Selective sensitivity to hydrocortisone of regulatory functions that determine the magnitude of the antibody response to type III pneumococcal polysaccharide.

Previous studies on the basis for the immunosuppressive potential of adrenal corticosteroids have stressed that the effects of these agents on immune functions depend on the animal species being considered, as well as the subpopulations of lymphocytes involved in the expression of immune functions examined. In the present work, we have evaluated the effect of a single dose of hydrocortisone on three different immunoregulatory functions that can influence the magnitude of an antibody response to Type III pneumococcal polysaccharide (SSS-III) in mice; these functions include suppressor, amplifier, and helper activity that are dependent upon the presence of distinct subpopulations of thymus-derived (T) cells. The results obtained show that a single injection of a relatively large dose of hydrocortisone, when given at the time of priming with carrier, eliminated all evidence of carrier-specific helper T cell activity; hydrocortisone was also found to eliminate a significant amount of helper T cell activity when given after such activity had been generated. But, under the same experimental conditions, suppressor and amplifier T cell activities were unaffected, even in this steroid-sensitive species. Such selective sensitivity may account for some of the immunosuppressive potency of steroids.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Novel leukocyte agonists are released by endothelial cells exposed to peroxide.

Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.     

Direct evidence for the involvement of T suppressor cells in the expression of low-dose paralysis to type III pneumococcal polysaccharide

Prior treatment (priming) with a subimmunogenic dose of type III pneumococcal polysaccharide results in the development of an antigen-specific state of unresponsiveness termed low-dose paralysis. Such unresponsiveness can be transferred by spleen cells obtained from mice within 5 to 24 hr after priming; the suppressive activity of transferred cells is abolished by treatment with monoclonal anti-Thy-1.2 antibody and complement. These findings show clearly that low-dose paralysis is mediated by T suppressor cells.     

The sarcolemma of Aplysia smooth muscle in freeze-fracture preparations

Smooth muscle cells in the sheath covering the visceral ganglion of Aplysia californica were examined with the techniques of freeze-fracture and conventional electron microscopy. The sarcolemma of these muscle cell invaginates to form myriad caveolae that have an intrinsic marker within their membrane. This intrinsic structure of the caveolar membrane is revealed by freeze-fracture and consists of rows of large particles in the outer half and matching grooves on the complementary inner half of the membrane. In thin plastic sections, parallel striations or shelves within the caveolar membrane appear to be the equivalent of the particles and grooves of the fractured membrane. Physical fixation of some specimens by rapid freezing in supercooled liquid nitrogen or in liquid helium suggests that in their natural state, the caveolar ostia are not uniform in size and that at any given moment a number of caveolae are flattened. When segments of the connective nerves which link the visceral ganglion to the cephalic ganglia are stretched in vitro two to three times their in situ length, the caveolae lose their invaginated shape and are fully exposed to the extracellular space. The caveolar membrane, so stretched, is pulled into the line of fracture with the result that the large particles rather than the ostia appear on the cleaved surface. This flattening of the caveolae is reversible and suggests that they might serve as miniature stretch-receptors within the membrane of the smooth muscle cells. The caveolae are accessible to extracellular horseradish peroxidase but do not appear to pinocytose the protein.