Cell biology of leukocyte abnormalities--membrane and cytoskeletal function in normal and ...

52 Pages·2007·7.58 MB·English

REVIEW
ARTICLE
CELL BIOLOGY
OF LEUKOCYTE
ABNORMALITIES
Cell Biology of Leukocyte Abnormalities-Membrane and Cytoskeletal Function in
Normal and Defective Cells
Ultratructure ofthe Human Neubtpl 22:3
Basic _bmisty of ke keon 22
Basic Pharmacology ofthe 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 andOidativeMet :237
Membrane and CytoskeletalAbrmnaties of Human Neutrophis 241
Neutrophil Dysfunction Linked to Structural Abnormalitiesof the Cytoskeleton 241
Neutrophil Dysfunction Linked to Regulatory Abnormalities ofthe Cvtoskeleton 242
Neutrophil Membrane and Cytoskeletal Dvsfunction Secondarv to Abnormal Oxidative
Metabolism 246
Disorders ofOxidant 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, neu-
trophil) has been studied since the days of Ehrlich, the precisemechanisms
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 opsoniza-
tion 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 influ-
enced not onlv bv the immune svstem and the availabilitv of cytoplasmic
and granule enzvmes but also by dvnamic properties of the cell mem-
brane 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 micro-
filaments. The motile and bactericidal functions of the neutrophil are
subsequently expressed.
From the lepartments of Pathology and Physiology. University ofConnecticut Health Center,
lFarmington. COnnecticuit.
Slupp)rted bb-Grants FS-01106andCA-15364fromtheNationalInstitutesofHealthandbN-Grant
BC-179 from the American Cancer Society. Dr. Oliver is an American Cancer Societv Facults
R(esarch Aw5ardee.
A\ccepte(l forpuibli.ation April 17. 1978.
..%ddrt-ss reprint requests to Janlet S1. Oliser, PhD, Department of Physiology, UniversityofCon-
oceticiat lealth (;enter Stho)ol of Medicine, IFarmington, CT 06032.
0002-9440/78/1010-0219$01.00 221
222 OLIVER AmericanJournal
ofPathology
PMN
Bacterial invasion
Chemotacticfactor (CF)generation - CFbindingtosurfacereceptors
CHEMOTAXIS
tositeofinfection
Opsonizationofbacteria _ Binding of bacteria to surface receptors
0
PHAGOCYTOSIS OXIDANTGENERATION
LYSOSOMALDEGRANULATION
BACTERIALKILLING
_ 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
lvsosomaldegranulation (capitalletters, Steps5through 8),dependmostimportantlyontheintegrit
of the plasma membrane and ofthecytoplasmic microtubules and microfilaments.
It has been recognized that the failure of bacterial surveillance in
specific diseases is associated directly or indirectly with defects in mem-
brane 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 pharmacologyofmicrotubules 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
October1978
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 sug-
gested. The analysis draws heavily from the concepts of modern cell
biology. Alternative approaches tonormal and pathologic leukocyte func-
tion have been presented in several recent monographs 14 and sym-
posiums.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 ofthe 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; theyaremembrane-bounded and
contain biochemically and cytochemicallydemonstable acid hydrolases as
well as myeloperoxidase which is involved in bacterial killingviaoxidative
mechanisms. The specific granules are not strictly lysosomes; their mem-
brane 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 micro-
tubules [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 approx-
imately 240 R in diameter that radiate from the center of the cell in all
directions toward the plasma membrane. At highermagnification (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 micro-
tubules 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 be-
cause their formation in cells from soluble tubulin dimer (see below)
224 OLIVER AmericanJournal
ofPathology
requires the interaction of the PMN with a surface or with a ligand.15
Thus, the centriole in Figure2A 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, approximately60
A in diameter, are present under the membrane of the round cell, occu-
pying protrusions and ruffled regions of the plasma membrane and pre-
venting access of cytoplasmic granules to the membrane. Their major
subunit component is the globular protein actin. Microfilaments become
more prominent in PMN during motileorendocvtic processesdue in large
part to their concentration in regions ofsurface activity.",-- Thus, thecells
in Figure 3 which are internalizing oil emulsion by phagocytosis show
marked polarization of microfilaments in pseudopods. A similar recruit-
ment 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 fila-
ments 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 lig-
ands, including Con A. Thus, these vesicles very likely reflect the occur-
rence of adsorptive pinocytosis induced by Con A. Massive membrane
deformation is involved in the internalization of large particles in phago-
cytic vesicles (PV) by PMN as illustrated in Figure 3. The fusion of
granule membranes with phagocvtic vesicle membranes, leading to dis-
charge of granule contents into the resulting phagolysosomes (PL), is also
readily observed in Figure 3.
Vol.93,No.1 LEUKOCYTEABNORMAUT1ES 225
October1978
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 micro-
tubule, 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 at37 C in the presence ofGTPanddisassemblyat4 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 bio-
chemical 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 proper-
ties 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 ahollow 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. Furtherdetails ofthese
226 OUVER AmericanJoumal
ofPaxhokgy
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 ofactin 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 aswell 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 ofATP, interact in various ways with actin, andare 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 func-
tion 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 similaramino
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
October1978
This alkaloid binds with tubulin and causes the rapid and complete
disassembly ofcytoplasmic microtubules in all mammalian cells. Its speci-
ficity 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 dis-
assembly but related, for example, to its inhibition ofmembrane transport
processes at high dose." However, the availability ofcolchicine 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 butylhydroper-
oxide.15
In contrast to this range of antimicrotubule drugs, pharmacologic dis-
ruption of the microfilament system of animal cells usually relies on a
single and notverysatisfactory agent, cytochalasin B. This fungal metabo-
lite usually causes structural disorganization of microfilaments in mam-
malian 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 macro-
phage 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 anesthet-
ics have more recently been employed to probe membrane-associated
microfilament functions. These agents maynot directly influence filament
organization but they may displace membrane-bound calcium, perturb
microfilament-membrane associations, and thus impair specific micro-
filament-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 lyso-
somaldegranulation in neutrophilsareall influenced by the cytoskeleton.
228 OLIVER American-Joumal
ofPathobogy
Cytochalasin B completely abolishes random cell movement, surface
ruffling activity, chemotaxis, and phagocytosis while promoting the extra-
cellular 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 gran-
ules and plasma membrane.
Colchicine is considered by most investigators to inhibit lysosomal
degranulation "'66 and to impair chemotactic but not random cell move-
ment.r7 Colchicine generally does not reduce the rate of phagocytosis by
neutrophils."-° However, as explained below, Berlin et al have estab-
lished 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 ofpharmacologic evidence,
microtubules have thus been assigned the rolesofprovidingorientation to
gross membrane activities, of associating directly or indirectly with gran-
ules 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 inourlaboratory '3'" haveestablished 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 immedi-
ately 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 micro-
filament 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 devel-
oped a unified view of cytoskeletal function in chemotaxis, phagocytosis,
and lysosomal degranulation based on these data.