Cell Biology of Leukocyte Abnormalities--Membrane and Cytoskeletal Function in Normal and Defective Cells. a Review

REVIEW ARTICLE

CELL BIOLOGY OF LEUKOCYTE ABNORMALITIES Cell Biology of Leukocyte Abnormalities-Membrane and Cytoskeletal Function in Normal and Defective Cells

Ultratructure of the Human Neubtpl 22:3

Basic _bmisty of ke keon 22 Basic Pharmacology of the Cyoseton 226b

Leukwyte Functo That Depen on CyosMkta Integrt 22-7 The Regulation of Miofdlament Disrbuon 232

The Regulto of Micobule Assaemb 2;34 Ligand-Membrane Interaction and Oidative Met :237 Membrane and Cytoskeletal Abrmnaties of Human Neutrophis 241 Neutrophil Dysfunction Linked to Structural Abnormalities of the Cytoskeleton 241 Neutrophil Dysfunction Linked to Regulatory Abnormalities of the Cvtoskeleton 242 Neutrophil Membrane and Cytoskeletal Dvsfunction Secondarv to Abnormal Oxidative Metabolism 246 Disorders of Oxidant Generation 246 Disorders of Oxidant Removal 249

Concluding Remarks 51 Cell Biology of Leukocyte Abnormalities-Membrane and Cytoskeletal Function in Normal and Defective Cells A Review

Janet M. Oliver, PhD

ALTHOUGH THE POLY\1ORPHONUCLEAR LEUKOCYTE (PMN, neutrophil) has been studied since the days of Ehrlich, the precise mechanisms by which it functions to seek out and inactivate foreign microorganisms are not totally clear. Analvses performed over the past 2 decades have greatlv advanced our understanding of the biochemistry of bacterial killing by neutrophils and have provided important insight into the molecular basis of inherited diseases associated with specific enzvme deficiencies. Simi- larlv, immunologic studies have illuminated the role of complement and antibodies in the generation of chemotactic factors and in the opsonization of bacteria. In parallel, the high susceptibility of certain patients to infection has been explained in terms of defects in the serum opsonic or chemotatic activity. Recentlv it has become clear that PMN function is importantly influenced not onlv bv the immune svstem and the availabilitv of cytoplasmic and granule enzvmes but also by dvnamic properties of the cell membrane and the cvtoplasniic microtubules and microfilaments, known col- lectivelI as the cvtoskeleton. The processes of chemotaxis, phagocytosis, oxidant generation, and Iysosomal degranulation (Text-figure 1, Steps 5 through 8) are central to neutrophil function. Their initiation depends on the existence on the plasma membrane of receptors that recognize and bind surface ligands (Text-figure 1, Steps 1 through 4), setting in motion a variety of events, including the activation of membrane enzyme systems and the specific assembly or mobilization of microtubules and microfilaments. The motile and bactericidal functions of the neutrophil are subsequently expressed. From the lepartments of Pathology and Physiology. University of Connecticut Health Center, lFarmington. COnnecticuit. Slupp)rted b-bGrants FS-01 106 and CA-15364 from the National Institutes of Health and bN- Grant BC-179 from the American Cancer Society. Dr. Oliver is an American Cancer Societv Facults R(esarch Aw5 ardee. A\ccepte(l for puibli.ation April 17. 1978. ..%ddrt-ss reprint requests to Janlet S1. Oliser, PhD, Department of Physiology, University of Con- oceticiat lealth (;enter Stho)ol of Medicine, IFarmington, CT 06032. 0002-9440/78/1010-0219$01.00 221 222 OLIVER American Journal of Pathology

PMN Bacterial invasion

Chemotactic factor (CF) generation - CF binding to surface receptors

CHEMOTAXIS to site of infection

Opsonization of bacteria _ Binding of bacteria to surface receptors 0

PHAGOCYTOSIS OXIDANT GENERATION

LYSOSOMAL DEGRANULATION

BACTERIAL KILLING

_ Digestion TEXr-FIGURE 1-The role of the neutrophil in defense against infection. The proper initiation of bacterial surveillance by PMN requires a competent immune system for generation (Steps 1 and 2) and recognition (Steps 3 and 4) of chemotactic and opsonic factors. The proper resolution of inactivated bacteria depends on the presence of a correct biochemical complement of granule enzymes (Step 9). The intermediate processes of chemotaxis, phagocytosis, oxidant generation, and lvsosomal degranulation (capital letters, Steps 5 through 8), depend most importantly on the integrit of the plasma membrane and of the cytoplasmic microtubules and microfilaments.

It has been recognized that the failure of bacterial surveillance in specific diseases is associated directly or indirectly with defects in membrane or cytoskeletal organization and function. In perhaps the most dramatic of disorders associated with cytoskeletal dysfunction, the Chediak-Higashi syndrome, it has also been possible to substantially improve the progress of patients by application of laboratory data to the therapy of the disease. I have tried in the following pages to present an integrated view of PMN functions that depend on the integrity of the cytoskeleton. This synthesis is preceded by a general overview of PMN ultrastructure and of the biochemistry and pharmacology of microtubules and microfilaments. I emphasize the role of the plasma membrane both as a transducer, signal- ling changes in cytoskeletal order, and as the generation site for oxidants required for bacterial killing. These basic data are applied to give insight Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 223 October 1978 into a series of diseases associated with defects in neutrophil chemotaxis, phagocytosis, lysosomal degranulation, and oxidant generation or dis- posal. In specific cases, some possible therapeutic approaches are suggested. The analysis draws heavily from the concepts of modern cell biology. Alternative approaches to normal and pathologic leukocyte function have been presented in several recent monographs 14 and symposiums.5'6 Ultrasbtucre of the Hwnan Neutophil The basic ultrastructure of human neutrophils is well known and has been especially finely illustrated by Bessis.7 Review of ultrastructure here will accordingly be brief and will emphasize features of the PMN that are not the major concem in textbook descriptions. As shown in Figure 1, PMN contain a multilobed, highly condensed, and metabolically inactive nucleus (N), an abundance of large, round granules (probably the azurophilic granules, AG), as well as smaller often elongated or dumbbell-shaped granules (probably specific granules, SG). The azurophilic granules are lysosomes; they are membrane-bounded and contain biochemically and cytochemically demonstable acid hydrolases as well as myeloperoxidase which is involved in bacterial killing via oxidative mechanisms. The specific granules are not strictly lysosomes; their membrane encloses alkaline phosphatase, lysozyme, a variety of bactericidal cationic proteins, and an iron-binding protein, lactoferrin.A13 In the present context the most important morphologic features of the PMN are its so-called cyoskeletal components (primarily the microtubules [MT] and microfilaments [MF]) and the plasma membrane. The origin and appearance of microtubules is illustrated in Figures 1 and 2. Examination at low magnification (Figure 1) reveals fibers approximately 240 R in diameter that radiate from the center of the cell in all directions toward the plasma membrane. At higher magnification (Figure 2) it is apparent that the central concentration of microtubules forms about the centrioles. These are paired cylindric structures 1500 R in diameter, some 3000 to 5000A long and made up of nine groups of three microtubules arranged in a spiral.7 A 600 to 900A diameter satellite (S) emerges from each of the microtubule groups, and cytoplasmic microtubules originate from the satellite region. The centrioles usually occupy the concavity of the horseshoe-shaped cell nucleus 7 (Figure 1). Micro- tubules were resistant to identification in PMN until recently,14 in part because their preservation for electron microscopy requires fixation of PMN in glutaraldehyde at room temperature or higher and in part because their formation in cells from soluble tubulin dimer (see below) 224 OLIVER American Journal of Pathology requires the interaction of the PMN with a surface or with a ligand.15 Thus, the centriole in Figure 2A was photographed from a section through a PMN that had simply been isolated from buffy coat, fixed in suspension, and processed for electron microscopy. The satellites of this centriole are particularly well preserved, but only two microtubules are present. In contrast, the abundant microtubules in the PMN illustrated in Figure 1 and associated with the centriole in Figure 2B reflect their assembly during a 5-minute exposure of the leukocyte suspensions respectively to the plant lectin concanavalin A (Con A) or to phagocytic particles (oil emulsion). The microfilaments in Figure 1 occupy the region of the cytoplasm immediately below the plasma membrane. These fibers, approximately 60 A in diameter, are present under the membrane of the round cell, occu- pying protrusions and ruffled regions of the plasma membrane and preventing access of cytoplasmic granules to the membrane. Their major subunit component is the globular protein actin. Microfilaments become more prominent in PMN during motile or endocvtic processes due in large part to their concentration in regions of surface activity.",-- Thus, the cells in Figure 3 which are internalizing oil emulsion by phagocytosis show marked polarization of microfilaments in pseudopods. A similar recruitment of filaments is shown below to accompany other motile events, eg, chemotaxis and Con A cap formation. The reticular appearance of the microfilaments in Figures 1 and 3 is typical of neutrophils: organized filament bundles reminiscent of the stress fibers of cultured fibroblasts 10 are not usually observed in leukocytes. A third category of cytoskeletal fibers is also visible in Figure 3: the intermediate filaments, designated IF or 100-k filaments.21 These filaments are not yet characterized either biochemically or functionally in leukocytes. Finally, the plasma membrane in Figure 1 appears as a typical bilayer structure. Its major feature from a functional viewpoint is its deformabil- ity. Small endocytic vesicles (EV) that form from invaginations of the cell surface are apparent in Figure 1. It is usually thought that PMN do not undergo pinocytosis, ie, ingestion of fluid by engulfing small droplets of medium. They do, however, pinocytize following binding of certain ligands, including Con A. Thus, these vesicles very likely reflect the occurrence of adsorptive pinocytosis induced by Con A. Massive membrane deformation is involved in the internalization of large particles in phagocytic vesicles (PV) by PMN as illustrated in Figure 3. The fusion of granule membranes with phagocvtic vesicle membranes, leading to discharge of granule contents into the resulting phagolysosomes (PL), is also readily observed in Figure 3. Vol. 93, No. 1 LEUKOCYTE ABNORMAUT1ES 225 October 1978 The relative absence from PMN of a number of typical ultrastructural features of other cell types should also be noted. Neutrophil granules are generated during cell differentiation in the bone marrow, utilizing rough endoplasmic reticulum and Golgi apparatus as described by Bainton and Farquhar.'0' 13 No further granule generation occurs following release into the circulation, and the endoplasmic reticulum and Golgi apparatus are correspondingly reduced in amount and activity in mature PMN. Similarly, aerobic metabolism is of minimal importance, as suggested by the infrequency of mitochondria (Figure 1, M) in circulating PMN. Bask Biochemisty of the Cytoskeleto Although this review is concerned primarily with membrane-related functions of the PMN cytoskeleton, a brief survey of key biochemical properties determined in vitro may provide useful orientation. Tubulin, the protein dimer that is the major component of the microtubule, is readily isolated from brain (where it constitutes 15% of the total protein) and other cells and tissues using a recycling method based on polymerization at 37 C in the presence of GTP and disassembly at 4 C and in the presence of calcium.2n24 Tubulin consists of two nonidentical protein subunits, each of molecular weight approximately 55,000.25', A group of higher-molecular-weight proteins, known variously as HMW (high-molecular-weight components), MAPs (microtubule-associated proteins), and "tau" proteins, copurify with tubulin. 21'29" Some or all of these proteins may be essential to promote microtubule assembly. Microtubule assembly in vitro is concentration-dependent. The critical concentation for assembly can, however, be lowered by the presence of nucleating centers such as microtubule rings in brain tubulin prepara- tions 29 or specific cell organelles, ie, isolated kinetochores, basal bodies, and centrioles,°'3 that nucleate microtubule assembly in vivo. The biochemical properties of tubulin appear to be highly similar over a range of phylogenetic groups: thus, bovine brain tubulin copolymerizes with the tubulin of flagellated protozoa. On the other hand, microtubules from different sources vary in susceptibility to disassembly by treatments such as cold, elevation of calcium, and nucleotide withdrawal.21"532 Part of these differences may relate to differences in the complement and properties of accessory proteins. Microtubules that form in vitro are closely similar to those observed in cell cytoplasm (Figures 1 and 2). The globular tubulin dimers pack into a tube with a hollow core whose diameter is approximately 240X and whose length can vary up to many microns. The typical fuzzy coat appears to be composed of accessory proteins.27 Each microtubule consists of a three- start right-handed helix with 13 subunits per turn. Further details of these 226 OUVER American Joumal of Paxhokgy biochemical investigations are available in various reviews 21,M,U-U and symposiums.7" Actin was the first protein of the microfilament system of nonmuscle cells to be purified.'"4 It is a globular protein of molecular weight approximately 42,000. It can polymerize in 0.1 M KCI to form 60-A diameter filaments consisting of a double helical array of actin molecules. These actin filaments are similar in appearance to the microfilaments illustrated in close approximation to leukocyte membranes in Figures 1 and 3. Actin appears to be a well-conserved protein, having a closely, but not absolutely, similar structure between phylogenetic groups and in skeletal and smooth muscle cells as well as in nonmuscle cells. All actins can activate the Mg-ATPase of muscle myosin. Nonmuscle cells also contain myosin (illustrated in a monocyte in Figure lOB), although at much lower concentrations than actin."34 In contrast to actin, the properties of myosins vary widely between tissues and species. However, all mammalian myosins consist of both heavy and light chains, catalyze the hydrolysis of ATP, interact in various ways with actin, and are capable of forming bipolar, thick filaments in vitro. In addition to these major components of the microfilament system, a wide range of associated proteins my be involved in microfilament function in nonmuscle cells. Thus, a protein known as cofactor is required for macrophage actin-myosin ATPase activity.7'41'42 Similarly, solutions of actin filaments can be induced to form gels in the presence of filamin (from smooth muscle),4 actin-binding protein (from macrophages),17 and four separate Acanthawmoeba proteins." The significant controversy over the properties and physiologic roles of these interesting proteins is beyond the scope of this review. Finally, it was previously noted that animals cells, including PMN, contain a third category of filaments, the intermediate or 100-A filaments (Figure 3). These filaments have been identified in and purified from a variety of mammalian cell types and appear to have closely similar amino acid compositions and molecular weights (approximately 54,000).21,45.4 The function of these filaments in nonmusele cells is unknown. However, it is particularly provocative that colchicine induces the proliferation of 100-A filaments in leukocytes and other cells.47 Basic Pharmaio of the Cybtoleton The role of microtubules and microfilaments in PMN and other animal cells has been inferred primarily from functional changes following the exposure of cells to pharmacologic agents that may be selective inhibitors or activators of the cytoskeletal system. The favored inhibitory drug in the case of microtubules is colchicine." Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 227 October 1978 This alkaloid binds with tubulin and causes the rapid and complete disassembly of cytoplasmic microtubules in all mammalian cells. Its specificity for microtubules has been questioned,16 and those investigators who persist in treating cells with doses above 10 M colchicine probably run the risk of observing effects of colchicine unrelated to microtubule disassembly but related, for example, to its inhibition of membrane transport processes at high dose." However, the availability of colchicine analogues such as lumicolchicine and isocolchicine that share certain non- microtubule effects but do not bind with or disrupt tubulin permits controlled use of colchicine. A range of alternative drugs that share no obvious common properties with colchicine besides their capacity to bind with and disrupt microtubules in vitro and in vivo can be employed to determine if a colchicine response reflects microtubule inhibition. These include the carbamate antitumor drug nocodazole or R17934, vinblastine, vincristine, griseofulvin, podophyllotoxin (all discussed in Reference 36) as well as glutathione (GSH)oxidizing agents such as diazene dicarboxylic acid (bis-N,N-dimethylamide) (diamide) and tertiary butylhydroperoxide. 15 In contrast to this range of antimicrotubule drugs, pharmacologic disruption of the microfilament system of animal cells usually relies on a single and not very satisfactory agent, cytochalasin B. This fungal metabolite usually causes structural disorganization of microfilaments in mammalian cells when observed by either immunofluorescence or electron microscopic techniques."7 Unfortunately it does not directly disassemble actin filaments in vitro although it may impair actin gelation by macrophage actin-binding protein.' As a further complication, cytochalasin B inhibits the active transport of sugars in PMN at lower concentrations than those required to affect microfilament organization.5' Local anesthetics have more recently been employed to probe membrane-associated microfilament functions. These agents may not directly influence filament organization but they may displace membrane-bound calcium, perturb microfilament-membrane associations, and thus impair specific microfilament-dependent functions of cells.5'-u No methods for pharmacologic disruption of 100-A filaments has been reported. In fact, they appear to be remarkably stable structures that can be isolated following dissolution of the majority of cellular proteins with strong detergents." Leukocyte Funcos That Depend on Cytoskeletal Integrity The results of pharmacologic studies have indicated that the essential, membrane-dependent processes of chemotaxis, phagocytosis, and lysosomal degranulation in neutrophils are all influenced by the cytoskeleton. 228 OLIVER American-Joumal of Pathobogy Cytochalasin B completely abolishes random cell movement, surface ruffling activity, chemotaxis, and phagocytosis while promoting the extracellular release of granule enzymes."6-'-6" This suggests a requirement for microfilaments to support all forms of mechanical cell movement and membrane deformation and to prevent spontaneous fusion between granules and plasma membrane. Colchicine is considered by most investigators to inhibit lysosomal degranulation "'66 and to impair chemotactic but not random cell movement.r7 Colchicine generally does not reduce the rate of phagocytosis by neutrophils."-° However, as explained below, Berlin et al have established that PMN normally show a segregative movement of membrane proteins and lipids a into or out of the membrane that encloses phagocytic vesicles. This partitioning of surface components does not occur in colchicine-treated cells. On the basis of pharmacologic evidence, microtubules have thus been assigned the roles of providing orientation to gross membrane activities, of associating directly or indirectly with granules to enable their contact and fusion with endocytic vesicles, and of directing molecular reorganization of neutrophil membranes. These analyses have been the subject of frequent review."-"-5, However, they contain several inherent difficulties. One is the lack of concensus on drug specificity (mentioned above); another is the problem of assigning a positive role to a particular structure based on events following its removal; another is the absence of integration between the respective effects ascribed to microtubules and microfilaments on the same basic cell functions. Recent ultrastructural studies in our laboratory '3'" have established the following general relationships between microtubules, microfilaments, and membranes in neutrophils: 1. Binding of soluble, eg, chemotactic factors or lectins, or particulate, eg, phagocytic particles or a solid surface, ligands to membrane receptors induces rapid assembly of microtubules from centrioles. 2. Microfilaments are specifically recruited to the cytoplasm immediately adjacent to membrane involved in binding events. This recruitment involves only local regions in the case of particulate ligands. 3. Microtubules are generally excluded from these areas of microfilament concentration. 4. Nevertheless, the distribution of microfilaments is regulated at least in part by the inducible system of cytoplasmic microtubules. Evidence for these relationships is summarized below. We have developed a unified view of cytoskeletal function in chemotaxis, phagocytosis, and lysosomal degranulation based on these data. Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 229 October 1978

The induction of centriole-associated microtubules in response to surface binding events was first described by Hoffstein and co-workers "4'I and has been quantified by direct counting of microtubules in variously treated cells both by Hoffstein and in our laboratory. Its occurrence in Con-A-treated and phagocytizing PMN was established above. Thus, Figure 2A illustrates the typical appearance by electron microscopy of the centriole region in a PMN that has not been subjected to any surface perturbation. Very few microtubules are present. In contrast, Figure 2B shows abundant microtubules radiating in all directions from the centriole region in a phagocytizing PMN. These microtubules are maintained as long as cells are actively ingesting. They disassemble when phagocytosis is complete (Text-figure 2).8 Similarlv, the extensive microtubule network in Figure 1 was induced by brief exposure of the cell to Con A. I emphasize that this microtubule induction appears to occur in response to a wide variety of physiologic stimuli. For example, binding of the C5a component of complement is as effective as Con A in promoting microtubule assembly." We have shown that microtubule induction accom- panies phagocytosis of nonopsonized (latex) as well as opsonized particles."," The recruitment of microfilaments to cytoplasm subtending regions of ligand-membrane interaction was also previously illustrated. When a ligand such as Con A is bound uniformly over the whole membrane, then microfilaments are continuously present under the membrane (Figure 1).""7O However, when the surface binding event is localized to specific regions of the cell, the surface-associated microfilaments develop a corresponding asymmetric distribution. During phagocytosis, for example,

uJ -_ -20) _UJU LLJ z -i-5 Q VI) TEXT-FIGRLE 2-Microtuble assembly during Z ;0 phagocytosis. Ingestion of opsonized zvmosan u at 30 C by human PMN (open circles) is associated with rapid induction of centriole-associ- u1o ated microtubules (solid circles). When D phagocytosis is complete, microtubule dis- £ -LJ assembli occurs rapidly. D 5 0 I~-z

TIME (MIN) 230 OLIVER American Journal of Pathology microfilaments concentrate in pseudopods that enclose attached particles.17" This is demonstrated in Figure 3 and is emphasized in Figure 4, where pseudopods are seen enveloping opsonized zymosan particles at discrete intervals over the surface. At higher magnification, it is clear that the pseudopods contain a dense network of microfilament bundles that excludes both granules and microtubules (Figure 5). Intervening areas of membrane are not enriched for filaments. Similarly, leukocytes under- going directed (chemotactic) movement up a concentration gradient of a chemotactic peptide show a specific polarization of actin filaments toward the advancing edge, which is the region of ligand-membrane interaction. This anterior polarization is demonstrated by immunofluorescence label- ing of PMN using antiactin antibody as in Figure 6.19,70 The exclusion of microtubules from areas of microfilament assembly'6 is also apparent in phagocytic cells. A monocyte in process of ingesting latex beads is illustrated in Figure 7. Microtubules are present throughout the cytoplasm except in those regions where microfilament-rich pseudopods are engulfing particles. Similarly, in Figure 4, microtubules can be observed near the centriole but not in association with the pseudopods. This exclusion is emphasized by antiactin and antitubulin immunofluorescence in Figure 8. Each pair of rabbit peritoneal macrophages is attempting to ingest a single opsonized erythrocyte. Label due to antiactin is extensively concentrated in pseudopods. In direct contrast, label due to antitubulin is not concentrated and may show a defect in the region of the pseudopod. Despite the clearly different topographic organization of the microtubule and microfilament systems in the cells illustrated above, we consider that microfilament distribution is significantly regulated by microtubules. This first became apparent during examination of the process of Con A cap formation." Capping is a phenomenon that follows microtubule disassembly. We and others have shown that Con-A-receptor complexes maintain a uniform surface distribution on human peripheral blood PMN, monocytes, and lymphocytes possessing an intact microtubule system (Figures 9a and 9b). However, if microtubule assembly is prevented by colchicine or other drugs, Con A moves to one pole of the cell to form a cap. The cap usually occupies a bulge or protuberance in the membrane. This phenomenon is readily observed by fluorescence microscopy using fluorescein-conjugated Con A (fluorescein-Con A) to label the cells (Fig- ures 9c and 9d) and forms the basis of a useful assay for the presence or absence of an intact microtubule system in a cell population. 8'"'"'71,n We " recently observed that Con A need not be present for develop- Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 231 October 1978

ment of the cell shape change associated with capping. Rather, a protuberance can develop in PMN suspensions exposed only to microtubule inhibitors such as colchicine or diamide. Con A added subsequently collects over the protuberant region, forming a cap. Latex beads also show an altered interaction with colchicine-treated (shape-changed) cells.' As noted above, latex binds over the entire membrane of microtubule-containing cells, inducing a series of local microfilament-rich pseudopods (Figures 7 and 9e). However, latex is concentrated over the protuberance of colchicine-treated cells from which it is removed by phagocytosis (Figures 9g and 11). In further analyses we determined that the protuberance is made up of a highly organized array of microfilaments."'70 These filaments underlie the extensively plicated membrane of the cap as shown in Figure 1OA. They contain not only actin but also myosin-like filaments (Figure lOB), and they tend to limit the passage of phagocytic vesicles into the bulk granule-containing cytoplasm (Figure 11). We concluded that surface ligands such as Con A and phagocytic particles are distributed to regions of the leukocyte membrane that are enriched for associated microfilaments. Cells containing microtubules show a regulated, uniform recruitment of microfilaments and hence bind Con A and particles over their entire surface. Cells lacking microtubules lose the capacity to restrain or direct the distribution of microfilaments. Hence, the filaments aggregate to one pole of the cell, which becomes the site of ligand concentration. These ultrastructural observations extend the views of cytoskeletal function in leukocytes derived from earlier pharmacologic studies, as previously discussed. The essential roles of microfilaments are probably not substantially different from those proposed in cytochalasin-based experiments. Thus, microfilaments most likely provide a contractile network enabling cell movement and phagocytosis to occur while providing a physical barrier to degranulation. On the other hand, ultrastructural analysis indicated a rather different role of microtubules in neutrophil function than did pharmacologic analysis. In particular, it was considered from experiments with colchicine that microtubule-granule association may be required for lysosomal degranulation and that microtubule-membrane interaction may be required for maintenance of cell shape and for directed cell movement. Our new observations suggest that the major role of microtubules is to control the specific recruitment and dissolution of microfilaments. This role could be achieved via direct microtubule-microfilament interaction but more likely occurs via microtubule regulation of cell surface properties, as described in the following section. This role is 232 OUVER American Joumal of Patholgy consistent with evidence based on studies with colchicine that microtubules provide direction to cell movement: they specify microfilament recruitment to the leading edge of the cell. This role is also consistent with pharmacologic evidence that an intact microtubule system regulates the region of membrane involved in phagocytosis: they provide for the local polarization of filaments at sites of ligand binding. Thus, the processes of cell movement and particle ingestion both lose orientation in colchicine- treated cells. This role also provides a simple partial explanation for colchicine inhibition of lysosomal degranulation: particles ingested via a protuberance remain physically separated from granules for longer peri- ods than normal, leading to delayed phagolysosome formation. The l on MicrofilamentM Distbo How do microtubules control the recruitment of microfilaments? Mor- phologic analyses per se do not provide any information about underlying mechanisms. However some biochemical and biophysical studies are available that supplement the ultrastructural data. These studies, which are reviewed below, indicate that microfilament recruitment is associated with specific changes in membrane properties at sites of ligand binding. Microtubules may regulate these membrane changes and so indirectly regulate the distribution of microfilaments. The concept that microfilaments associate with specialized membrane regions is based on evidence that the membrane subtended by microfilaments in phagocytic cells (which is, of course, the membrane that surrounds the particles and is removed during phagocytic vesicle formation) is probably different in composition and properties from the bulk membrane. In 1971, Tsan and Berlin 1 demonstrated that the plasma membranes of post-phagocytic cells maintain their full complement of transport carriers for nonelectrolytes (amino acids, purine bases) despite loss of membrane by endocytosis. These same post-phagocytic cell membranes were subsequently shown to contain a greatly reduced density of receptors for Con A." Their "microviscosity," measured by fluorescence polarization techniques, is substantially reduced compared with that of prephagocytic cell membrane. " The most likely explanation for these data is that the membrane that surrounds the phagocytic particle and is intemalized carries along disproportionately few transport carriers and disproportionately many lectin receptors and membrane components that tend to elevate microviscosity. This can only occur if the membrane that contacts a phagocytic particle and subsequently becomes the focus for microfilament recruitment assumes a verv different composition from the nonphagocytic membrane. Vol. 93, No. 1 LEUKOCYTE ABNORMAUT1ES 233 October 1978 The concept that microtubules are involved in the development of these local specialized membrane domains associated with microfilament polarization has also gained experimental support. I showed in the section Leukocyte Functions that Depend on Cytoskeletal Integrity that microtubule disruption by colchicine prevents the selective polarization of microfilaments during phagocytosis in PMN. Massive filament polarization can occur, forming a protuberance that defines the subsequent region of phagocytosis. Berlin has established that colchicine also perturbs membrane remodeling during phagocytosis. A relatively random removal of surface markers (transport carriers and lectin receptors) is observed when colchicine-treated cells ingest oil emulsion and polystyrene latex particles.'i60 In addition, the microviscosity of membranes from colchicine- treated cells is not substantially different before or after phagocytosis.*,'" These data indicate that an intact system of microtubules is required to regulate leukocyte membrane composition and hence to control microfilament assembly. We have speculated that the altered composition of the phagocytic vesicle membrane in colchicine-treated cells may contrib- ute to their impaired lysosomal degranulation. For example, substances that normally accumulate in phagocytic membrane and enhance fusion may be depleted in the phagocytic membrane of colchicine-treated cells." The essential surface change(s) that are regulated by microtubules and lead to filament recruitment cannot as yet be defined. Con A and chemotactic substances have been reported to induce a rapid increase in Ca2+ fluxes in PMN 71'.7 that may be localized 7 to the region of ligand- receptor interaction. These flux changes could be limited via microtubule-membrane interaction and could represent a specific signal to filament concentration. Alternatively, the physical change in membrane properties that was detected by Berlin and Fera's * studies of membrane microviscosity is just as likely to cause filament recruitment as a biochemical or metabolic change. For example, a common feature between the processes of phagocytosis and chemotaxis in PMN is the rapid occurrence of membrane outflow to form,, respectively, the pseudopods that enclose particles and the lamellopodia that occupy the leading edge of moving cells.7 We"" proposed that such flow could be explained in terms of a local reduction in surface tension that results from a ligand-induced change in membrane composition, enables the extrusion of membrane, and is restrained by microfilament recruitment. The area of membrane involved would normally be limited by direct or indirect microtubule- membrane association. Cells lacking microtubules behave as if unregu- lated partitioning of membrane and recruitment of filaments have oc- curred. 234 OLIVER American Journal of Pathology The Regulation of Mcrotubule Assembly Whereas the study of microfilament regulation can be importantly centered around the control of their distribution, microtubules appear to assume a very similar distribution, ie, radiating from the centriole region, under a variety of conditions. On the other hand, the number of microtubules in leukocvtes appears to be subject to strict control. In particular, two classes of metabolic "messengers" have been proposed to be involved in microtubule modulation: cyclic nucleotides and metabolites of glutathione. Cyclic nucleotides were first implicated in microtubule regulation by Porter.8" Their involvement was subsequently extended to leukocytes by Weissman and co-workers."-"-" These latter investigators established that human PMN exposed to cytochalasin B can recognize and bind ligands such as zymosan or the C5a component of complement. No endocytosis can occur in such drug-treated PMN, but cytoplasmic granules fuse with regions of plasma membrane associated with ligand, releasing granule enzymes such as f-glucuronidase and lysozyme into the incubation medium. In this system, colchicine was shown to inhibit zymosan or C5a- induced extracellular lysosomal degranulation, most likely because microtubule integrity is required for fusion to occur. Cyclic AMP and agents that promote cyclic AMP generation also reduced degranulation, suggesting an inhibition of microtubule function by these agents. On the other hand, lysosomal degranulation was enhanced by D20, a putative stabilizer of microtubules in vitro, and by cyclic GMP and agents promoting its generation. Hence, cyclic GMP was suggested as an in vivo promotor of microtubule function in PMN. To support their data, Weissmann's group determined microtubule assembly by direct counts of the number of microtubules visible in electron micrographs within a defined distance from a centriole of cytochalasin-treated cells.", (Control studies have shown that the number of peripheral microtubules in cytochalasin-treated PMN is proportional to the number of centriole-associated microtubules provided that sufficient numbers of sections are counted to achieve a statistically significant result.") More centriole-associated microtubules were counted in cytochalasin-treated PMN that had been exposed to C5a-containing serum in the presence of cyclic GMP than in similar cells treated with only C5a- containing serum. Fewer microtubules were present in cells treated with cyclic AMP. These data supported the proposal based on degranulation studies that cyclic AMP may inhibit and cyclic GMP may promote the assembly of microtubules in leukocytes. Since their original publication, the Weissmann group's conclusions Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 235 October 1978 have been criticized in part from their own evidence that colchicine delays and reduces lysosomal degranulation but does not abolish it.' In addition, Malawista and co-workers s," reported that increased cyclic AMP levels may develop as a consequence of microtubule disassembly rather than being a cause of depolymerization under physiologic conditions. The former observation is consistent with our morphologic evidence that colchicine-treated cells show a reduction but not absence of phagolysosome formation due in part to the prolonged entrapment of phagocytic vesicles within a microfilament mesh that excludes granules (Figure 11). In addition, we suggested that an abnormal membrane composition of phagocytic vesicles that form in colchicine-treated cells could reduce but probably not abolish degranulation.7 Malawista's observation indicates in addition that the cyclic nucleotide-microtubule relationship may be more complex than originally envisaged. Nevertheless, Weiss- mann's original hypothesis of microtubule regulation in PMN has enabled investigators to approach several puzzling clinical situations (see below) and so remains a highly significant contribution whatever the actual detailed mechanism. The possibility that metabolites of glutathione may influence microtubule assembly first arose from demonstration in fertilized sea urchin eggs by Rebhun and co-workers that a GSH-oxidizing agent, diazene dicarboxylic acid (bis-[N,N-dimethylamide]) (diamide), inhibits mitotic spindle formation and depolymerizes assembled spindle microtubules.'7 ' We proposed 15 that these agents, as well as the GSH peroxidase substrate tertiary butylhydroperoxide (BHP) 9 might similarly inhibit cytoplasmic microtubule assembly in leukocytes. This hypothesis was tested by direct counting of centriole-associated microtubules in electron micrographs of cells treated with Con A in the presence of the oxidants. In addition, the Con A capping phenomenon previously discussed (Figure 9) was employed to screen for microtubule disruption. Diamide and BHP appeared to cause a rapid and reversible inhibition of microtubule assembly as determined by their induction of Con A capping (Table 1). Direct counts of microtubules supported this con- clusion. Thus, cells treated with Con A alone showed an increase from 2 to approximately 20 microtubules per centriole. Diamide and BHP, like colchicine, prevented this increase (Table 2). It was also shown that exogenous H202 can cause reversible microtubule disassembly via glutathione oxidation.'1 The effects of diamide and BHP were expressed when GSH levels were decreased between 20 and 50% and recovery of microtubule integrity was coincident with return of GSH to approximately 90% of control levels. 236 OLIVER American Journal of Pathology

Table 1-Effects of Agents Known to Inhibit Microtubule Assembly and Agents That Oxidize GSH on Con A Capping on Human PMN Percent Line Treatment Time (minutes) capped cells 1 Buffer 10 6 2 Buffer 30 7 3 Colchicine (104 M) 30 88 4 Nocodazole (5 x 10-7 M) 30 87 5 Diamide, 50 nmole/lOcells 10 87 6 BHP, 50 nmole, 10 cells 10 73 Cells were incubated with drugs at 37 C for the times indicated. FITC-Con A (10 4g/ml) was present during the last 5-minute period of incubation. The cells were fixed with para- formaldehyde, washed, wet mounted, and examined by fluorescence microscopy. The percent capped cells (as in Figure 9) was determined from counts of 100 cells per condition in five experiments. Of the noncapped cells, approximately 10{% showed a patchy distribution mostly due to internalization of lectin and the remainder were uniformly fluorescence- labeled (Figure 9).

Further analyses have indicated that microtubule disassembly is most importantly correlated with an increase in GSSG concentration rather than with a decrease in GSH level or of the GSH: GSSG ratio. Thus, PMN from a patient with GSH synthetase deficiency and only 20% of normal GSH were capable of Con-A-induced microtubule assembly. This indicates that normal (high) GSH is not essential for assembly. The same mutant cells were resistant to butylhydroperoxide-induced microtubule inhibition, thereby implicating GSSG in disassembly." These PMN are discussed below. We consider that microtubule disassembly driven by products of GSH oxidation may be important during phagocytosis.' As noted before (Text-

Table 2-Microtubule Counts in Untreated and Con-A-Treated Human Neutrophils Line Treatment Microtubules ± SEM 1 Buffer 2.6±0.4(11) 2 Con A 17.7 ± 2.5 (17) 3 Codchicine-Con A 1.2 ± 0.6 (11) 4 Diamide-Con A 2.8 ± 0.6 (8) 5 BHP-Con A 3.6 ± 1.1 (9) 6 Diamide-buffer (10 min)-Con A 25.0 ± 1.4 (7) Cells were incubated with drugs and Con A as described in Table 1. They were subsequently fixed and processed for electron microscopy as described in Reference 15. Random sections containing a centriole were photographed at a magnification of x 14,000 and printed to a final magnification of x 56,000. The number of microtubules was counted within an area corresponding to a 2-M square centered on a centriole. The number of cells counted is given in parentheses. Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 237 October 1978 figure 2), the uptake of opsonized zymosan by PMN is accompanied by very rapid assembly of centriole-associated microtubules. When uptake is complete (after approximatelv 5 minutes under the conditions emploved in this experiment) an equallv rapid disassemblv of centriole-associated microtubles is observed, returning to tvpical levels found in unstimulated cells by 9.5 minutes. No marked changes in GSH or GSSG levels occur during the period of microtubule assembly. However, the disassemblv of microtubules coincides with a doubling in soluble GSSG levels and with a marked (10-fold) increase in the level of oxidized glutathione that is complexed with protein in the form of mixed disulfide (protein-SSG). These data are summarized in Table 3. Thev support the view based on Con-A-induced assemblv and its inhibition bv GSH oxidants that products of GSH oxidation may be involved in the physiologic regulation of microtubule assemblv status. Thev extend these data bv suggesting that one important metabolite or group of metabolites promoting disassemblv mav be mixed disulfides that form between glutathione and protein. Ligand-Membrane Interaction and Oxidative Metabolism Bacterial killing requires the integrity of the cvtoskeleton-dependent processes of chemotaxis, phagocvtosis, and Ivsosomal degranulation. These events are not, however, sufficient to ensure killing. In addition, a pyridine nucleotide-dependent enzyme system(s) that consumes oxygen and generates oxidants such as hydrogen peroxide (H202) must be acti-

Table 3-The Concentrations of GSH, GSSG, and Protein-SSG in Phagocytizing Neutrophils Protein-SSG (nmoJes GSH GSSG GSH released Percent (nmoles/ (nmoles/ from protein/ recovery of Incubation 10 cells) 10' cells) 10' cells) glutathione Buffer, 4 C 1.49 0.015 0.003 100 Zymosan, 30 C, 0.5 min 1.43 0.018 0.005 96.6 Zymosan, 30 C, 1.5 min 1.28 0.019 0.004 86.8 Zymosan, 30 C, 3.5 min 1.23 0.024 0.015 84.9 Zymosan, 30 C, 3.5 min 1.15 0.028 0.033 81.4 Zymosan, 30 C, 9.5 min 1.13 0.029 0.050 81.3 Buffer, 30 C, 10 min 1.41 0.013 0.004 94.6 Human PMN (107 cells per 1 ml incubation) were incubated with serum-opsonized zymosan (0.3 mg) for the indicated times, collected by rapid centrifugation, and extracted in cold trichloracetic acid as described in Reference 68. GSH and GSSG were measured in the soluble extracts. Protein-SSG was determined from GSH released from the protein pellet during borohydride reduction. Data are the average of results obtained in duplicate in three experiments. The corresponding time course of phagocytosis and microtubule assembly is given in Text-figure 2. (From Burchill et al.") 238 OLIVER American Journal of Pathology

Medium Membrane Cytoplasm

NADPH_ C

NADP

HMS TEXT-FIGURE .3-Outline of the generation and detoxification of oxidants by PMN. Interaction of soluble or particulate ligands (large arrows) wsith membrane receptors activates an oxidase that generates superoxide (O2 and H202. This generation most likely occurs at the membrane and continues within phagolysosomes (PL) where the oxidants participate in bacterial killing. Oi- and H202 that penetrate the cytoplasm are removed in large part via glutathione peroxidase (GP). Regeneration of GSH by glutathione reductase (GR) consumes NADPH derived from the hexose monophosphate shunt (HMS). The first and regulatory enzyme of the HMS, glucose-6phosphate dehydrogenase (G6PD), increases in activitv to meet the increased demand for reducing equisalents.

vated. This oxidase system is under intensive study by biochemists and clinicians. Great controversv exists about which of several leukocvte enzymes is "the oxidase" required for bacterial activity; about precise mechanisms by which molecular 02 is converted to reactive products; about which reduced pyridine nucleotide (NADH or NADPH) is involved in oxidant generation; about whether the nucleotide is a direct or indirect source of reducing equivalents; and about which of the various products are of major importance in the oxidative inactivation of bacteria within phagolysosomes. These studies are the subject of numerous excellent reviews.92"100 In particular, the recent reviews of Cheson et al9" and Segal 100 set out manv of the controversies that have been the subject of recent debate. From the viewpoint of the cell biologist it is particularlv significant that oxidant generation is an activatable process most likely occurring at the plasma and/or phagocytic vesicle membrane. It may also be significant that the agents commonly used to induce oxidant formation are substances that also influence the assembly and distribution of cytoskeletal components. The evidence noted in the section The Regulation of Micro- tubule Assembly, that products of the oxidase system such as H202 can Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 239 October 1978 promote microtubule disassembly both by direct (poorly reversible) oxidation and indirectly via GSH oxidation, raises another important possibility: mechanisms regulating oxidant generation and removal may protect or regulate cytoskeletal function. In the following section the general process of oxidant generation and removal is outlined. The inducing agents and the probable site of oxidant formation are then briefly discussed. The consequences of abnormal metabolism of oxidants in neutrophils are emphasized in a subsequent section (page 246). The basic facts about oxidant generation and removal are illustrated in Text-figure 3 and can be summarized as follows: Binding to the PMN membrane of phagocytic particles as well as a range of surface ligands is followed without appreciable delay by a burst of oxygen consumption and generation from this oxygen of the superoxide anion (0f), hydrogen peroxide (H202), and probably hydroxyl radicals (OH-). In the case of phagocytosis of opsonized microorganisms, these substances are generated in and/or diffuse into phagolysosomes, where they participate in bacterial killing. Killing can result from direct oxidation of the ingested microorganism, from establishment of free radical chain reactions possibly involving lipid peroxides, from the use of H202 to provide substrate for myeloperoxidase-dependent halogenation and oxidative decarboxylation of bacterial protein, and possibly from other interactions of oxidants with ingested particles." In all cases, oxidants are also released into the cell incubation medium (where the assays for oxidant production are usually performed) and into the cytoplasm, from which they must be rapidly removed. Their major route of detoxification involves glutathione and the hexose monophosphate shunt.10`3" Excess H202 entering the cytoplasm reacts with reduced glutathione (GSH) in a GSH peroxidase-mediated reaction to produce oxidized glutathione (glutathione disulfide, GSSG) and water. GSSG is reduced back to GSH utilizing a coupled enzyme, GSH reductase, and NADPH provided from the hexose monophosphate shunt path- way of glucose metabolism. The activity of this shunt is geared to the cytoplasmic ratio of NADP to NADPH and possibly to GSSG levels."0' Thus, a rapid activation follows ligand binding. The level of GSH in normal neutrophils is usually high (approximately 4 mM) and the GSH: GSSG ratio is poised at approximately 100: 1. The reduced pyridine nucleotides are also relatively abundant (between 0.1 and 0.5 mM). Thus, the normal PMN is well equipped to protect its cytoplasm while utilizing H202 and other oxidants segregated within phagocytosomes for bacterial killing. 240 OLIVER American Journal of Pathology

A wide range of surface ligands share the ability to activate oxygen consumption and oxidant formation. These include both opsonized and nonopsonized phagocytic particles,"° Con A,",'1" the chemotactic peptide F-Met-Leu-Phe,1* and a variety of physiologic ligands such as immunoglobulins and complement components.'" Microtubule assembly and microfilament concentration at regions of ligand-membrane association are induced bv these same agents. In addition, the oxidase is activated by phorbol mvristate acetate 110 and D20,111 which are thought to promote microtubule assemblv, and by calcium ionophores "12 and cvtochalasin E,113 which may modify microfilament function. No direct activation is produced by cytochalasin B, but this putative inhibitor of microfilament function greatly enhances oxidant generation induced by soluble ligands.'"*'09 Assuming the enzyme to be associated with the plasma membrane (see below), these data indicate that cvtoskeleton-regulated changes in membrane properties following ligand binding may permit expression of the enzyme system. It is attractive to speculate further that the activated oxidase may be selectivelv included in the membrane enveloping phagocvtic particles. However, this idea can be only indirectly supported via some older experiments showing that oxvgen consumption during phagocytosis is reduced by high doses of colchicine. 14 How good is the evidence that the enzvme is plasma membrane (and phagolysosomal membrane) bounded? No clear concensus for a surface location exists for the major oxidase(s) that generates 0;- and H202 and activates the GSH cycle and hexose monophosphate shunt of human PMN. This may in part reflect the multiplicity of enzvmes that are capable of catalyzing the oxidation of NADH or NADPH in PMN. These various enzymes are discussed by Cheson et al," who argue for a particulate NADPH oxidase as "the oxidase" of human PMN. Other investigators have asserted a specific plasmalemmal localization for "the oxidase." In particular, Segal and Peters 115 (see Reference 100) presented evidence that "the oxidase" exists in isolated plasma membrane fractions and catalvzes nitroblue tetrazolium reduction bv both NADH and NADPH at high (2.4 mM) substrate concentrations but only by NADH at low (24 AM) concentrations. This membrane location was supported by Briggs et al,116 who- used an electron microscope technique to demonstrate the NADH-dependent precipitation of cerium ions by H202 on the plasma and phagolysosomal membranes of phagocytizing PMN. In further support of a surface location, Goldstein et al 117 found that 0a- and H202 generation can be inhibited by the nonpenetrating protein reagent p- diazobenzene sulfonate, and Tsan and co-workers 118,119 as well as Tak- anaka and O'Brien '" have similarlv inhibited oxidant generation with the Vol. 93, Noo. 1 LEUKOCYTE ABNORMAUTIES 241 October 1978 nonpenetrating sulfhydryl reagents p-chloromercuribenzene sulfonate and p-chloromercuribenzoate and with neuraminidase. Finally, it was noted above that "'the oxidase" is activated not only during phagocytosis but also following the binding of soluble ligands to surface receptors and following the binding of particulate ligands to cytochalasin-B- treated (nonphagocytic) cells. These latter observations are consistent with a membrane location for the enzyme system. Assuming a cell surface location, the mechanism of enzyme activation is still not clear. Segal l'o as well as Takanaka and O'Brien 1" suggest that enzyme expression follows a conformational change in the membrane as it invaginates to form the wall of the phagocytic vacuole. This explanation does not account for enzyme activation by soluble ligands (Con A, C5a, F- Met-Leu-Phe) nor for activation by phagocytic particles and soluble ligands in cytochalasin-treated cells. It seems more likely that activation could be another consequence of the changes in membrane composition at sites of ligand-receptor interaction that we consider to be regulated at least indirectly by microtubules (see The Regulation of Microfilament Distribution). Membrane and Cytoskeletal Abnormalities of Human Neutrophis So far in this review we have considered the human PMN from the viewpoint of its cellular biology. A synthesis of microtubule and microfilament function has been developed; it is hoped that this clarifies their respective roles in chemotaxis, phagocytosis, and lysosomal degranulation. It has been proposed that generation of the oxidants required to kill ingested microorganisms depends on dynamic properties of the plasma membrane. I have emphasized that surface binding events provide the trigger to microtubule assembly, microfilament recruitment, and oxidant generation: motile and bactericidal cell functions are subsequently expressed. In the remaining sections, emphasis is directed toward the cellular pathology of the human neutrophil. In particular, a series of diseases is discussed in which neutrophil functional abnormalities can be linked directly or indirectly to defects at the level of the membrane or cytoskeleton. In many of these disorders, affected individuals are abnormally susceptible to overwhelming infection and early death. Neutropti Dysfunt to StructW Abnomaltis of the Cy _ton A single disorder attributed to an abnormality in the protein constitu- ents of the neutrophil cytoskeleton has been described. This is a syndrome named "neutrophil actin dysfunction" by Boxer, Hedley-Whyte, and 242 OLIVER American Journal of Pahology

Stossel."l Only 1 patient with the defect has been identified, although the severity of the defect and subtlety of the diagnosis may impair its recognition. The case in Boston involved a child who presented with recurrent bacterial infection associated with severely impaired neutrophil chemotaxis, reduced phagocytosis, and increased extracellular release of neutrophil granule enzymes. No immunologic defects could be identified in the cells or serum, and oxidative metabolism appeared to be normal. Electron microscopic analysis revealed a scarcity of microfilament-containing pseudopods. On the other hand, acrylamide gel electrophoresis showed that the amounts of actin were similar between control and patient cells. This discrepancy was further analyzed by comparing the ability of normal and patient cell actin to polymerize in the presence of ATP and 0.6M KCI. Extracts of patient cells contained only 12.5% as much actin that could be polymerized and collected by ultracentrifugation as did extracts of normal cells. Thus, it was concluded that the functional disorders resulted from an abnormality in the actin or in factors regulating actin polymerization. Unfortunately, the child's death precluded further study of these cells. No therapeutic approaches to this type of disorder are immediately apparent. Neuropi Dsfwctin ke to R.lto, Abnmalties of the Cytoske-eton No disease has been linked to structural changes in PMN microtubules. However, two disorders have been described in which at least one mani- festation is abnormal microtubule regulation apparently linked to abnormal cyclic nucleotide metabolism. The first of these, Chediak-Higashi syndrome (CH syndrome),"' 2 is a disease of children and of a variety of animal species, characterized by partial albinism, the presence of giant granules in all granule-containing cells, and marked susceptibility to infection linked to two major defects in PMN function: impaired chemotaxis and impaired lysosomal degranulation following phagocytosis. It was believed for many years that the functional abnormalities were linked to the granule abnormality. How- ever, giant granules are rare in PMN compared with the relative abundance of morphologically normal granules. The giant granules we have seen in electron micrographs of PMN from 5 patients with CH syndrome all resemble secondary granules, derived perhaps from autophagic or endocytic activity of the cells (Figure 12). Furthermore, the granule abnormality can be tremendously exaggerated by maintaining CH fibroblasts in tissue culture for a few days after reaching confluence; this treatment has no adverse effects on cell viability or growth of replated cells.'" These observations led us to propose that the ultrastructural abnormality may be a reflection of some other basic problem. However, Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 243 October 1978

Table 4-Microtubule Assembly in Normal and Chediak-Higashi Neutrophils Normal Patient 1 Patient Z Patient 2 Buffer 2.6 ± 0.4(11) 1.0 ± 0.6 (5) - 3.3 ± 0.5 (100) Buffer,ConA 17.7 ± 2.5(17) 1.7 ± 1.2 (9) 2.9 0.4(8) 5.2 ± 1.0 (8) Cyclic GMP (103 M), - 26 ± 4.3 (4) - - Con A Carbachol (10-5 M), - 28 ± 1.3 (6) - Con A Bethanechol (104 M), - 21 ± 3.1 (9) 16 ± 1.6 (10) - Con A Ascorbate (1 0-2 M), - - 13 1.6 (10) - Con A Ascorbate (200 mg/day) - - - 9.2 ± 1.7 (9) in vivo Ascorbate in vivo, - - - 11.4 ± 2.2 (6) Con A Cells (5 x 104/ml) were incubated for 30 minutes in buffer with or without added drugs (except 20 minutes for ascorbate) followed by 5 minutes with Con A (100 Jg/ml). They were collected by centrifugation, fixed with glutaraldehyde, and processed for electron microscopy. Microtubules were counted within a 2-p, square centered on randomly selected centrioles. The number of cells examined is given in parentheses. (Results obtained in collaboration with Drs. R. B. Zurier, D. F. AJbertini, L. A. Boxer, and C. B. Pearson.) (From Oliver n and Boxer et al.1") none was immediately obvious. For example, no immunologic defects had been detected in CH cells or serum; no consistent enzvme defects had been detected; and no metabolic abnormalities were known besides the abnormality reported by Root et al,""'22 ie, resting CH cells showed abnormally high hexose monophosphate shunt activitv. On the other hand, the characteristic abnormal properties of CH leukocytes are remarkably similar to the properties of colchicine-treated normal cells. Both show impaired chemotaxis and Iysosomal degranulation.5'4.7 Trump and others have observed giant (CH-like) granules in liver and in human PMN following chronic exposure to colchicine.72124,25 Hence, we 1"'1V proposed that the altered function of CH neutrophils might reflect a microtubule abnormalitv. Experiments that support this proposal were recently reviewed in detail in this journal 72 and so do not require extensive repetition. To encapsulate the data, it was found that CH neutrophils form Con A caps spontaneouslv, whereas normal neutrophils do not cap except when pretreated with drugs that prevent Con-A-induced microtubule assemblv. Ultrastructural analyses showed that Con A also failed to induce centriole-associated microtubule assemblv in CH cells (Table 4; Figure 12A). We were unable to link this failure of microtubule assemblv to the absence of tubulin or to structural changes in CH tubulin: cells from the 244 OLIVER American Journal of Pathology

CH mouse analogue (C576J bg/bg) showed similar amounts of colchicine-binding protein to normal (C576J + /+) mouse cells and CH mouse brain tubulin copolvmerized with rabbit tubulin to the same extent as did normal mouse brain tubulin. Thus, a microtubule regulatory defect was suspected in CH cells. This suspicion was strengthened by the identification of a number of agents that could inhibit spontaneous Con A capping in CH cells. The first such agents identified were cyclic GMP and two analogues of acetylcholine (carbamylcholine [carbachol] and carba- mvl B-methylcholine [bethanechol] '127) that are thought to elevate cyclic GMP levels in leukocvtes. Direct counts of centriole-associated microtubules confirmed that these substances restored Con-A-induced microtubule assembly (Table 4). The cholinergic agonists were subsequently found also to correct the chemotactic and bactericidal defects of CH PMN in vitro,'" to correct the abnormal morphology of granules in CH cells,'" and to normalize the rate of exogenous protein turnover in CH mouse liver."29 Thus, considerable support could be drawn for our suggestion that a microtubule defect correctable by manipulation of cyclic GMP levels was associated with the clinically important dvsfunction of CH syndrome neutrophils. An additional observation of central importance was made by Boxer and co-workers."'m These investigators found that the chemotactic and bactericidal defects of CH cells were correctable in vitro and also in vio not onlv bv cvclic GMP and bethanechol but also by ascorbic acid. This vitamin, a known stimulus to cvclic GMP generation in normal human leukocvtes,13' also prevented spontaneous Con A capping and enabled the assembly of centriole-associated microtubules in CH cells when given in vivo or in vitro (Table 4).1*f Two patients with CH syndrome were accordingly treated with ascorbate (200 mg/day) and have been free of abnormal infection for approximately 2 vears.133 Reports of several other patients showing marked clinical improvement in response to ascorbate have come to our attention. Thus, CH syndrome provides a remarkable example of the successful transfer of information directly from the labora- torv to the clinic. Whether the patients will be protected against the accelerated lymphoma-like phase of the disease remains to be determined. Despite the clinical success achieved in CH svndrome, it is clear that much more work is required to definitively establish the basic lesion in this disease. The correction of the microtubule abnormality by agents that elevate cvclic GMP suggests that a clinic nucleotide imbalance may be a primary defect. Boxer and colleagues '30 supported this possibility bv reporting a 10-fold elevation of cyclic AMP levels in CH neutrophils Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 245 October 1978 compared with normal levels. However, no abnormality in cyclic AMP or cyclic GMP was detected in CH mouse tissues by Lyons and Pitot;'2' Malawista and co-workers found that cyclic AMP is elevated following microtubule disassembly in normal human PMN.'"^ Thus, high levels of cyclic AMP in CH neutrophils could be a consequence and not a cause of the microtubule abnormality. The possibility exists that a circulating factor that acts synergistically with surface ligands is required for microtubule induction and is absent from CH cells. Serotonin is an obvious candidate: it raises cyclic GMP in human leukocytes 131 and is missing from CH platelets.`-"36 Alternatively, a membrane-level abnormality may impair transmembrane information transfer in CH cells such that Con A binding fails to elicit a microtubule assembly response. Evidence from Kanfer et al 136 indicates abnormalities in CH cell membrane lipid composition. Recent studies in our laboratory 137 support and extend these observations. In particular, we have established an increased "microviscosity" of CH membranes (measured according to Berlin and Fera '2) associated with elevated sphingomyelin levels. It should also be noted that ascorbate is a powerful reducing agent that can interact with H202 to kill bacteria in vitro."'*6," Root 19 has observed abnormally high hexose monophosphate shunt activity in CH cells, possibly reflecting higher than normal activity of H202 generation. Micro- tubule assembly in normal PMN is prevented by agents such as H202 that can lower GSH levels and can also directly oxidize tubulin-SH in vivo and in vitro.'5"1 In our experience, GSH levels are not abnormal in CH PMN (mouse and human). However, accelerated GSH turnover is difficult to eliminate. Thus, the possibility exists that ascorbate protects microtubules in CH cells in part because it corrects a subtle defect in oxidative metabolism. An interesting complication is the discoverv of an enzyme defect in both human and mouse CH neutrophils: a severe reduction in levels of the azurophilic granule enzyme elastase.'3' Vassali and co-workers 19 suggested that this enzyme may be importantly involved in the normal killing of specific microorganisms to which CH patients are especially susceptible. A second dysfunction in which a microtubule abnormality and a cyclic nucleotide abnormality coexist and may be causally related has been reported by Gallin et al.'40 These investigators studied a 7-year-old girl who presented with a history of repeated infection that could be correlated with a series of leukocvte functional abnormalities: diminished adherence to nylon, diminished bactericidal activity, diminished extracellular lysosomal enzyme release, and, in particular, inability to show random or 246 OLIVER American Journal of Pathology

chemotactic movement in the conventional Boyden chamber assay or to show selective cell orientation in the electron microscopic assay for chemotactic competence developed by Gallin and co-workers.141 The most marked ultrastructural abnormality associated with the failure to orient in a gradient of chemotactic factor was a striking increase in the number of microtubules and a failure of the centrioles to consistently locate between the posterior nucleus and advancing pseudopods. The microtubule abnormality was associated with a 400% increase in levels of cyclic GMP in mononuclear cells. Unfortunately, no functional improvement was observed following treatment of the cells with prostaglandin E1, which raises cyclic AMP, or with colchicine; the patient died before critical tests of membrane properties such as receptors for chemotactic substances could be performed. Nevertheless, the parallel between this analysis and the Ch&diak-Higashi analysis provides further support for a relationship of functional importance to human neutrophils between cvclic nucleotide regulation and microtubule assembly status. Neutoplil Mimang an u Dysfiiction Secondary to AobalOMe As described in the section Ligand-Membrane Interaction and Oxida- tive Metabolism and in Text-figure 3, leukocyte oxidative metabolism is rigorously controlled by interdependent enzyme systems involved in the generation of oxidants, their utilization in microbicidal reactions, and the removal of excess of the same oxidants from the cytoplasm. Individuals whose PMN fail either to generate oxidants or to remove oxidant excesses show reduced bactericidal activity and heightened susceptibility to infection. In the following section, I review a series of relatively well-studied diseases in which failure of bacterial surveillance is associated directly or indirectly with the failure of surface binding events to trigger oxidant generation. This is followed by discussion of two unusual situations in which the inability to remove excess oxidants leads to oxidative damage to structures such as microtubules and hence to inadequate bactericidal activity of the affected cells.

Disorders of Oxidant Generation Only one example of an apparent membrane level structural change has been described that is associated with neutrophil neutrophil functional impairment. The defect is the absence of the oxidase system for O; and H202 generation in neutrophils from patients with chronic granulomatous disease (CGD). Holmes, Quie, Good, and co-workers 42,"" first established that CGD neutrophils can ingest but not kill catalase-positive bacteria. It was subsequently found by Baehner and Nathan 1" and by Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 247 October 1978

Holmes and co-workers 145 that these neutrophils fail to show a burst of 02 consumption, H202 production, or hexose monophosphate shunt activation during phagocytosis. More recent studies have shown that CGD cells also fail to generate O- in response to binding of phagocytic particles 1" or other ligands, eg, Con A.91 These data are all consistent with a missing oxidase system. No absolute concensus exists concerning the absence of NAD(P)H- dependent oxidase from CGD cells. For example, Baehner and co-workers originally proposed that CGD cells may lack a soluble oxidase, and Curnutte and co-workers consider that a particulate, probably granule- associated, oxidase is absent from CGD cells (reviewed by Cheson et al 99 and Baehner ). However, the recent studies of Segal and Peters,100'115"147 showing that NAD(P)H-dependent nitroblue tetrazolium reduction fails to occur in isolated CGD leukocyte membranes whereas other oxidases can be demonstrated in CGD cells, strongly suggest the important absence of a surface enzyme activity. The absence of oxidase activity most likely reflects deletion of a functional enzyme protein from the membrane of CGD cells. However, several interesting alternatives exist. One possibility is based on the proposal by Segal 100 that the membrane oxidase generates superoxide anions which are reduced to H202 by SH groups on the surface. The resulting oxidized membrane sulfhiydryls must be regenerated to continue the reaction. Regeneration is very likely mediated via GSH and NADPH supplied from the hexose monophosphate shunt. Recent studies by Graf and Berlin 145 indicate that, at least in erythrocytes, a transmembrane system exists for the reduction of surface sulfhydryls. A defect in this disulfide reduction could account for the biochemical properties of CGD cells. An abnormality in the mechanism that triggers oxidase activity could also be basic to the disease. I proposed above that membrane oxidase activation following ligand binding may occur via a ligand-induced local change in membrane properties. According to this model, the absence of enzyme activation in CGD cells could reflect not the absence of enzyme but failure of a particular change in the membrane following ligand binding that is required for expression of the enzyme. Such failure could be linked to an abnormal membrane composition or to abnormal cytoskeleton-membrane interactions. A triggering abnormality was proposed by Weening and co-workers 149 to underly an unusual abnormality of neutrophil function in two siblings with recurrent infection. PMN from these particular patients showed normal phagocytosis in serum-containing media of a range of particles: latex, zvmosan, immunoglobulin G (IgG)-coated latex, IgG aggregates. 248 OLIVER American Journal of Paftogy

The serum plus IgG-opsonized particles also induced normal oxidase activation as measured by 02 consumption, O; and H202 production, and hexose monophosphate shunt activation. However, the particles that were simply incubated with serum (presumably opsonized mainly with the C3b components of complement) failed to stimulate oxidant production. No defect in C3 or Fc receptor activity could be determined. Hence, the primary abnormality in this rare disorder may be the failure of information transfer from one type of occupied receptor to the activatable oxidase. Such failure could result from the absence or inactivity of a transducing component of the receptor or from a physical or chemical change in the properties of the plasma membrane. In addition to these failures of oxidase activation correlated with direct membrane abnormalities, several other disorders lead indirectly to the failure of adequate triggering of the surface oxidase of human PMN. Glucose-6-phosphate dehydrogenase deficiency is a well-studied example. Several independent groups whose work is summarized by Cheson et al "9 reported that absence from neutrophils of glucose-6-phosphate dehydrogenase, the first enzyme of the hexose monophosphate shunt, is associated with failure of 02 consumption, oxidant generation, and bacterial killing. The shunt is the major source of reducing equivalents in the form of reduced pyridine nucleotides required to initiate and/or propagate oxidant generation as well as to detoxify excess oxidant. Thus, the apparent failure of oxidase activation can be rationalized in terms of substrate limitation. The disease can accordingly be considered a membrane dysfunction secondary to a metabolic defect in PMN. The failure of the same series of events has been reported in patients with neutrophil GSH peroxidase deficiency. Holmes et al 150 described several patients lacking GSH peroxidase, who fail to show increased 02 consumption or HMS activation during phagocytosis. Recently, Matsuda et al 151 also reported such a patient. All these patients show impaired microbicidal activity. One explanation is that these cells not only lack GSH peroxidase but also directly lack the membrane oxidase. Another explanation is that initiation or propagation of oxidant formation at the membrane requires parallel rapid activation of the HMS. This activation is dependent not only on increasing NADP but also on increasing GSSG, as proposed by Eggleston and Krebs.0'" Thus, GSSG limitation due to peroxidase insufficiency could produce CGD-like symptoms by effectively preventing the activity of the membrane oxidase despite its presence in normal amounts. It is perhaps worth noting that confusion over GSH peroxidase deficiency has been caused by an older report that normal human PMN may lack the enzyme.152 Since butylhydroperoxide, a spe- Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 249 October 1978 cific substrate for GSH peroxidase, causes GSH oxidation and hex- osemonophosphate shunt activation in normal human PMN,15 it is clear that intact cells possess GSH peroxidase.

Disorders of Oxidant Removal Deficiencies of glutathione peroxidase and glucose-6-phosphate dehydrogenase are associated with inadequate oxidant generation. On the other hand, deficiencies of glutathione reductase and glutathione synthetase produce a situation in which the oxidase is not inactivated but rather functions to the detriment of the cells. Loos and co-workers 0,103,W described a family with generalized GSH reductase deficiency associated with periodic hemolytic crises although without pronounced subsceptibility to infection. Neutrophils from these patients showed normal chemotactic and phagocytic activity. However, the metabolic accompaniments of phagocvtosis in the cells were distinctly abnormal. For example, enhanced 02 consumption, H202 production, and HM S activation during phagocytosis began normally but ceased after 5 to 10 minutes. On the other hand, O- production continued normally in the same cells when measured continuously by including cvtochrome C in the phagocvtosis medium. These apparently contradictory data could be rather simply explained. GSH reductase deficiency did not impair oxidant generation. However, no GSH-dependent detoxification of oxidant that penetrated the cell cytoplasm was possible. Hence, cytoplasmic components were rapidly oxidized and inactivated by H202 and other reactive species during phagocvtosis. The cvtochrome C employed to trap 0- prevented this accumulation of oxidant and so protected against oxidative damage to the cells. Closely similar abnormalities of neutrophil function have been observed in a patient with oxoprolinuria resulting from a generalized deficiency of GSH synthetase.1" Analyses of these latter cells indicated that cvtoplasmic microtubules may be an important target of H202 oxidation in cells with this and related defects in GSH metabolism. The GSH- svnthetase-deficient patient is a child who presented with hemolysis and metabolic acidosis as a neonate and has continue to show persistent compensated hemolytic anemia.1" Alkalinizing agents have been admin- istered since birth for correction of acidosis. Activity of GSH svnthetase has been less than 4% of normal in all cells tested, and GSH levels range from less than 10% of normal (ervthrocytes) to approximately 20 to 25% of normal (leukocytes). Studies of his neutrophils were prompted by several unexplained episodes of severe neutropenia accompanying otitis media at the age of approximately 2 years. 250 OLIVER American Joumal of Patology

We first established that certain ligands produce normal responses in these cells."," For example, Con A binding was accompanied by the assembly of centriole-associated microtubules and by a normal small increase in H202 production to twice resting levels. The early events of phagocytosis were also normal. Thus, the initial rate of uptake of opsonized oil particles was the same in control and patient cells, and rapid oxidase activation accompanied the onset of phagocytosis. However, the products of the oxidase were not adequately controlled; therefore, H202 levels in the medium of the patient's cells quickly (within 5 minutes after beginning phagocytosis) reached twice the levels measured in normal cells. A variety of events that normally accompany phagocytosis did not occur. For example, the patient's cells were defective in both the iodination of zymosan and killing of Staphlococcus aureus. These data suggested a failure of lysosomal degranulation. Subsequent ultrastructural analyses revealed that no microtubule assembly accompanied the phagocytosis of oil emulsion in these cells. The most likely explanation for these results is that cells deficient in GSH can remove limited amounts of H202, ie, the amounts generated by Con A binding. However, the greater amount of H202 generated during phagocytosis cannot be adequately removed. Hence, H202 accumulates in the cytoplasm and oxidizes cellular components whose integrity is required for bacterial killing within phagolysosomes. One early target appears to be the cytoplasmic microtubule system. Continued uncontrolled oxidant production most likely causes irreversible cellular damage and hence is responsible for neutropenia during infection in this patient. These studies with GSH reductase and synthetase-deficient cells point out a potential problem associated with present attempts to "cure" CGD bv introduction of H202-generating systems. Success in restoration of oxidant generation, shunt activation, and bacterial killing has been achieved in vitro by use of latex beads coated with glucose oxidase as phagocytic stimuli for CGD neutrophils.'5'157 Similarly, iodination of zymosan and killing of bacteria has been demonstrated in CGD cells exposed to exogenous H202." It is thus considered that CGD cells can function adequatelv when provided with H202. However, normal neutrophils go to great trouble to minimize H202 levels via tightly regulated synthesis and rapid inactivation of excess oxidant. Microtubule inhibition is one rapid consequence of increases in both exogenous 91 and endoge- nous " H202 in PMN. Thus, careful monitoring is needed to ensure that motile functions of CGD neutrophils are not compromised by treatments planned to boost oxidant levels in the vicinity of these cells. Conversely, it has been proposed that PMN that generate excess oxidants may show improved motile function and bactericidal activity when Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 251 October 1978

treated with antioxidant drugs, due to protection of the cytoskeleton by such agents. Preliminary support for this attractive possibility has been obtained by studying the patient with oxoprolinuria. In particular, Repine et al I" recently reported normal phagocytosis but failure of lysosomal degranulation in PMN incubated with the oxidant traps nitroblue tetrazolium and vitamin E. This result again emphasizes the need for careful monitoring of representative neutrophil functions during chemotherapeu- tic trials.

Concluding Remarks In this review I have attempted to explain the processes of chemotaxis, phagocvtosis, oxidant generation, and lysosomal degranulation in normal and genetically abnormal human PMN. In my view these leukocyte functions are most importantly dependent on the integrity of three cellular components: the plasma membrane, the submembranous microfilaments, and the cytoplasmic microtubules. These components are often discussed in isolation, and the biochemical and pharmacologic aspects of their function are analyzed separately here. However, PMN motile and bactericidal activities require the interdependent functioning of membranes, microtubules, and microfilaments. I have therefore tried to provide an integrated view of cytoskeleton-membrane organization and function in human PMN. I have particularly emphasized dynamic aspects of the cytoskeleton and membranes, eg, the induction of microtubule assembly and membrane enzyme activation by surface ligands and the reorganization of microfilaments in response to the same ligands. With this background established, I have selected for discussion a series of diseases in which abnormalities of chemotaxis, phagocytosis, Iysosomal degranulation, and/or oxidant generation can be explained directly or indirectly by abnormalities in dymanic properties of PMN membranes, microtubules, or microfilaments. I emphasize that even preliminary insight into the basis of these disorders has sometimes been sufficient to suggest useful clinical approaches to the management of patients. In several of these neutrophil abnormalities, ie, neutrophil actin dysfunction, Chediak-Higashi syndrome, and its "antithesis" described by Gallin and co-workers, the cellular dysfunctions were well documented but the molecular basis was completely obscure prior to cell biologic analysis. Sny- derman and Pike 159 and Chusid and co-workers 160 emphasized the existence of a large number of other neutrophil bactericidal abnormalities resulting from as yet unexplained cellular defects. Further analyses of the functional interactions between membranes and cytoskeletal components in neutrophils may not only clarify the molecular bases of the disorders 252 OLIVER American Journal of Pathology described here but also may prov-ide insight into the origins and proper therapeutic approach to other granulocyte dysfunctions.

References 1. Humbert JR, Miescher PA, Jaffe ER (editors): Neutrophil Physiology and Pathol- ogy. New York, Grune & Stratton. 1975 2. Bellanti JA, Dayton DH (editors): The Phagocytic Cell in Host Resistance. Ne- York, Raven Press. 1975 3. Greenwalt TJ, Jamieson GA (editors): The Granulocyte: Function and Clinical Utilization. New- York, Alan R. Liss, Inc., 1977 4. Murphy P: The Neutrophil. New York, Plenum Medical Book C., 1976 3. Becker EL, Lagunoff D: Symposium: The cell biology of inflammation. Am J Pathol 85:384-478, 1976 6. Ward PA: Sy-mposium: Disorders of leukocv-te function. Am J Pathol 88:700-752. 1977 7. Bessis MI: Living Blood Cells and Their Ultrastructure. New- York, Springer- V'erlag New York Inc., 1973 8. Bainton DF, Farquhar MG: Origin of granules in polvmorphonuclear leukocvtes: Two types derived from opposite faces of the Golgi complex in developing granulocvtes. J Cell Biol 28:27 -301, 1966 9. Bainton DF, Farquhar MG: Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocvtes. II. Cytochemistry and electron microscopy of bone marrow- cells. J Cell Biol 39:299-317, 1968 10. Bainton DF, UIIv-ot JL, Farquhar MG: The development of neutrophilic polymorphonuclear leukocvtes in human bone marrow\. J E.xp Med 134:907-934, 1971 11. Spitznagel JK, Dalldorf FG, Leffell MS, Folds JD, W'elsh IRH, Cooney- NH. NMartin LE: Character of azurophil and specific granules purified from human polymorphonuclear leukocvtes. Lab Invest 30:774-785. 1974 12. Holtzman E: Lvsosomes: A survey. New York, Spring-Verlag New York Inc., 1975 13. Bainton DF: Differentiation of human neutrophilic granulocytes: Normal and abnormal.' pp 1-27 14. NMalawista SE, Bensch KG: Human polymorphonuclear leukocytes: Demonstra- tion of microtubules and effect of colchicine. Science 156:521-529. 1967 15. Oliver JMI, Albertini DF, Berlin RD: Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils. J Cell Biol 71:921-932, 1976 16. Allison AC, Davies P: NMechanisms of endocvtosis and exocytosis. Symp Soc E.xp Biol Nied 28:419-446, 1974 17. Stossel TP, Hartwig JH: Interactions of actin, mvosin. and a new- actin-binding protein of rabbit pulmonar macrophages. II. Role in cytoplasmic movement and phagocytosis. J Cell Biol 68:602-619, 1976 18. Berlin RD, Oliver JNM: Analogous ultrastructure and surface properties during capping and phagocytosis in leukocvtes. J Cell Biol 77:789-804, 1978 19. Oliver JNI, Krawiec JA, Becker EL: The distribution of actin during chemotaxis in rabbit polymorphonuclear leukocvtes. J Reticuloendothel Soc (In press) 20. Lazarides E, Weber K: Actin antibody: The specific visualization of actin filaments in non-muscle cells. Proc Nat] Acad Sci USA 71:2268-2272. 1974 21. Gaskin F, Shelanski NIL: Nlicrotubules and intermediate filaments. Essays in Biochemistry, V'ol 12. Edited by PN Campbell. WNNN Aldridge. 1976, pp 115-146 22. Shelanski NIL, Gaskin F, Cantor CR: NMicrotubule assembly in the absence of added nucleotides. Proc Natl Acad Sci USA 70:765-768. 1973 23. Borisv GG, Olmsted JB, Niarcum JN\I Allen C: N\icrotubule assembly in -itro. Fed Proc 33:167-174, 1974 Vol. 93, No. 1 LEUKOCYTE ABNORMAUTIES 253 October 1978

24. Weingarten MD, Suter MM. Littman DR, Kirschner MNW: Properties of the depolymerization products of microtubules from mammalian brain. BiochemistrY 13:5529-5537, 1974 25. Weisenberg RC: Microtubule formation in vitro in solutions containing low calcium concentrations. Science 1774:1104-1105, 1972 26. Olmsted JB, Borisy GG: Microtubules. Annu Rev Biochem 42:507,504. 1973 27. Dentler WNL, Granett S, Rosenbaum JL: Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. J Cell Biol 65:237-241, 1973 28. Cleveland DW, Hwo SY, Kirschner MNW: Phy-sical and chemical properties of purified Tau factor and the role of Tau in microtubule assembly. J Mol Biol 116:227-247, 1977 29. Borisv GG, Olmsted JB: Nucleated assembly of microtubules in porcine brain extracts. Science 177:1 196-1197, 1972 :30. Telzer BR, Moses MJ, Rosenbaum JL: Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells. Proc Natl Acad Sci USA 72:4023-4027, 1973 :31. Snyder JA, McIntosh JR: Initiation and growth of microtubules from mitotic centers in lvsed mammalian cells. J Cell Biol 67:744-760, 1975 32. johnson KA, Borisy GG: The equilibrium assembly of microtubules in vitro. Molecules and Cell Movement. Edited by S Inoue, RE Stephens. New York. Raven Press, 1975, pp 119-141 :33. Snyder JA, McIntosh JR: Biochemistry and physiology of microtubules. Annu Rev Biochem 45:699-720, 1976 :34. Stephens RE, Edds KT: NMicrotubules: Structure, chemistry, and function. Phvsiol Rev 56:709-777, 1976 &35. Soifer D (editor): Biology of Cvtoplasmic Microtubules, V'ol 253. New- York, New- York Academy of Sciences, 1973 36. Borgers M, de Brabander NI (editor): Microtubules and M1icrotubule Inhibitors. Amsterdam, North-Holland Publishing Co., 1975 37. Goldman R, Pollard T, Rosenbaum J (editors): Cell Motility. Cold Spring Har- bor. N.Y., Cold Spring Harbor Laboratory, 1976 :38. Pollard TD, Weihing RR: Actin and mvosin and cell movement. CRC Crit Rev Biochem 2L-465, 1974 39. Pollard TD: Functional implications of the biochemical and structural properties of cytoplasmic contractile proteins.' pp 259-285 40. Clarke MI, Spudich JA: Nonmuscle contractile proteins: The role of actin and mvosin in cell motility and shape determination. Annu Rev Biochem 46:797-822, 1977 41. Stossel TP, Hartwig JH: Interactions between actin, myosin and an actin-binding protein from rabbit alveolar macrophages: Alveolar macrophage mvosin Mg2+- adenosine triphosphatase requires a cofactor for activation by actin. J Biol Chem 250:5706-5712, 1975 42. Stossel TP: Contractile proteins in phagocvtosis: An example of cell surface-to- cytoplasm communication. Fed Proc 36:2181-2184, 1977 4:3. Shizuta Y, Shizuta H, Gallo NM, Davies P, Pastan I: Purification and properties of filamin, an actin binding protein from chicken gizzard. J Biol Chem 251:6562-6567, 1976 44. NMaruta H. Kom ED: Purification from Acanthameoba castellanii of proteins that induce gelation and syneresis of F-actin. J Biol Chem 252:399-402, 1977 45. Cooke P: A filamentous cvtoskeleton in vertebrate smooth muscle fibers. J Cell Biol 68:539-556, 1976 46. Starger JNI, Goldman RD: Isolation and preliminary characterization of IOnm filaments from baby hamster kidney (BHK-21) cells. Proc Natl Acad Sci USA 74:2422-2426. 1977 254 OUVER American Journal of Pathology

47. Hv-nes RO, Destree AT: 10nm filaments in normal and transformed cells. Cell 13:151-163, 1978 48. Wilson L, Bryan J: Biochemical and pharmacological properties of microtubules. Adv Cell Mol Biol 3:21-72, 1974 49. Berlin RD: Temperature-dependence of nucleoside membrane transport in rabbit alveolar macrophages and pol-morphonuclear leukocytes. J Biol Chem 248:4724- 4730, 1973 30. Hartwig JH, Stossel TP: Interactions of actin, myosin, and an actin-binding protein of rabbit pulmonary macrophages. III. Effects of cytochalasin B. J Cell Biol 71:295-303, 1976 51. Zigmond SH. Hirsch JG: Cy-tochalasin B: Inhibition of D-2-deoxygylucose transport in leukocytes and fibroblasts. Science 176:1432-1434, 1972 32. Nicolson GL, Smith JR, Poste G: Effects of local anesthetics on cell morphology and membrane-associated cv-toskeletal organization in BALB/3T3 cells. J Cell Biol 68:395-402, 1976 53. Nicolson GL: Transmembrane control of the receptors on normal and tumor cells. I. Cvtoplasmic influence over cell surface components. Biochim Biophys Acta 457:57-108, 1976 54. Weissmann G, Goldstein I, Hoffstein S, Tsung P-K: Reciprocal effects of cAMP and cGMP on microtubule-dependent release of lvsosomal enzymes. Ann NY Acad Sci 2553:750-762, 1973 55. Becker EL: Some interrelations of neutrophil chemotaxis, lysosomal enzy-me secretion and phagocytosis as revealed by synthetic peptides. Am J Pathol 85:385- 394, 1976 36. Zurier RB, Hoffstein S, Weissmann G: Mechanisms of lvsosomal enzyme release from leucocytes. I. Effect of cyclic nucleotides and colchicine. J Cell Biol 58:27-41. 1973 37. Bandmann U. Rydgren L, Norberg B: The difference between random movement and chemotaxis: Effects of antitubulins on neutrophil granulocvte locomotion. Exp Cell Res 88:6.3-73, 1974 38. Malawista SE: Microtubules and the mobilization of ly-sosomes in phagocytizing human leukocytes. Ann NY Acad Sci 253:7.38-749, 1975 59. Ukena TE, Berlin RD: Effect of colchicine and vinblastine on the topographical separation of membrane functions. J Exp Med 136:1-7, 1972 60. Oliver JM, Ukena TE, Berlin RD: Effects of phagocy-tosis and colchicine on the distribution of lectin-binding sites on cell surfaces. Proc Natl Acad Sci USA 71:394- 398, 1974 61. Tsan MF, Berlin RD: Effect of phagocytosis on membrane transport of nonelectrolytes: J Exp Med 134:1016-1035, 1971 62. Berlin RD, Fera JP: Changes in membrane microviscosity associated with phagocvtosis: Effects of colchicine. Proc NatI Acad Sci USA 74:1072-1076, 1977 63. Berlin RD: Mlicrotubules and the fluidity of the cell surface. Ann NY Acad Sci 253:445-454, 1975 64. Berlin RD: Microtubule-membrane interactions: Fluorescence techniques." pp 327-43:39 65. Malawista SE: Effects of colchicine and vinblastine on the mobilization of lysosomes in phagocytizing human leukocytes." pp 199-206 66. Albertini DF, Berlin RD, Oliver JM: The mechanism of Concanavalin A cap formation in leukocytes. J Cell Sci 26:37-75, 1977 67. Hoffstein S, Soberman R, Goldstein I, Weissmann G: Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocvtes. J Cell Biol 68:781-787., 197-6 68. Burchill BR, Oliver JM, Pearson CB, Leinbach ED, Berlin RD: Microtubule dv- Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 255 October 1978

namics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes. J Cell Biol 76:439-447, 1978 69. Oliver J\1, Spielberg SE, Pearson CB, Schulman JD: Microtubule assembly and function in normal and glutathione svnthetase deficient human polyrmorphonuclear leukocytes. J Immunol 120:1181-1186, 1978 70. Oliver J\1, Lalchandani R, Becker EL: Actin redistribution during Concanavalin A cap formation in rabbit neutrophils. J Reticuloendothel Soc 21:359-365 19,7 71. Oliver JN: Concanavalin A cap formation on human polyrmorphonuclear leukocvtes induced by R17934, a new antitumor drug that interferes with microtubule assembly. J Reticuloendothel Soc 19:389-395, 1976 72. Oliver JM: Impaired microtubule function correctable by cyclic GNIP and cholinergic agonists in the Chediak-Higashi syndrome. Am J Pathol 85:395-418, 1976 73. Gallin JI, Rosenthal AS: The regulatory role of divalent cations in human granulocvte chemotaxis: Evidence for an association between calcium exchanges and microtubule assembly. J Cell Biol 62:394-609, 1974 74. Naccache PH, Showell HJ, Becker EL, Sha'afi RI: Transport of sodium, potassium and calcium across rabbit polymorphonuclear leukocyte membranes: Effect of chemotactic factor. J Cell Biol 73:428-444, 1977 5z. Gallin JI, Gallin EK, NMalech HL, Cramer EB: Structural and ionic events during leukocvte chemotaxis. Leukocyte Chemotaxis: Methods, Physiology and Clinical Implications. Edited by JI Gallin, PG Quie. New York, Raven Press, 1978, pp 123- 142 76. Bessis \1, de Boisfleurv A: A catalogue of white blood cell movements (normal and pathologic). Blood Cells 2:365-410, 1976 7 Senda N, Tamura H, Shibata N, Yoshitake J, Kondo K, Tanaka K: The mechanism of the movement of leukocytes. Exp Cell Res 91:393-407, 1975 78. Ramsey WS: Analysis of individual leukocvte behavior during chemotaxis. Exp Cell Res 70:129-139, 1972 79. Ramsey WS: Locomotion of human polymorphonuclear leukocvtes. Exp Cell Res 72:489-501 1972 80. Ramsey WS, Harris A: Leukocyte locomotion and its inhibition by antimitotic drugs. Exp Cell Res 82:262-270, 1972 81. Porter KR: Cytoplasmic microtubules and their functions. In Principles of Biomo- lecular Organization. Edited by GE W7olstenholme, NI O'Connor. Boston, Little, Bro-n & Co., 1966, pp 308-345 82. Zurier RB, Weissmann G, Hoffstein S, Kammerman S. Tai HH: Nechanisms of Ivsosomal enzyvme release from human leukocv-tes. II. Effects of cAM1P and cGNIP, autonomic agonists, and agents which affect microtubule function. J Clin Invest 5:3:297---309. 1974 83. Goldstein I, Hoffstein S, Gallin J, W7eissmann G: \iechanisms of lysosomal en- zvrme release from human leukocvtes: licrotubule assembly and membrane fusion induced by a component of complement. Proc Natl Acad Sci USA 70:2916-2920, 1973 84. Hoffstein S. Goldstein IN1. WNeissmann G: Role of microtubule assemblv in lvsosomal enzy-me secretion from human polymorphonuclear leukocytes: A reevalua- tion. J Cell Biol 73:242-256, 1977 8D. Rudolph SA, Greengard P. N\alawista SE: Effects of colchicine on cyclic ANIP levels in human leukocytes. Proc Natl Acad Sci USA 74:3404_3408, 197 86. \Nalaw-ista SE, Oliver J\1 Rudolph SA: Nlicrotubules and cyclic ANIP in human leukocytes: On the order of things. J Cell Biol 77:881-86, 1978 87. Nath J, Rebhun LI: Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs: Involvement on NADP+ and glutathione. J Cell Biol 68:440-450, 1976 256 OLIVER American Journal of Pathology

88. Mellon NMG, Rebhun LI: Sulfhvdrnls and the in vitro polymerization of tubulin. J Cell Biol 70:226-238, 1976 89. Rebhun LI, Nath J. Remillard SP: Sulfhydryls and the regulation of cell division." pp 1343-1366 90. Beutler E, Duron 0, Kelly BM1: Improved method for the determination of blood glutathione. J Lab Clin Med 61:882-888, 1963 91. Oliver JM, Berlin RD, Baehner RL, Boxer LA: Mechanisms of microtubule disassembly in vivo: Studies in normal and chronic granulomatous disease leucocytes. Br J Haematol 37:311-322, 1977 92. DeChatelet LR: Oxidative bactericidal mechanisms of polymorphonuclear leukocytes. J Infect Dis 131:295-303, 1973 93. Klebanoff SJ: Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocvtes.' pp 127-152 94. Karnovsky NML: Biochemical aspects of the functions of polymorphonuclear and mononuclear leukocvtes.2 pp 25-44 95. Klebanoff SJ: Antimicrobial systems of the polyrnorphonuclear leukocytes.2 pp 45-0 96. Johnston RB, Keele BB, Misra HP, Webb LS, Lehmeyer JE, Rajagopalan KV: Superoxide anion generation and phagocvtic bactericidal activity-.2 pp61-76 97. Baehner RL. The growth and development of our understanding of chronic granulomatous disease.2 pp 173200 98. Root RK: Comparison of other defects of granulocvte oxidative killing mechanisms with chronic granulomatous disease.2 pp 201-226 99. Cheson BD, Curnutte JT, Babior BM: The oxidative killing mechanisms of the neutrophil. Prog Clin Immunol 3:1-65, 1977 100. Segal AW: Chronic granulomatous disease: Biochemistry and oxygen metabolism. Inbom Errors of Immunity and Phagocytosis. Edited by F Guttler. Lancaster, England, MTP Press Ltd., 1978 (In press) 101. Reed PW7: Glutathione and the hexose monophosphate shunt in phagocytizing and hydrogen peroxide-treated rat leukocytes. J Biol Chem 244:2459-2464, 1969 10-2. Weening RS, Roos D, Van Schaik MULJ, V'oetmar AA, de Beer NI, Loos HA: The role of glutathione in the oxidative metabolism of phagocs-tic leukocvtes. Studies in a family with glutathione reductase deficiency. Proceedings of the First European Conference on Phagocytic Cells. Edited by F Rossi. Padova, Italy. Piccin NMedical Books, 1978 (In press) 103. Roos D, WAeening RS, Loos JA: The protective role of glutathione.'" 104. Eggleston LV, Krebs HA: Regulation of the pentose phosphate cycle. Biochem J 138:425-435, 1974 105. Sbarra AJ, Karnovsky NIL: The biochemical basis of phagocvtosis. I. Metabolic changes during the ingestion of particles by polyrmorphonuclear leukocvtes. J Biol Chem 234:1,355-1362, 1959 106. Iver GYN, Islam MF, Quastel JH: Biochemical aspects of phagocytosis. Nature 192:535-541, 1961 107. Romeo D, Zabucchi G, Rossi F: Reversible metabolic stimulation of pol-morphonuclear leukocytes and macrophages by Concanavalin A. Nature (New Biol) 243:111-112, 1973 108. Goldstein INM, Roos D, Kaplan HB, W7eissmann G: Complement and immunoglobulins stimulate superoxide production by human leukocvtes independently of phagocvtosis. J Clin Invest 56:1155-1163, 1975 109. Becker EL, Sigman NI, Oliver JM: Superoxide production induced in rabbit polymorphonuclear leukocy%tes by synthetic chemotactic peptides and AZ3187: The nature of the receptor and the requirement for Ca2+. (Submitted) Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 257 October 1978 110. DeChatelet LR, Shirley PS, Johnston RB: Effect of phorbol msristate acetate on the oxidative metabolism of human polymorphonuclear leukocytes. Blood 47:545- 534, 1976 111. Nakamura NM, Nakagawara A, Minakami S: Mimicking of phagocytic metabolism by leukocvtes suspended in heavy water (D2O). Exp Cell Res 102:429-431, 1976 112. Romeo D, Zabucchi G, NMiani N, Rossi F: Ion movement across leuckocyte plasma membrane and excitation of their metabolism. Nature 253:542-544, 1975 11.3. Nakagawara A, Kakinuma K, Shin H, Misazaki S. Minakami S: Lack of c-t(- chalasin E-induced superoxide release by polymorphonuclear leukocytes of patients with chronic granulomatous disease: A new diagnostic test. Clin Chim Acta 70:1.3:3- 137, 1976 114. Malawista SE, Bodel PT: The dissociation by colchicine of phagocytosis from increased oxygen consumption in human leukocytes. J Clin Invest 46:786-796, 1967 115. Segal AW, Peters TJ: Analytical subeellular fractionation of human granulocvtes with special reference to the localization of enzymes involved in microbicidal mechanisms. Clin Sci NMol Med 52:429-442 116. Briggs RT, Drath DB, Karnovsky NL, Karnovsky IJ: Localization of NADH oxidase on the surface of human polvmorphonuclear leukocytes by a new cyto- chemical method. J Cell Biol 67:366-586, 1973 117. Goldstein INI, Cerqueira NI, Lind S, Kaplan HB: Evidence that the superoxide- generating system of human leukocytes is associated with the cell surface. J Clin Invest 59:249-254, 1977 118. Tsan N F, Newman B, N clntyre PA: Surface sulphvdrvl groups and phagocytosis- associated oxidative metabolic changes in human polymorphonuclear leukocytes. Br J Haematol 33:189-204, 1976 119. Tsan NI F, NMcIntyre PA: The requirement for membrane sialic acid in the stimulation of superoxide production during phagocvtosis by human polvmorphonuclear leukocvtes. J Exp NMed 143:1308-1316, 1976 120. Takanaka K, O'Brien PJ: Nlechanisms of H202 formation bv leukocvtes: Evidence for a plasma membrane location. Arch Biochem Biophys 169:428-435, 1975 121. Boxer LA, Hedlev-Whyte ET, Stossel TP: Neutrophii actin dysfunction and abnormal neutrophil behavior. N Engl J NMed 291:1093-1099, 1974 122. Wolff SNI, Dale DC, Clark RA, Root RK, Kimball HR: The Chediak-Higashi syndrome: Studies of host defenses. Ann Intern NMed 76:293306, 1972 123. Oliver J\1, Krawiec JA, Berlin RD: Carbamylcholine prevents giant granule formation in culture fibroblasts from beige (Chediak-Higashi) mice. J Cell Biol 69:205-210, 1976 124. Hirsimaki Y, Arstila AU, Trump BF: Autophagocytosis: in vitro induction by microtubule poisons. Exp Cell Res 92:11-14, 1973- 125. Powell HC, Wolf PL: Neutrophilic leukocvte inclusions in colchicine intoxication. Arch Pathol Lab Med 100:136-138, 1976 126. Oliver JNI, Zurier RB, Berlin RD: Concanavalin A cap formation on polvmorphonuclear leukocvtes of normal and beige (Chediak-Higashi) mice. Nature 253:471- 473, 1975 127. Oliver JNM, Zurier RB: Correction of characteristic abnormalities of microtubule function and granule morphology in Chediak-Higashi syndrome with cholinergic agonists: Studies in vitro in man and in rivo in the beige mouse. J Clin Invest a7:1239-1247, 1976 128. Boxer LA, Rister NI, Allen JNM, Baehner RL: Improvement of Chediak-Higashi leukocvte function by cyclic guanosine monophosphate. Blood 49:9-17, 1977 129. Lyons RT, Pitot HC: Protein degradation in normal and beige (Chediak-Higashi) mice. J Clin Invest 61:260-268, 1978 1.30. Boxer LA, WTatanabe ANt, Rister M, Besch HR, Allen J. Baehner RL: Correction 258 OLIVER American Journal of Pathology

of leukocy-te function in Chediak-Higashi syndrome by ascorbate. N Engl J Med 295:1041-1045, 1976 131. Sandler JA, Gallin JI, Vaughan NI: Effects of serotonin, carbamrlcholine, and ascorbic acid on leukocyte cyclic GMP and chemotaxis. J Cell Biol 67:480-484, 19735 132. Boxer LA, Albertini DF, Baehner R, Oliver JNM: Correction in vitro and in vivo of the microtubule assembly defect in Chediak-Higashi syndrome neutrophils: An ultrastructural analysis. (Submitted) 13.3. Check WA: Cell organelle defects again associated with genetic disease. JAMA 238:461-462, 19 47 1.34. Bell TG, Meyers KMN, Prieur DJ, Fauci AS, Wolff SM, Padgett GA: Decreased nucleotide and serotonin storage associated with defective function in Chediak- Higashi syndrome cattle and human platelets. Blood 48:175-184, 1976 135. Boxer GJ, Holmsen H, Robkin L, Bang NU, Boxer LA, Baehner RL: Abnormal platelet function in Chediak-Higashi syndrome. Br J Haematol 35:321-a333, 1977 136. Kanfer JN, Blume RS, Yankee RA, Wolff SM: Alteration of sphingolipid metabolism in leukoc tes from patients with the Chediak-Higashi syndrome. N Engl J Med 279:410-413, 1968 137. Berlin RD, Fera JMt, Oliver JM, Graf P: Altered membrane lipid composition in Chediak-Higashi syndrome. (In preparation) 138. Lewin S: Vitamin C: Its molecular biology and medical potential. N'ew York. Academic Press Inc., 197-6 139. Vassali J-D, Granelli-Pipemo A, Griscelli C, Reich E: Specific protease deficiency in polymorphonuclear leukocytes of Chediak-Higashi syndrome and beige mice. J Exp Med 147:1283-1290, 1978 140. Gallin JI, Malech HL, Wright DG, Whisnant J, Kirkpatrick CH: Recurrent severe infections in a child with abnormal leukocvte function: Possible relationship to increased microtubule assembly. Blood 31:919, 1978 141. NMalech HL, Root RK, Gallin JI: Structural analysis of human neutrophil migra- tion: Centriole, microtubule and microfilament orientation and function during chemotaxis. J Cell Biol 73:666-693, 1977 142. Holmes B, Quie PG, Windhorst DB, Good RA: Fatal granulomatous disease of childhood: An inbom abnormality of phagocytic function. Lancet 1:1223-1228. 1966 143. Quie PG, White JG, Holmes B, Good RA: In vitro bactericidal capacity of human polvmorphonuclear leukocvtes: Diminished activity in chronic granulomatous disease of childhood. J Clin Invest 46:668-679, 1967 144. Baehner RL, Nathan DG: Leukocyte oxidase: Defective activity in chronic granulomatous disease. Science 135:8.35-836, 1967 143. Holmes B, Page AR, Good RA: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 46:142-2-1432, 1967 146. Cumutte JT, Kipnes RS, Babior BM: Defect in pyridine nucleotide dependent superoxide production by a particulate fraction from the granulocytes of patients with chronic granulomatous disease. N Engl J Med 293:628-632, 1973 147. Segal AW, Peters TJ: Characterization of the enzyme defect in chronic granulomatous disease. Lancet 1:1363-1363, 1976 148. Graf P, Berlin RD: Cyclic regeneration of surface sulfhvdryl groups. Fed Proc 39:316, 1978 149. Weening RS, Roos D, Weemaes CMR, Homan-Mfrller JWNT, van Schaik MLJ: Defective initiation of the metabolic stimulation in phagocytizing granulocytes: A new congenital defect. J Lab Clin Med 88:737-768, 1976 130. Holmes B, Park BH, Ntalawista SE, Quie PG. Nelson DL, Good RA: Chronic Vol. 93, No. 1 LEUKOCYTE ABNORMALITIES 259 October 1978

granulomatous disease in females: A deficiency of leukocyte glutathione peroxidase. N Engl J M1ed 283:217-221, 1970 131. Matsuda I, Oka Y, Taniguchi N, Furuyama NI, Kodama S, Arashima S, NMitsuyama T: Leukocvte glutathione peroxidase deficiency in a male patient with chronic granulomatous disease. J Pediatr 88:381-583, 1976 152. Baehner RL, Gilman N, Karnovsky NIL: Respiration and glucose oxidation in human and guinea pig leukocytes: Comparative studies. J Clin Invest 49:692-700, 1970 153. Loos H, Roos D, Weening RS, Houwerzijl J: Familial deficiency of glutathione reductase in human blood cells. Blood 48:33-62, 1976 154. Spielberg SP, Kramer LI, Goodman SI, Butler J, Tietze F, Quinn P, Schulman JD: 5-Oxoprolinuria: Biochemical observations and case report. J Pediatr 91:237- 241, 1977 15. Spielberg SP, Boxer LA, Oliver JMI, Butler EJ, Schulman JD: Altered phagocytic and microtubule function in leukocytes from a patient with severe glutathione syntherase deficiency (5-oxoprolinuria). Proceedings of the Intemational Sym- posium on Inbom Errors of Metabolism in Man. Basel, S, Karger, AG, 1978 (In press) 136. Baehner RL, Nathan DG, Kamovskv M\L: Correction of metabolicdeficiencies in the leukocytes of patients with chronic granulomatous disease. J Clin Invest 49:865-870, 1970 157. Johnston RB, Baehner RL: Improvement of leukocyte bactericidal activity in chronic granulomatous disease. Blood '35:330-355, 1970 138. Repine JE, Rao G, Beall GD, White JG: Inhibition of human neutrophil oxidative metabolism and degranulation in vitro by nitroblue tetrazolium and vitamin E. Am J Pathol 90:659-674, 1978 139. Snvderman R, Pike MC: Disorders of leukocyte chemotaxis. Pediatr Clin North Am 24:377-393, 1977 160. Chusid NMJ, Gallin JI, Dale DC, Fauci AS, Wolff SM: Defective polymorphonuclear leukocvte chemotaxis and bactericidal capacity in a boy with recurrent pyo- genic infections. Pediatrics 38:313-520, 1976 260 OUVER American Joumai of Pabtoo

[Illustrations follow] EV i A\

I-

Fiure 1-Uftrastructure of the human PMN. A section through a Con-A-treated human PMN reveals a lobed nucleus (N); two categories of granules, the specific granules (SG) and azurophilic granules (SG); a centriolar region (CR) in the cell center from which microtubules (MT) radiate; and a peripheral area of granule exclusion that contains microfilaments (MF). Numerous endocytic vesicles (EV) are seen at the cell periphery. These probably contain Con A-receptor complexes in process of intemalization. This mature cell contains a single mito- chondrion (M). (x 14,000) Fgu 2-The centriolar region of normal human PMN before (A) and after (B) initiation of phagocytosis. Two (of three) microtubules are indicated by arrows in the unstimulated cell. The nine groups of three microtubules with their associated satellite structures (S) that make up the centiole are readily observed. In the stimulated cell, six (of 25) microtubules are indicated by arrows. Bar = 0.5 ju. (Reprinted from Oliver et al " with permission from the Journal ot Immunology.)

Fqe 3-Phagocytosis of oil emulsion by PMN. Phagocytosis is initiated by enclosure of oil droplets within microfilament (MF)-rich pseudopods. Phagocytosis is complete when the particles are completely inside the cell within a phagocytic vesicle (PV). Engulfment is associated with the disappearance of membrane-associated microfilaments and with fusion of cytoplasmic granules with the vesicle membrane to produce a phagolysosome (PL). The fibers marked IF are intermediate or 100-A filaments. Bar = 1.0 it. (x 11,600) 2 4

Fue 4-Phagocytosis of zymosan by human PMN. The cell was exposed to opsonized zymosan (Z) for 0.5 minutes as described by Burchill et al." Microfilament-rich paeudopods enclose the particles. A centriole (C) with associated microtubules is visible. (x 11,600) (Adapted from Berlin and Oliver "I with permission from the Joumal of Cell Biology.) Fgu 5-Detail from the cell in Figure 4, emphasizing the microfilamentous network that excludes granules and microtubules in the advancing pseudopod. Lysosomal degranulation is apparent in the filament-free base of the developing phagocytic vesicles. (x 51,900) (Adapted from Berlin and Oliver " with permission from the Joumal of Cell Biology.) 00~E

U.00

0 c Ecm o W000

E' 0 CL

e co0 0C WUW _ 2! -

C--0 0

0 0CD0 00 ' 0.0 0 v 0 >D E : .0 M

-CO 0 0. 0 CD00Q z0003 0:c b05O

0 > nODD C to

.0ao c0=0 E0t;00 0 o

a0- X- W_ W-

C C 0 0. =-0 D C Ca QO*-- - - 00 .0 C0.0

- E wO .j

Ilk-- ibE'

.- -ft Jr . -..Lw- ;b.-1

I A--

Fiure 7-Phagocytosis of latex by human peripheral blood monocyte. Phagocytosis via microfilamient-rich pseudopods is occuring over the whole membrane. Lysosomal degranulation is apparent when the phagocytic vesicles move out of the filamentous region (arrows). Microtubules (MT) are present in all regions of the cell (arrowheads) except in the filament-rich areas of active phagocytosis. (x 17,800) (From Berlin and Oliver 18 with permission from the Joumal of Cefl Biology.) I .... .o .. Z-L. z:

Fgure 8-Phagocytosis d opsonized erythrocytes by rabbit peritoneal macrophages. Macro- phages were incubated with antibody-coated sheep erythrocytes and subsequently immunofluorescence-labeled with antiactin (a and b) or antitubulin (c and d) antibodies. Fluorescence due to antiactin is concentrated in pseudopods from each of two cells in the process of engulfing a single erythrocyte. In contrast, no accumulation of fluorescence due to antitubulin occurs at the region of pseudopod formation. (Initial magnification x 1250) (From Berlin and Oliver 1I with permission from the Journal of Cell Biology.) Fie 9-The distribution of polystyrene beads and fluorescein-Con A on human PMN. In a through d, PMN were incubated with fluorescein-Con A without or with previous exposure for 30 minutes to 10' M colchicine. A random distribution of fluorescence is maintained on cells that can assemble microtubules (a and b). Microtubule- depleted cells develop a protuberance, and Con A collects over this region in a typical surface cap (c and d). I n e through h, cells were incubated with carboxylated polystyrene beads as well as with Con A. PMN that can assemble microtubules internalize latex over the whole cell. Fluorescence appears to be concentrated in the membranes of these phagocytic vesicles (e and f). In contrast, colchicine-treated (microtubule-depleted) PMN concentrate both latex and Con A in the same cap region. (Initial magnification x 1250) (From Berlin and Oliver " with permission from the Joumal of Cell Biol- ogy.) i6:

Fgu 1O-Ultrastructure of Con-A-capped leukocytes. A-The human blood monocyte was capped by incubation with diamide and Con A. Paired centrioles (C) lacking microtubules are present The cap region consists of highly plicated membrane that is underlaid by a dense microfilament network. B-A cross- section through the cap. The microfilament-rich core contains many myosin-like fibers (F). (A, x 12,300; B, x 55,800) (Reproduced from Albertini et a]i with permission from the Joumal of Cell Science.) i 1

42~

Figure 1 I-Phagocytosis in a colchicine-treated human blood monocyte. Ingestion is proceeding via the microfilame rich protuberance, causing an apparent delay in lysosomal degranulation. (x 15,300) (From Berlin and Oliver "I w permission from the Journal of Cell Biology.) Fure 12-Ultrastructure of Con-A-treated Chediak-Higas syndrome neutrophils without (A) and with (B) prior exposure to carbamylcholine (104 M, 30 minutes). Both cells shi a typical giant granule (g), numerous apparently normal granules, and centrioles which are either depleted microtubules (A) or associated with numerous microtubules (B). (A, x 9300; B, x 13,100) (From Oliver.")