Expression of specific granule markers on the cell surface of neutrophil cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6- fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12- binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.     

Differential responses to adjuvants of macrophages from young virgin, aging virgin and aging breeder mice

Age related differences in macrophage responsiveness to adjuvants seen previously with thioglycollate induced cells were also found with resident macrophages. Thus, activation of phagocytosis by lipopolysaccharide, polyadenylic-polyuridylic acid complexes and muramyl dipeptides occurred when exposed to macrophages removed from young but not aging C58 and C3H/He mice. However, breeding status differences were observed in that aging virgin mice were unresponsive to these adjuvants, while aging breeder mice responded similarly to young virgin mice. Analysis of any changes in membrane phospholipids was carried out to determine their association with the age related and breeding status differences observed. Significant differences occurred in 32P labelling of phosphatidyl inositol between unstimulated macrophages from young and aging C58 mice. No differences were evident, however, after LPS stimulation. Analysis of macrophage plasma membranes from young and aging mice for cholesterol revealed significant age related differences in their ability to undergo increases in cholesterol content after LPS activation.     

Association between gelatinase release and increased plasma membrane expression of the Mo1 glycoprotein

Mo1, a glycoprotein heterodimer (gp 155,95) that functions as an adhesion promoting molecule and as the C3bi receptor of human myeloid cells, is expressed in increased amounts in the plasma membrane after exposure of polymorphonuclear leukocytes (PMNs) to various stimuli. Previous studies have suggested that secondary granules represent an intracellular pool of Mo1 that, upon degranulation, fuse with the plasma membrane resulting in a tenfold increase in surface expression of Mo1. To determine the intracellular location of Mo1, we monitored Mo1 expression by immunofluorescence and compared it to the release of myeloperoxidase (MPO, a marker for the primary granules), vitamin B12 binding protein (B12BP, secondary granules), and gelatinase (gelatinase- containing organelles) following exposure to various stimuli. Human neutrophils stimulated with 20 mmol/L fluoride for 16 minutes exhibited a twofold increase in Mo1 expression and gelatinase release but no enhanced release of primary or secondary granular contents. In a similar fashion, incubation of cells at 37 degrees C for five minutes with 7.5 X 10(-9) to 10(-6) mol/L N-formyl-methionyl-leucyl- phenylalanine (FMLP) resulted in significant increases in both surface Mo1 expression (three- to fivefold) and gelatinase release (five- to eightfold) without significant release of either MPO or B12BP. In addition, both the fluoride and FMLP experiments demonstrated that Mo1 up-modulation alone is not sufficient to activate superoxide (O2-) production. These data indicate that at least one intracellular storage pool of Mo1 is the gelatinase-containing organelles and that their fusion with the plasma membrane results in increased expression of Mo1 on the cell surface.     

Macrophage activation by adjuvants in aging mice

Peritoneal macrophages from young (3-8 mo) and aging (12-29 mo) mice of the C58, BALB/c, C3H/He, C57BI/6J, and B6D2F1 stains were compared for their capacity to become activated by various adjuvants in four assays. In chemiluminescence, activation by phorbol myristic acetate or zymosan of macrophages from aging mice of the C58, BALB/c, and C3H/He strains was increased approximately twofold greater than that of cells from young mice. A reversal of this was seen in the same three strains when measuring activation of phagocytosis by lipopolysaccharide, polyadenylate:polyuridylate (polyA:poly U), or muramyl dipeptide in that increased activity was induced readily in macrophages from young but not aging mice. Similarly, tumoricidal activity of macrophages from young but not aging mice was stimulated 6.0- and 4.4-fold by lipopolysaccharide and poly A:poly U, respectively, in the C58 strain (the only strain studied). Activation by lipopolysaccharide and poly A:poly U of the hexose monophosphate shunt in macrophages from the C58 and C3H/He strains also was significant in young but not aging mice, whereas it occurred in both age groups of the BALB/c and C57B1/6J mice. A reversal of response patterns was observed between aging female virgin and breeder C58 mice in the chemiluminescence and hexose monophosphate shunt assays in that the breeding mice mimicked the young virgin mice.