Stanley Yachnin

Further studies on the mechanism of suppression of human lymphocyte transformation by human alpha fetoprotein

Abstract We investigated the possibility that noncovalent binding of negatively charged molecules such as prostaglandins (PGs) might be partly responsible for human alpha fetoprotein (HAFP) heterogeneity with respect to charge and the suppression of human lymphocyte responses, since PGs are potent suppressors of lymphocyte transformation. Indomethacin, an inhibitor of PG synthesis, had no effect upon the transformation of adherent-cell-depleted human lymphocytes, nor did it interfere with the capacity of HAFP to inhibit lymphocyte transformation. When a partially suppressive dose of HAFP was added to mitogen-stimulated lymphocytes together with varying doses of prostaglandin E 1 or E 2 (PGE 1 or PGE 2 ), no evidence of inhibitory synergy was demonstrable; their combined suppressive action was either less than or approximately equal to the sum of their respective inhibitory effects. Addition of PGE 2 to a nonsuppressive dose of an impotent HAFP preparation resulted in no greater lymphocyte suppression than that achieved by PGE 2 alone. Analysis of the kinetics of suppression of lymphocyte transformation by PG and HAFP yielded unequivocal evidence for their disparate mechanisms of action. Suppression of lymphocyte transformation by PGE 2 is evident by 24 to 40 hr of culture, persists throughout the entire culture period (including peak deoxyribonucleic acid [DNA] synthesis at 72 to 88 hr and beyond), and does not vary in profundity at any time. By contrast, HAFP-induced suppression of lymphocyte DNA synthesis is lacking at 24 to 40 hr, reaches a peak at 72 to 88 hr, and is waning by 96 to 112 hr. Cultures containing HAFP synthesize more DNA than control cultures during the 120 to 136 hr culture period. These observations, together with our earlier demonstration of a subpopulation of human lymphocytes resistant to HAFP inhibition, suggest that, after the first 24 to 40 hr of culture, HAFP may enhance the proliferation of a suppressor population which gradually dampens the proliferative response. HAFP is capable of suppressing the proliferative response of periodate-treated lymphocytes; this confirms that it does not act via competition with the cell membrane for a mitogen-binding site. Immunofluorescence studies indicate that human peripheral blood lymphocytes lack membrane-associated HAFP or HAFP receptors of high affinity. Our results indicate that PG, either present as a putative contaminant in HAFP isolates or endogenously synthesized by lymphocytes (or monocytes), plays no role in lymphocyte suppression by HAFP.

Studies on human alpha-fetoprotein: isolation and characterization of monomeric and polymeric forms and amino-terminal sequence analysis.

Human alpha-fetoprotein has been isolated from the serum and ascitic fluid of a patient with hepatoma by a combination of immunoadsorbent column chromatography and Sephadex G-150 gel filtration. Human alpha-fetoprotein is a sialylated glycoprotein with an estimated molecular weight of 67 500, composed of a single-chain polypeptide of approximately 580 amino acid residues and 3.6% carbohydrate. It is a negatively charged protein with an acid isoelectric point (pH 4.57). In addition to the monomeric form of alpha-fetoprotein, we have identified human alpha-fetoprotein polymers, including dimeric and trimeric forms, which dissociate to the monomer only upon exposure to disulfide-reducing reagents, implying that their formation is dependent upon intermolecular disulfide bonds. These polymers are found in human alpha-fetoprotein isolated by isoelectric focusing in both the major (pI 4.57) and minor (pI 5.2) alpha-fetoprotein fractions. The first 17 residues of the NH2-terminal amino acid sequence of the hepatoma-derived human alpha-fetoprotein have been identified. Fetal alpha-fetoprotein is indistinguishable from hepatoma alpha-fetoprotein by several criteria, including immunoelectrophoresis, acryalmide gel electrophoresis, and proclivity for dimerization.

The effects of cholesterol oxidation products in sickle and normal red blood cell membranes

The oxysterol content in normal and sickle red blood cell (RBC) membranes was assessed using thin-layer chromatography and capillary gas chromatography/mass spectrometry. Several more oxysterols were present in sickle RBCs compared to normal RBCs. Sickle RBC membranes had a higher concentration of 5 alpha,6 alpha-epoxycholesterol, 5 alpha-cholestane-3 beta,5,6 beta-triol, 7-ketocholesterol and 19-hydroxycholesterol than normal RBC membranes. The increased oxysterols in sickle RBC may be an effect of the increased oxidative stress which occurs in sickle RBC membranes. Physical characteristics of normal and sickle RBC membrane ghosts with and without inserted oxysterols were examined by Fourier transform infrared spectroscopy. The data are consistent with a greater sterol content in sickle cells compared to normal RBC membranes, and a possible oxysterol-cholesterol synergism.

Inhibition of NK cell-mediated cytotoxicity by oxysterols.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.

The influence of oxygenated sterol compounds on dipalmitoylphosphatidylcholine bilayer structure and packing

Fourier Transform Infra-red and Raman Spectroscopies indicate that 7 alpha-hydroxycholesterol and 7-ketocholesterol have a diminished capacity to condense (increase the packing order of) fluid-state dipalmitoylphosphatidylcholine (DPPC) acyl chains when compared with the effects of cholesterol and the other oxidized sterols studied. DPPC head groups were also more ordered by 7-ketocholesterol over the temperature range 10 degrees - 70 degrees C. Primary effects of these sterols appear to be associated with the hydrophillic regions of the DPPC bilayer, although packing arrangements with acyl chains are also involved. Phosphate and acyl chain ester groups were observed to possess a packing order which was invariant which indicates that these may be the target groups in the interaction with 7-ketocholesterol. A surprising observation was the synergistic amplification of the effects of 7-ketocholesterol by the presence of cholesterol in the DPPC bilayer.

Oxygenated cholesterols synergistically immobilize acyl chains and enhance protein helical structure in human erythrocyte membranes

Fourier transform infrared spectroscopy revealed that insertion of 20 alpha-hydroxycholesterol into human erythrocyte membranes (10% of total membrane sterol) immobilized the lipid acyl chains to a degree equivalent to enriching total membrane cholesterol by 50% (Rooney, M.W., Lange, Y. and Kauffman, J.W. (1984) J. Biol. Chem. 259, 8281-8285). Raman spectroscopy showed that the amount of acyl chain rotamers was not significantly altered by the presence of 20 alpha-hydroxycholesterol, indicating that acyl chain immobilization was limited to an inhibition of lateral motion. The presence of 20 alpha-hydroxycholesterol may synergistically enhance the acyl-chain-immobilizing behavior of membrane cholesterol. In addition, protein helical structure was not altered by 20 alpha-hydroxycholesterol. The insertion of 7 alpha-hydroxycholesterol into erythrocyte membranes resulted in an increase in protein helical structure which was comparable to that observed for erythrocyte membranes enriched with pure cholesterol by 50%. However, both acyl chain mobility and conformation were unchanged. These results suggest a synergistic behavior between oxysterols and cholesterol in modifying erythrocyte membrane packing.

Inhibition of human lymphocyte transformation by oxygenated sterol compounds

Abstract Oxygenated sterol compounds (OSC), potent inhibitors of sterol synthesis in both resting and mitogen-stimulated human lymphocytes, are capable of suppressing the DNA-synthetic response of human peripheral blood lymphocytes to mitogenic lectins, anti-human thymocyte antiserum, and the mixed lymphocyte culture. The most potent OSC are 20α-hydroxycholesterol and 25-hydroxycholesterol, which inhibit DNA synthesis in mitogenstimulated lymphocytes at 7.4 and 3.9 × 10 −6 M , respectively. Lymphocytes which have been exposed to OSC for 18 hr and washed free of inhibitor are fully capable of DNA synthesis when subsequently challenged with mitogen. Suppression of DNA synthesis by OSC is not apparent during the first 24–40 hr of culture. The inhibition of lymphocyte DNA synthesis by OSC can be partially reversed by the addition of 10 −2 M mevalonate to the culture. Sterol synthesis by mitogen-stimulated lymphocytes is enhanced by culturing them in medium supplemented with lipoprotein (and cholesterol-)-depleted serum. In such medium, the 50% inhibitory doses of 25-hydroxycholesterol for suppression of both mitogen-stimulated lymphocyte DNA and sterol synthesis are approximately equal (−3 × 10 −7 M ). Sterol synthesis is a necessary, but not sufficient, part of the program of lymphocyte proliferative response to mitogens; the evidence presented suggests that lymphocytes can utilize exogenous sterol at least partially for the purpose of cell replication.

Effect of inserted oxysterols on phospholipid packing in normal and sickle red blood cell membranes

Fourier transform infrared (FTIR) spectroscopy was used to examine the effect of oxysterol insertion into normal and sickle RBC membranes and the total lipid extracts of the membranes. Examination of the FTIR C-H stretch and fingerprint regions reveal that the insertion of 7 alpha- and 7 beta-hydroxycholesterol has the greatest effect on the fluidity of RBC membranes and lipid extracts. The results confirm the observation that sterol molecules are oriented in the membrane so that the 7 position is located in the phospholipid head group region at the lipid/water interface. The substitution of a keto for a hydroxy group at the number seven position decreases the effect of the sterol on membrane packing.

Inhibition of cytolytic T lymphocyte activity by oxysterols

The objective of this study was to investigate the effects of oxysterols (OS), namely 5α-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10−5 M 5α-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h51Cr release. At a concentration of 5×10−6 M, 5α-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.

Inhibition of oxygenated sterol entry into human red cells and lymphocytes by isolated serum lipoproteins.

Abstract We studied the uptake of certain oxygenated sterols by human lymphocytes during their incubation in lipoprotein-depleted medium and found that it resembles closely the uptake of oxygenated sterols by human red-cell membranes. Entry of oxygenated sterols into lymphocytes is virtually complete within 5 min of exposure of the cells to the oxygenated sterol-containing medium, is related to the concentration of oxygenated sterols and is impeded by low temperature. The presence of free cholesterol does not alter the amount of oxygenated sterol entering lymphocytes. However, substitution of lipoprotein-containing medium for lipoprotein-depleted medium reduces the amount of oxygenated sterol entering lymphocytes by 65–75%. Oxygenated sterol uptake by lymphocytes from individuals with homozygous familial hypercholesterolemia does not differ from that of normal lymphocytes. Oxygenated sterols bind to all density classes of human lipoproteins, but do not alter their hydrodynamic properties of their cholesterokprotein mass ratios. These compounds also have little or no effect on cholesterol exchange between incubation medium and red-cell membranes. Both LDL and HDL, when added to oxygenated sterol-containing medium, effectively diminish the amount of oxygenated sterol taken up by red cells and lymphocytes; LDL is approximately 2.5–4 times more effective than HDL in preventing oxygenated sterol entry into cells. Lipoproteins can also act as acceptors of oxygenated sterols previously inserted into lymphocytes. Since oxygenated sterols have been reported to be atherogenic, the modulating effects of lipoproteins on oxygenated sterol uptake by cells, and the alterations in membrane structure and function which ensue, may be a useful model for further study.