Venous blood provides a ready source of large numbers of unstimulated granulocytes and mononuclear cells. Exploiting the differences in the relative densities of the leukocytes circulating in venous blood, one can separate leukocytes from erythrocytes as well as isolate the individual leukocyte populations in high purity for use in ex vivo studies.

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MPO activity is critical for optimal microbicidal activity of normal PMNs. In the absence of MPO, auxiliary mechanisms protect most MPO-deficient hosts from clinically significant sequelae, except for some persons with diabetes mellitus who suffer severe candidal disease. However, given our limited knowledge of the clinical impact of MPO deficiency, histochemical staining of peripheral blood smears or MPO activity of isolated leukocytes should be assessed in patients with unexplained fungal disease or with suspected impaired host defenses. Recently isolated cDNA probes provide important tools for dissecting the molecular and cell biology underlying hereditary MPO deficiency and the link between MPO gene expression and myeloid differentiation.

Polymorphonuclear leukocytes (PMNs) represent a prominent cellular element in the innate immune system, serving to ingest exogenous particles and microbes and to kill phagocytosed microorganisms. The microbicidal activity of PMNs depends on the interactions of a broad array of potent systems, including relatively stable degradative proteins as well as labile reactive radicals. These systems can be categorized as oxygen-dependent and nonoxidative mechanisms, although the physiologically relative activity depends on the precisely orchestrated interplay between both systems. The enzyme complex responsible for the activity of the oxygen-dependent system is the respiratory burst oxidase and its important contribution to host defense is best illustrated by the frequent and severe infections seen in individuals whose PMNs lack oxidase activity, namely patients with chronic granulomatous disease (CGD). Multiple elements comprise the oxygen-dependent system, and significant advances have been made in the past decade in understanding the protein components of the respiratory burst oxidase, their subcellular distribution in resting PMNs, and their agonist-dependent assembly into a functional system at phagosomal and plasma membranes. In parallel, substantial insights into the molecular bases of CGD have likewise been made. Nonetheless there remain significant gaps in our understanding of the precise functional contributions of particular components of the system, the molecular mechanisms that regulate their coordinated assembly, and the role of related proteins in nonphagocytic cells.

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈5 minutes). These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery.

This unit describes the isolation of high-purity neutrophils (or polymorphonuclear leukocytes; PMN) and the assays that can be performed to assess their function. An assay for the in vitro study of phagocytic capacity is presented, and microbicidal assays using lysostaphin and differential centrifugation are also described. During phagocytosis or on exposure to soluble agonists, neutrophils exhibit a marked increase in oxidative metabolism which is mediated by activation of NADPH oxidase and results in the formation of superoxide anion as well as hydrogen peroxide and other reactive species. The nitroblue tetrazolium reduction slide test is presented, and is a nonquantitative screening test for the presence of reactive oxygen products. The quantitation of superoxide by the kinetic and static assays are detailed, and a "broken cell" assay is also outlined.Finally, measurement of hydrogen peroxide formation is described.

Biosynthesis of myeloperoxidase (MPO), a myeloid lysosomal hemoprotein critical for the optimal oxygen-dependent microbicidal activity of human neutrophils, is incompletely understood. The primary translation product undergoes cotranslational N-linked glycosylation with subsequent insertion of the Fe-containing prosthetic group into the peptide backbone, thereby converting the enzymatically inactive, heme- free apoproMPO into the peroxidatively active precursor, proMPO. Eventually, proMPO undergoes proteolytic processing into native, lysosomal MPO, with subunits of 59 and 13.5 Kd. We studied three unanswered questions regarding MPO biosynthesis: (1) At what point during MPO biosynthesis is the heme moiety inserted into the apoenzyme? (2) What consequences does heme-insertion have on subsequent processing events? (3) What role does the mannose-6-phosphate receptor (M6PR) system play in the delivery of MPO to the lysosome? Disruption of Golgi by brefeldin A (BFA) produced two major changes in MPO biosynthesis: (1) processing of the 89-Kd precursor to mature MPO was blocked and (2) constitutive secretion of the MPO precursor was inhibited. Inhibition of heme synthesis with succinyl acetone (SA) reduced peroxidase activity and profoundly blocked processing of proMPO to mature MPO. This inhibition of processing was not a generalized effect on all lysosomal enzymes, because the maturation of a non-heme-containing lysosomal enzyme, beta-glucuronidase, was not altered. Electron microscopy showed that, although the normal peroxidase staining of endoplasmic reticulum was absent in SA-treated cells, there were MPO- related peptides in the ER. The role of the M6PR system was assessed by immunoprecipitating fractions obtained from M6PR affinity column chromatography. The 89-Kd proMPO failed to adhere to the M6PR affinity column, whereas the 59-Kd heavy subunit of mature MPO was specifically eluted from the column. We interpret these data to indicate that: (1) processing of proMPO to mature MPO occurs in a post-ER compartment that is itself BFA-sensitive or is distal to a BFA-sensitive compartment and (2) heme insertion into apoproMPO precedes and may be a prerequisite for proteolytic processing to enzymatically active mature MPO. Our analysis of the M6PR system in MPO biosynthesis led to the unanticipated finding that there were phosphomannosyl residues on mature MPO, but none on proMPO. We suggest that the bulk of proMPO at any time is not phosphorylated, but, when generated, the phosphorylated proMPO is quickly processed to the phosphorylated 59-Kd subunit of mature MPO. Thus, if the M6PR is important in the intracellular transport of MPO, it is the phosphorylated mature MPO that is directed to the lysosomal compartment by this system.(ABSTRACT TRUNCATED AT 400 WORDS)

The biosynthesis and processing of myeloperoxidase (MPO), a cationic enzyme present in the azurophilic granules of human polymorphonuclear leukocytes (PMNs), were studied in the human promyelocytic leukemia cell line, HL-60. HL-60 cells produce large quantities of enzymatically active MPO that has the same electrophoretic behavior as MPO isolated from normal PMNs. Mature MPO is a glycoprotein of approximately 150,000 molecular weight (mol wt) composed of two heavy-light protomers (alpha 2 beta 2) with subunits of 59,000 and 13,500 mol wt, respectively, under reducing conditions. The primary translation product of MPO messenger RNA (mRNA) isolated from HL-60 cells was a single polypeptide of mol wt 80,000. In HL-60 cells labeled with [35S]-methionine, the labeled MPO isolated by immunoprecipitation had a mol wt of 89,000. Treatment of this 89-kilodalton (kDa) species with endoglycosidase H produced a 79-kDa peptide, suggesting that the 89-kDa protein contained high-mannose side chains. The 89-kDa species had no detectable peroxidase activity. During chase experiments some of the 89-kDa peptide was processed to smaller species of mol wt 39,000, 59,000, and 13,500, although a fraction of the 89-kDa peptide remained unprocessed after a chase of 100 hours. In addition, a small amount of the 89-kDa peptide appeared in the medium without any of the processed smaller peptides. These studies suggest that the primary translation product in MPO biosynthesis is an 80-kDa peptide that undergoes cotranslational cleavage of the signal peptide and glycosylation to produce an 89-kDa pro-MPO, that pro-MPO is a single polypeptide containing the alpha and beta subunits of MPO and contains endoglycosidase H-susceptible high- mannose side chains, and that posttranslational modification of pro-MPO results in targeting to the lysosome and proteolytic maturation of pro- MPO to active enzyme. In light of the previous observation that MPO- deficient and normal PMNs contain an 89-kDa protein immunochemically related to MPO, these studies on MPO biosynthesis indirectly support the hypothesis that defective posttranslation processing by pro-MPO may underlie hereditary MPO deficiency.