that spontaneous and ATPMg-dependent phosphatase activities described in these studies are probably ascribable to different enzymes. Since both phosphorylase and myosin light chains are phosphorylated when smooth muscle contracts these phosphatases may participate in coordinating arterial contractility and metabolism.