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Neutrophil granules in health and disease
Postgraduate course Neutrophil Judith
granules
in health
and disease
Falloon, M.D., and John 1. Gallin, M.D. Bethesda, Md.
Once staining and microscopy techniques made the granules of the neutrophil visible, the pursuit of their function challienged those interested in the neutrophil and its role j.n health and in myriad disease states. Both a secretory function of the granules postulated by Paul Ehrlich’ in the late 1800s whereby neutrophils modify their environment and an intracellular digestive or lysosoimal function postulated by Ehrlich’s contemporary Elie Metchnikoff’ appear to be of importance. Current understanding of neutrophil granules has separated these functions according to granule type with the specific granules (also called secondary granules) serving the environmental modification or external secretory function and the azurophilic granules (also called primary granules) acting as lysosomes by fusing with phagosomes to form phagolysosomes with a predominantly intracellular site of action.3 Although this is, of course, a conceptual oversimplification, since specific granule contents are found in phagolysosornes and azurophilic granules are clearly released into the extracellular milieu through degranulation and cell death,4 the concept of the separation of functions lhas proved useful. GRANULE CONTENTS AND FUNCTIONS The two main neutrophil granules, specific and azurophilic, are separated on the basis of differences in content, morphology, density, and appearance at different stages of neutrophil maturation (Fig. 1). During myelopoiesis, the first granules become visible at about the promyelocyte stage and stain azurophilic in the bone marrow; these are the primary or azurophilic granules. Later on during maturation, synthesis of these granules ceases, and they are distributed to daughter cells during cell division, thus decreasing From the Bacterial Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda. Received for publication Nov. 21, 1985. Accepted for pu,blication Jan. 9, 1986. Reprint requests: Judith Falloon, M.D., Bacterial Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bldg. 10, Rm. llN112, Bethesda, MD 20892.
~1 FMLP:
CGD:
N-formylmethionylleucylphenylanine Chronic granulomatous
disease
the azurophilic granule content of the cells.” The granules produced second during neutrophil maturation, called secondary or specific granules, then come to predominate, comprising about two thirds of the granules of the mature ce11.5The differences between these granules have permitted the development of subcellular fractionation techniques leading to the separation of disrupted cells into granule-enriched fractions that have been used to probe further into granule content and function. The azurophilic granules sediment heterogeneously on sucrose gradient9 and contain microbicidal enzymes, proteases, acid hydrolases, and bactericidal cationic proteins (Table I).6-” These contents correlate with the postulated lysosomal function of azurophilic granules. The specific granule, a less dense granule that stains heavily for glycoprotein, contains lysozyme, collagenase, lactoferrin, vitamin B,,-binding protein, and possibly histaminase and sialidase.6-‘3 Recently, the specific granule-enriched fraction of neutrophils has been demonstrated to contain a unique b-type cytochrome’4. ” and possibly a flavoprotein’6 believed to be of importance in the formation of the complete NADPH oxidase enzyme responsible for the respiratory burst that produces the toxic oxygen products important in neutrophil function, including killing.17, ‘* Organelles that contain a preformed intracellular pool of receptors important in the response of the phagocyte to chemoattractants and interaction with serum complement proteins also cosediment with specific granules in gradients.“, ‘O A third granule type, a gelatinase containing tertiary granule, has been described as a separate entity with a density slightly less than that of specific granules on sucrose gradients.” Further knowledge of this granule is scanty; it most likely sediments with the specific granule-enriched fraction in most gradients and is potentially the site 653
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and
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J. ALLERGY CLIN. IMMUNOL. MAY 1986
FIG. 1. A human neutrophil stained for peroxidase. Five nuclear lobes (1 to 5/ are visible in the cytoplasm. Small, unstained specific granules (SJ are present throughout the cytoplasm. The larger, darkly stained structures are azurophilic granules that stain positively for peroxidase. irregularly shaped channels (at arrow] represent invaginations of the cell surface and are continuous with the external environment. Fixation in aldehydes and osmium. Scale bar = 0.5 pm. (Micrograph courtesy of Dr. Marc Friedman, Georgetown University.)
of certain contents currently attributed to specific granules. Neutrophil degranulation occurs in the midst of many other events during the complex response of the cell to inflammatory stimuli. As a neutrophil is exposed to chemoattractants such as N-formylated peptides released by bacteria,22 complement proteins, or leukotriene products, it develops a ruffled membrane and changes from a round to a polarized cell with anterior broad lammellipodium and posterior-narrow uropod. Cytoskeletal microtubules and microfilaments reorganize, and there is anterior movement of granules while the nucleus moves toward the rear of the ce11.23Cells aggregate and adhere to endothelial surfaces. There is subsequent disaggregation, and cells become increasingly motile (chemokinetic) with directed movement (chemotaxis) toward the more concentrated source of chemoattractant. These events are accompanied by ion fluxes, changes in membrane potential and surface charge, increased glycolysis, alterations in phospholipid and arachidonic acid metabolism, protein phosphorylation, and production of toxic oxygen radicals.24. 25 In this way these cells are directed to tissue sites where they can alter and destroy bacteria and other target cells. At sites of inflammation, both phagocytosis and internal digestion of microorganisms or other particles occur, and the finely tuned exocytosis of granules, prompted also by chemoattractants, occurs. In order to understand further this eomplex neutrophil response, the role
of granule contents in inflammatory and plasma membrane events has been the focus of recent investigations . In keeping with their different functions, the extracellular release of specific and azurophilic granules is under separate control. In vitro, for example, chemoattractants and other substances can be used to cause specific granule secretion under conditions whereby azurophilic granule enzymes will not be released.3. 4. lo. 26 Migration through filters in response to chemotactic stimuli results in preferential specific granule discharge,27 as does migration through tissues in vivo, as demonstrated in exudate cells harvested from human dermal suction blisters. The strongest stimuli for azurophilic granule degranulation also stimulate concomitant specific granule exocytosis.3 Specific granules, then, and probably the tertiary granules2’ appear readily released during cell stimulation so that their contents are available to modify the response of the cell to stimuli as well as to effect function. Investigations into the functions of granule contents have proceeded through isolation and characterization of granule proteins and through observations made on the neutrophils of patients with abnormal granules. In the specific granule, lactoferrin is an iron-chelating glycoprotein that probably has an antimicrobial function via iron chelation or by direct bactericidal effect2* and may function in neutrophil adhesiveness,*’ hydroxyl radical formation,30 and in control of myelo-
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TABLE
Neutrophil
I. Neutrophil Class
granule
0f constituent
Neutral serine proteases
Metalloproteinases Acid hydrolases
Other
references
in health
and
disease
655
contents*
Microbicidal enzymes
*From
granules
Azurophilic
granules
Myeloperoxidase Lysozyme Elastase Cathepsin G Proteinase3
Specific
granules
Lysozyme
Collagenaset
N-acetyl-P-glucosaminidase Cathepsin B Cathepsin D P-Glucuronidase P-Glycerophosphatase a-Mannosidase Bactericidal cationic proteins$
Lactoferrin Vitamin B,? binding proteins Cytochrome b Histaminase FMLP receptors5 C3bi receptors (CR3)S
3 and 10 with modifications.
TReleased asa latentenzyme. $Note added in proof: defensins, antimicrobial peptides in azurophilic granules, have recently Szklorek D, et al: J Clin Invest 76:1427, 1985). 9Present in a subcellular fraction cosedimenting with specific granules in sucrose gradients.
poiesis by inhibition of the production of granulocytemacrophage colony-stimulating factor.” Lysozyme,
present in both granules, digests glycopeptide debris in the cell,32 and hydrolyzes cell wall components of some bacteria.33 It may also modulate inflammation by suppressi:ng neutrophil chemotaxis and oxidative metabolism.‘4 The vitamin B,, binding protein of the specific granule remains enigmatic; its function is unknown. Cytochrome b, probably a specific granule membrane component, is believed to be a constituent of the membrane-associated electron transport chain comprising the NADPH oxidase responsible for the enhanced oxygen consumption and hydrogen peroxide generation of the respiratory burst of the neutrophil. I73I8 Unstimulated cells contain an inactive oxidase, which may consist of several subunits requiring assembly and then activation for activity. One subunit appears to be a cytochrome b, and one appears to be a flavoprotein, both believed to be partially located in the specific granule.14-16.35 Fusion of specific granule membrane containing cytochrome b w:ith the plasma membrane may be a key step in the formation of a complete oxidase enzyme,36 since the complete enzyme complex appears to be plasma membrane associated. Other specific granule functions include amplification of the inflammatory response by activation of the complement cascade, generation of the chemoattractant C5a,37 and release
been described
(Gang
T, Selsted
ME,
of a monocyte chemoattractant.3, ‘* Specific granule contents also promote neutrophil adherence to endothelium. It has recently been postulated that the specific granule membrane contains receptors important in neutrophi1 function and that fusion of granule membrane
with plasma membrane during exocytosis may provide membrane and receptors to the cell surface.3, 4o Since membrane turnover may be important in chemotaxis4’ and new membrane synthesis appears unnecessary, the specific granule membrane pool may be a requirement for membrane replacement during chemotaxis. By correlating specific granule degranulation with increased surface expression of receptors and through binding assays to subcellular fractions enriched for specific granules, some plasma membrane receptors have been demonstrated to exist in a preformed pool that may be on the specific granule membrane. These receptors include the chemotactic peptide receptor, often called the FMLP receptor for its identification by the binding of this synthetic peptide, and a complement receptor called CR3 that binds the C3b hydrolysis product called C3bi. 19,*O,42.43Activated neutrophils *have an asymmetric distribution of FMLP receptors on their cell surfaceti that could in part be caused by the discharging of specific granules at the leading edge of the cell during chemotaxis.3 This asymmetry, along with receptor turnover, may be cru-
666
J. ALLERGY CLIN. IMMUNOL. MAY 1996
Falloon and Gallin
TABLE II. Models
of neutrophil Neutrophil
specific
granule
deficiency Congenital-specific granule deficiency
function
Aggregation Disaggregation C3bi receptors Increased expression Chemotaxis FMLP receptors Increased expression Chemoattractant generation from serum by secretedproducts Phagocytosis Bactericidal activity NL
= normal;
NT
= not tested;
& = decreased.
Reproduced
from reference
cial for optimal sensing of a gradient of chemoattractant. The specific granule membrane thus may play a central role in the responsiveness of the human neutrophil to inflammatory stimuli. Azurophilic granule proteins have predominantly digestive and bactericidal functions. Elastase along with specific granule collagenase may permit penetration through tissues,45 and other enzymes serve to degrade phagocytosed material. Lysozyme and cationic proteins have bactericidal activity.46*47 MPO, a heme protein, has cytotoxic and antimicrobial activity and can inactivate humoral agents.48 It forms a part of the MPO-halide-hydrogen peroxide bactericidal system whereby the MPO-catalyzed oxidation of a halide by hydrogen peroxide results in the production of a number of potent oxidants. These provide the ability to injure or kill via oxidation or halogenation of important mo1ecules.48 MPO also appears to contribute to regulation of the respiratory burst enzyme activity.49 In addition, it and other azurophilic granule proteins may modulate the inflammatory response by inactivating or digesting chemoattractants and lysosomal enzymes.50 For example, an azurophilic granule product is able to destroy C5a activity,27 and the MPO system has been demonstrated to oxidize chemoattractant peptides and neutrophil secretory products.5’ The ability of the cell to digest chemoattractants may be important in chemotaxis.52 Azurophilic granules, then, serve lysosomal functions and are important in target cell death and in modulation of the inflammatory response. A novel role of neutrophil granules in the regulation of intracellular calcium has recently been proposed.53 Stimulation of the neutrophil by chemoattract&t stimuli results in rapid rises in cytosolic free calcium believed to represent release from nonmitochondrial in-
Neutrophil
NL Absent NL
NL
Absent NL Absent 1 NL Absent Absent NL 3 with
cytoplasts
J NL
Absent NT NL
modifications.
tracellular stores.54 These calcium rises are important in regulating functional responses to stimuli. Since both specific and azurophilic granule-enriched subcellular fractions contain a magnesium-adenosine triphosphate-dependent calcium pump promoting calcium uptake, these granules may be an intracellular calcium storage site, thus linking granules with cell activation in yet another way. MODELS OF GRANULE Cytoplasts
DEFICIENCY
Our understanding of the role of neutrophil granules in the inflammatory response has been strengthened by experimental models of granule-deficient cells and by patients whose cells are lacking in granule contents (Table II). The model cell, the enucleated cytoplast, is a plasma membrane-bound bag of cytoplasm pinched off from a neutrophil and contains less than 10% of the original granules of the ce11.55,56 It has various functional abnormalities including poor chemotaxis, slower bactericidal activity, abnormal shape change in response to chemoattractants, and abnormal disaggregation. The cytoplast preparation, unlike the neutrophil, is unable to increase the plasma membrane expression of FMLP or C3bi receptors in response to degranulating stimuli. *O*56 Some of these abnormalities may relate to the absence of specific granule membrane to provide for the membrane turnover and receptor replenishment needed for cellular responses such as chemotaxis, shape change, and disaggregation. Congenital
specific granule
deficiency
The model of specific granule deficiency, patients with recurrent bacterial infections (Table III) and a severe deficiency in the specific granule markers vitamin B,2 binding protein and lactoferrin, has been
VOLUME 77 NUMBER 5
TABLE
Neutrophil
III. Clinical
Granule
type
Specific granules
presentations
of patients
B, Baggiolini
Screening
Early onset recurrent infections with catalase-positive bacteria and fungi, granulomatous lesions Delayed separation of umbilical stump, leukocytosis, recurrent bacteria1 infections, periodontal disease Bacterial infections, infectious mortality (relates to extent of burn) Generally none; some patients with other host defense defects such as diabetes have bacterial or fungal infection (particularly Candida albicans) Recurrent bacterial infections, partial oculocutaneous albinism, progression to peripheral neuropathy, accelerated phase (organomegaly, coaguloplymphoma-like athy, neutropenia, syndrome)
Chediak-Higashi syndrome
described.57 The cells of these patients are morphologically abnonnal with misshapen nuclei. No granules are visible on Wright’s stain, but myeloperoxidase staining reveals azurophilic granules. The presence of empty specific granules has been suggested by electron microscopy studies5* Other abnormalities such as a reduction in the plasma membrane marker alkaline phosphatase, altered azurophilic granule complex carbohydrate staining by electron microscopy, and abnormal granule sedimentation in subcellular fractionation studies57-59are present. In vitro, these neutrophils demonstrate abnormal chemotaxis and bacterial killing. Like cytoplasts, these cells do not increase receptors after stimulation.“. 6o In vivo, one patient has demonstrated deficient neutrophil and monocyte recruitment to skin windows, although monocyte chemotaxis was not deficient in vitro. In addition, secretory products from the cells of this patient were unable to generate chemoattractants from serum.@’ Disaggregation was also abnormal.@’ These neutrophils were demonstrated to be lacking in gelatinase activity, suggesting absence of tertiary granules as well, * Although the underlying defect in this disorder is complex and not well understood, the abnormalities documented point to a role of the specific
*Dewald
manifestations
CGD (cytochrome deficient)
b
M, Gallin
J: Unpublished
observation.
in health and disease
657
abnormalities
Recurrent severe bacterial infections
MPG deficiency
and
granule
Specific granule deficiency
Thermal injury
Azurophilic specific granules
neutrophil
Clinical
Disorder
C3bi receptor (CR3) deficiency
Azurophilic granules
with
granules
test
Peripheral blood smear (misshapen nucleus, no granules on Wright’s stain) and enzyme contents of neutrophils Nitroblue tetrazoleum reduction test
Deficient binding of monoclonal antibodies against the C3bi receptor to neutrophils
MPG stain of neutrophils
Peripheral blood smear (characteristic large lysosomal granules in white cells)
granule and possibly the tertiary granule in chemotaxis, receptor upregulation, chemoattractant generation, disaggregation, and control of bacterial infections. Specific granule thermal injury
deficiency
secondary
to
Another model for specific granule-deficient cells is that of thermal injury. In bum patients a decrease in neutrophil lysozyme with increase in blood lactoferrin and lysozyme has been reported with neutrophil P-glucuronidase being normal, suggesting preferential specific granule degranulation.6’.62 Davis et a1.62correlated this decrease in neutrophil lysozyme with abnormalities of chemotaxis in bum patients. These abnormalities have, in turn, been correlated with increased mortality.63 After a bum, preferential specific granule exocytosis may interfere with chemotaxis, perhaps by lack of membrane and preformed receptors for tumover and, along with defects in oxidative metabolism,bl may contribute to compromised host defenses. Neonatal
neutrophils
Neonatal neutrophils comprise another system with decreased neutrophil lactoferrin but normal neutrophil MPO content.65 This suggests a relative specific granule deficiency in comparison with adult cells. In addition, neonatal neutrophils manifest a host of com-
668
Falloon and Gallin
plicated abnormalities when they are compared to adult cells. These include abnormalities of chemotaxis,@ adherence, 66. 67 disaggregation,68 membrane potential,69 phagocytosis and microbicidal activity,” and oxidative metabolism.@ In particular, hydroxyl radical production appears diminished, which has been attributed to lactoferrin deficiency.65 The specific granule deficiency in these cells may be in part responsible for the increased susceptibility of neonates to infection. C3bi receptor
(CR3) deficiency
Other patients have been described who appear to be deficient in a group of leukocyte membrane glycoproteins including the C3bi receptor,” a receptor normally found in neutrophil plasma membrane and specific granule-enriched cell fractions. Patients with this abnormality present with delayed separation of the umbilical stump, recurrent and severe bacterial and fungal infections, impaired wound healing, diminished pus formation, severe periodontal disease, and persistent leukocytosis. The severity of the clinical manifestations appears to be related to the degree of deficiency of the membrane glycoproteins.” In vitro, slightly differing abnormalities have been described, generally involving abnormalities of neutrophil aggregation, adherence, spreading, chemotaxis, and defective phagocytosis of some opsonized particles. Bactericidal activity is generally normal.” This defect involves deficiencies in a family of multimeric highmolecular-weight glycoprotein surface antigens with a common beta subunit and different alpha chains. The C3bi receptor is one of these molecules.74 Work in which the in vitro abnormalities of this disease are mimicked by adding a monoclonal antibody against the antigen to normal cells75,76suggests that the functional abnormalities are related to the missing glycoprotein. Studies of these patients suggest that the C3bi receptor functions in neutrophil adherence, aggregation, and chemotaxis and that its absence from the granules and plasma membrane of the neutrophil is responsible for severe human disease. CGD
Another patient group with abnormal granules is a subset of patients with CGD. CGD is a group of related disorders characterized by recurrent infections and granulomatous lesions.77-79The infections are due to an inability of the neutrophil to kill most bacteria and fungi because of an abnormality in oxidative metabolism with inability to produce toxic oxygen metabolites, including hydrogen peroxide. This abnormality is believed to result from various genetically absent or defective constituents of the membrane-associated
J. ALLERGY CLIN. IMMUNOL. MAY 1996
oxidase that normally triggers the respiratory burst or from defective activation of this oxidase. In one subset with X-linked inheritance, the patients’ specific granule-enriched fractions appear deficient in cytochrome b, ‘O,” and another subset may be deficient in a flavoprotein. 3s In a subset of patients with autosomal recessive inheritance, there is an absence of stimulusinduced phosphorylation of a protein distributed in plasma membrane and granule fractions that may interact with cytochrome b.82 In this way, abnormal specific granules may lead to an inability to mount a normal respiratory burst so that one of the important antibacterial mechanisms of the neutrophil is crippled. Abnormalities of azurophilic Myeloperoxidase deficiency
granules:
No patients lacking azurophilic granules have been described, but the inherited or acquired absence of azurophilic granule MPO is a relatively common neutrophil abnormality occurring in about 1 in 2000 persons.83 It is interesting that the lack of this protein, believed to play a key role in bacterial killing by normal neutrophils, does not usually result in illness, although some patients have been described with Candida albicans infections. The patients with infections generally have another illness such as diabetes mellitus contributing to compromised host defenses. In vitro, MPO-deficient cells undergo an augmented respiratory burst,49 and although bacterial killing is delayed, it is eventually normal, perhaps the result of the compensatory role of unusually vigorous production of toxic oxygen metabolites. The benign nature of MPO deficiency points out the complexity of neutrophil bacterial killing and the importance of killing mechanisms other than the MPO-halide-hydrogen peroxide system such as oxidative killing by other products of oxygen and nonoxidative killing by exposure of bacteria to the interior of the phagolysosome and to granule contents.&, 47,84,” The Chbdiak-Higashi
syndrome
The Chediak-Higashi syndrome is an inherited abnormality characterized by morphologically abnormal granules in neutrophils and other granule-containing cells.86 The giant granules of the cells are believed to be products of abnormal granule fusion,*’ perhaps related to abnormal microtubule metabolism88~89 or to altered membrane properties.” The neutrophils are functionally abnormal with decreased chemotaxis, abnormal lysosomal degranulation, and delayed and deficient bactericidal capacity.‘lT 92The patients have increased susceptibility to bacterial infection as well as variable complicated systemic manifestations, including neutropenia, blood coagulation abnormalities,
VOLUME 77 NUMBER 5
Neutrophil
neuropathy, partial albinism, adenopathy, hepatosplenomegaly, and an increased incidence of lymphoproliferative disorders.86 Since the underlying defect in this syndrome is unknown, the role of the abnormal granules in the neutrophil defect is unclear. Granule deficiency collection
secondary
to leukocyte
Granule exocytosis may be involved in the diminished functional capacity of neutrophils intended for transfusion that were collected by filtration leukapheresis.“’ In this method cells are collected by reversible adhesion to nylon fibers. Neutrophils become partially degranulated during this process with loss of specific granules being greater than that of azurophilic granules. These cells are functionally abnormal with abnormal posttransfusion kinetics, chemotaxis, phagocytosis, and bactericidal activity. Because of these defects, as well as increased recipient side effects with filtration leukapheresis cells, alternative collection methods are now prefemed.94 POSSIBLE ROLE OF NEUTROPHIL GRANULES IN SYSTEMIC DISEASE There is growing evidence that neutrophils and their granules contribute to a variety of systemic diseases through local release of damaging granule enzymes or toxic oxygen products. In particular, the neutrophil is believed important in the pathogenesis of both acute and chronic lung disease. In an acute lung disease, the adult respiratory distress syndrome, it is believed that activation of neutrophils by blood stream complement results in neutrophil trapping in the lung. The local release of toxic neutrophil products such as oxygen metabolites, proteolytic enzymes, and arachidonic acid products results in damaged alveolar endothelium and plasma exudation. 95-97In some animal models lung damage is dependent on production of reactive oxidants and can be diminished by inactivators of these oxidants, particularly by scavengers of hydrogen peroxide or hydroxyl radical.98, 99 In a chronic lung disease, emphysema, it is believed that lung irritants induce alveolar macrophages to release neutrophil chemoattractants. Neutrophils arriving in the lung degranulate, releasing enzymes such as elastase that contribute to the disruption of alveolar elastic fibers that results in emphysema. ‘O”Other systemic illnesses potentially related to neutrophil granule products include rheumatoid and crystalline arthritis, serum sickness, immune vasculitis and glomerulonephritis, myocardial infarction, and even malignancies at sites of chronic inflammation. ‘01-‘04The neutrophil and its granule components are thus potentially involved in many human disease states involving inflammation.
granules
in health
and
disease
659
SUMMARY The granules of the neutrophil, in addition to contributing to its distinctive morphologic appearance, are critical to its unique functions. Specific granules appear necessary for neutrophil recruitment to sites of inflammation, for upregulation of receptors important in the control of chemotaxis and the respiratory burst, for disaggregation, for bactericidal activity, and for chemoattractant generation. The azurophilic granules supply enzymes for digestive and bactericidal functions and supply MPO to the MPO-halide-hydrogen peroxide bactericidal system. Azurophilic granule contents also regulate inflammation by degrading inflammatory products. Both granules may play a role in intracellular calcium regulation. In addition to these activities that protect the host from infection, granules also, under certain circumstances, contribute to disease processes. For these reasons, greater knowledge about granule contents, control of degranulation, inactivation of toxic granule contents and products, and the role of granules in neutrophil membrane events and function has widespread implications for treatment of patients with neutrophil dysfunction syndromes and patients with multiple other systemic diseases.
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31. Broxmeyer HE, DeSousa M, Smithyman A, Ralph P, Hamilton J, Kurland JI, Bognacki J: Specificity and modulation of the action of lactoferrin, a negative feedback regulator of myelopoiesis. Blood 55:324, 1980 32. Chipman DM, Sharon N: Mechanism of lysozyme action. Science 165:454, 1969 33. Strominger JL, Ghuysen J-M: Mechanisms of enzymatic bacteriolysis: cell walls of bacteria are solubilized by action of either specific carbohydrases or specific peptidases. Science 156:213, 1967 34. Gordon LI, Douglas SD, Kay NE, Yamada 0, Osserman EF, Jacob HS: Modulation of neutrophil function by lysozyme. Potential negative feedback system of inflammation. J Clin Invest 64:226, 1979 35. Gabig TG, Lefker BA: Deficient tlavoprotein component of the NADPH-dependent O;-generating oxidase in the neutrophils from three male patients with chronic granulomatous disease. J Clin Invest 73:701, 1984 36. Higson IX, Durbin L, Pavlotsky N, Tauber A: Studies of cytochrome b-245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase. J Immunol 135:519, 1985 37. Wright DG, Gallin JI: A functional differentiation of human neutrophil granules: generation of C5a by a specific (secondary) granule product and inactivation of C5a’by azurophil (primary) granule products. J Immunol 119:1068, 1977 38. Ward PA: Chemotaxis of mononuclear cells. J Exp Med 128:1201, 1968 39. Harlan JM: Leukocyte-endothelial interactions. Blood 65: 513, 1985 40. Hoffstein ST, Friedman RS, Weissmann G: Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils. J Cell Biol 951234, 1982 41. Namere I, Kupfer A, Singer SJ: Reorientation of the golgi apparatus and the microtubule-organizing center inside macrophages subjected to a chemotactic gradient. Cell Moti15: 17, 1985 42. Fletcher MP, Seligmann BE, Gallin JI: Correlation of human neutrophil secretion, chemoattractant receptor mobilization, and enhanced functional capacity. J Immunol 128:941, 1982 -43. Berger M, O’Shea J, Cross AS, Folks TM, Chused TM, Brown EJ, Frank MM: Human neutrophils increase expression of C3bi as well as C3b receptors upon activation. J Clin Invest 74:1566, 1984 44. Sullivan SJ, Daukas G, Zigmond SH: Asymmetric distribution of the chemotactic peptide receptor on polymorphonuclear leukocytes. J Cell Biol 99:1461, 1984 45. Weiss SJ, Regiani S: Neutrophils degrade subendothelial matrices in the presence of alpha,-proteinase inhibitor: cooperative use of lysosomal proteinases and oxygen metabolites. J Clin Invest 73:1297, 1984 46. Spitznagel JK, Shafer WM: Neutrophil killing of bacteria by oxygen-independent mechanisms: a historical summary. Rev Infect Dis 7:398, 1985 47. Spitznagel JK: Nonoxidative antimicrobial reactions of leukocytes. Contemp Top Immunobiol 14:221, 1984 48. Clark RA: Extracellular effects of the myeloperoxidase-hydrogen peroxide-halide system. In Weissmann G, editor: Advances in inflammation research. New York, 1983, Raven Press, vol 5, p 107 49. Nauseef WM, Metcalf JA, Root RK: Role of myeloperoxidase in the respiratory burst of human neutrophils. Blood 61:483, 1983 50. Clark RA, Szot S: Chemotactic factor inactivation by stim-
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57. 58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
mated human neutrophils is mediated by myeloperoxidasecatalyzed methionine oxidation. J Immunol 128:1507, 1982 Clark RA. Borregaard N: Neutrophils autoinactivate secretory products by myeloperoxidase-catalyzed oxidation. Blood 65.375, 1985 Nunoi H, Endo F, Chikazawa S, Matsuda I: Regulation of receptors and digestive activity toward synthesized formyl chemotaaic peptide in human polymorphonuclear leukocytes. Blood 66 106, 1985 Klempner MS: An adenosine triphosphate-dependent calcium uptake pump in human neutrophil lysosomes. J Clin Invest 76:303, 1985 Prentki M, Wollheim CB, Lew PD: Ca*+ homeostasis in permeabilized human neutrophils: characterization of calcium sequestering pools and the action of inositol I ,4,5-trisphosphate. J Biol Chem 259: 13777, 1984 Roes D, Voetman AA, Meerhof LJ: Functional activity of enucleated human polymorphonuclear leukocytes. J Cell Biol 97:368, 1983 Gallin JI, Metcalf JA, Roos D, Seligmann B, Friedman MM: Organelledepleted human neutrophil cytoplasts used to study fmet-leu-phe receptor modulation and cell function. J Immunol 133:415, 1984 Gallin JI: Neutrophil specific granule deficiency. Annu Rev Med 36:263, 1985 Parmley RT, Tzeng DY, Baehner RL, Boxer LA: Abnormal distribution of complex carbohydrates in neutrophils of a patient wit1 lactofenin deficiency. Blood 62:538, 1983 Borregaard N, Boxer LA. Smolen JE, Tauber AI: Anomalous neutrophil granule distribution in a patient with lactoferrin deficiency: pertinence to the respiratory burst. Am J Hematol 18255, 1985 Gallin JI, Fletcher MP, Seligmann BE, Hoffstein S, Cehrs K. Mounessa N: Human neutrophil specific granule deficiency: a model to assess the role of neutrophil specific granules in tile evolution of the inflammatory response. Blood 59:1317, 1982 Wolach Et, Coates TD, Hugli TE, Baehner RL, Boxer LA: Plasma lactoferrin reflects granulocyte activation via complement in bum patients. J Lab Clin Med 103:284, 1984 Davis JM, Dineen P, Gallin JI: Neutrophil degranulation and abnormal chemotaxis after thermal injury. J Immunol 124: 1467, 19130 Warden GD, Mason AD, Pruitt BA: Evaluation of leukocyte chemotaxis in vitro in thermally injured patients. J Clin Invest 54:1001, 1974 Duque RE, Phan SH, Hudson JL, Till GO, Ward PA: Functional defects in phagocytic cells following thermal injury: application of flow cytometric analysis. Am J Path01 118: 116, 1985 Ambruso DR, Bentwood B, Henson PM, Johnston RB Jr: Oxidative metabolism of cord blood neutrophils: relationship to content and degranulation of cytoplasmic granules. Pediatr Res 18:1148, 1984 Andersorl DC, Hughes BJ, Smith CW: Abnormal mobility of neonatal polymotphonuclear leukocytes. J Clin Invest 68:863, 1981 Harris MC, Levitt I, Douglas SD, Gerdes JS, Polin RA: Effect of fibronectin on adherence of neutrophils from newborn infants. J Clin Microbial 21:243, 1985 Oison TA, Ruymann FB, Cook BA, Burgess DP, Henson SA, Thomas PJ: Newborn polymorphonuclear leukocyte aggregation: a study of physical properties and ultrastructure using chemotactic peptides. Pediatr Res 17:993, 1983 Sacchi F, Hill HR: Defective membrane potential changes in
Neutrophil
granules
in health
and
disease
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neutrophils from human neonates. J Exp Med 160: 1247,1984 70. Miller ME: Phagocyte function in the neonate: selected aspects. Pediatrics 64:709, 1979 71. Gallin JI: Leukocyte adherence-related glycoproteins LFA-1, Mol, and p150,95: a new group of monoclonal antibodies, a new disease, and possibly an opportunity to understand the molecular basis of leukocyte adherence. J Infect Dis 152:661, 1985 72. Anderson DC, Schmalsteig FC, Finegold MJ, Hughes BJ, Rothlein R, Miller LJ, Kohl S, Tosi MF, Jacobs RL, Waldrop TC, Goldman AS, Shearer WT, Springer TA: The severe and moderate phenotypes of heritable Mac-l, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. J Infect Dis 152:668, 1985 73. Buescher ES, Gaither T, Nath J, Gallin JI: Abnormal adherence-related functions of neutrophils, monocytes, and Epstein-Barr virus-transformed B cells in a patient with C3bi receptor deficiency. Blood 65:1382, 1985 74. Sanchez-Madrid F, Nagy JA, Robbins E, Simon P, Springer TA: A human leukocyte differentiation antigen family with distinct o-subunits and a common B-subunit: the lymphocyte function-associated antigen (LFA-I), the C3bi complement receptor (OKMliMac-l), and the ~150.95 molecule. J Exp Med 158:1785, 1983 75. Harlan JM, Killen PD, Seneca1 FM, Schwartz BR, Yee EK, Taylor RF, Beatty PG, Price TH, Ochs HD: The role of the neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro. Blood 66: 167, 1985 76. Schwartz B, Ochs HD, Beatty PG, Harlan JM: A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation. Blood 65: 1553, 1985 77. Gallin JI, Buescher ES, Seligmann BE, Nath J, Gaither T, Katz P: Recent advances in chronic granulomatous disease. Ann Intern Med 99:657, 1983 78. Tauber Al, Borregaard N, Simons E, Wright J: Chronic granulomatous disease: a syndrome of phagocyte oxidase deficiencies. Medicine 62:286, 1983 79. Segal AW: Variations on the theme of chronic granulomatous disease. Lancet 1:1378, 1985 80. Ohno Y, Buescher ES, Roberts R, Metcalf JA, Gallin Jl: A reevaluation of cytochrome b and flavin adenine dinucleotide in neutrophils from patients with chronic granulomatous disease and description of a family with probable autosomal recessive inheritance of cytochrome b deficiency. Blood (in press) 81. Segal AW, Cross AR, Garcia RC, Borregaard N, Valerius NH, Soothill JF, Jones OTG: Absence of cytochrome b-245 in chronic granulomatous disease: a multicenter European evaluation of its incidence and relevance. N Engl J Med 308:245, 1983 82. Segal A, Heyworth PG, Cockcraft S, Barrowman MM: Stimulated neutrophils from patients with autosomal recessive chronic granulomatous disease fail to phosphorylate a Mr44,000 protein. Nature 316:547, 1985 83. Parry MF, Root RK, Metcalf JA, Delaney KK, Kaplow LS, Richar WJ: Myeloperoxidase deficiency: prevalence and clinical significance. Ann Intern Med 95:293, 1981 84. Root RK, Cohen MS: The microbicidal mechanisms of human neutrophils and eosinophils. Rev Infect Dis 3:565, 1981 85. Elsbach P, Weiss J: A reevaluation of the roles of the O,-dependent and O,-independent microbicidal systems of phagocytes. Rev Infect Dis 5:843, 1983 86. Blume RS, Wolff S: The Chediak-Higashi syndrome: studies in four patients and a review of the literature. Medicine 51:247. 1972
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87. White JG, Clawson CC: The Chediak-Higashi syndrome: the nature of the giant neutrophil granules and their interactions with cytoplasm and foreign particulates. Am J Pathol98: 15 1, 1980 88. Nath I, Flavin M, Gallin II: Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome. J Cell Biol 95519, 1982 89. Oliver JM: Cell biology of leukocyte abnormalities-membrane and cytoskeletal function in normal and defective cells. A review. Am J Path01 93:221, 1978 90. Haak RA, Ingraham LM, Baehner RI., Boxer LA: Membrane fluidity in human and mouse Chediak-Higashi leukocytes. J Clin Invest 64:138, 1979 91. Wolff SM, Dale DC, Clark RA, Root RK, Kimball HR: The Chediak-Higashi syndrome: studies of host defenses. Ann Intern Med 76:293, 1972 92. Clawson CC, Repine JE, White JG: The Chediak-Higashi syndrome: quantitation of a deficiency in maximal bactericidal capacity. Am J Pathol 94539, 1979 93. Wright DG, Klock JC: Functional changes in neutrophils collected by filtration leukapheresis and their relationship to cellular events that occur during adherence of neutrophils to nylon fibers. Exp Hematol 7:11, 1979 94. Schiffer CA: Current status of granulocyte transfusion therapy. In Remington JS, Swartz MN, editors: Current clinical topics in infectious diseases. New York, 1984, McGraw-Hill Book Co, vol 5, p 189
95.
96.
97. 98.
99.
100. 101.
102. 103. 104.
Jacob HS, Craddock PR, Hammerschmidt DE, Moldow CF: Complement-induced granulocyte aggregation: an unsuspected mechanism of disease. N Engl J Med 302:789, 1980 Rinaldo JE, Rogers RM: Adult respiratory-distress syndrome. Changing concepts of lung injury and repair. N Engl J Med 306900, 1982 Tate RM, Repine JE: Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 128:552, 1983 Fox RB: Prevention of granulocyte-mediated oxidant lung injury in rats by hydroxyl radical scavenger, dimethylthiourea. J Clin Invest 74:1456, 1984 Till GO, Hatherill JR, Tourtellotte WW, Lutz MJ, Ward PA: Lipid peroxidation and acute lung injury after thermal trauma to skin: evidence of a role for hydroxyl radical. Am J Pathol 119:376, 1985 Janoff A: Biochemical links between cigarette smoking and emphysema. J Appl Physiol55:285, 1983 Weitzman SA, Weigburt AB, Clark EP, Stossel TP: Phagocytes as carcinogens: malignant transformation produced by human neutrophils. Science 227: 123 1, 1985 Mullane K, Bednar M: Contribution of leukocytes to ischemia-induced myocardial damage. Clin Res 32:475A, 1984 Cochrane CG: Role of granulocytes in immune complexinduced tissue injuries. Inflammation 2:319, 1977 Snyderman R: Mechanisms of inflammation and tissue destruction in the rheumatic diseases. In Wyngaarden JB, Smith LH, editors: Cecil textbook of medicine. Philadelphia, 1982, WB Saunders Co, p 1832
granules
in health
and disease
Falloon, M.D., and John 1. Gallin, M.D. Bethesda, Md.
Once staining and microscopy techniques made the granules of the neutrophil visible, the pursuit of their function challienged those interested in the neutrophil and its role j.n health and in myriad disease states. Both a secretory function of the granules postulated by Paul Ehrlich’ in the late 1800s whereby neutrophils modify their environment and an intracellular digestive or lysosoimal function postulated by Ehrlich’s contemporary Elie Metchnikoff’ appear to be of importance. Current understanding of neutrophil granules has separated these functions according to granule type with the specific granules (also called secondary granules) serving the environmental modification or external secretory function and the azurophilic granules (also called primary granules) acting as lysosomes by fusing with phagosomes to form phagolysosomes with a predominantly intracellular site of action.3 Although this is, of course, a conceptual oversimplification, since specific granule contents are found in phagolysosornes and azurophilic granules are clearly released into the extracellular milieu through degranulation and cell death,4 the concept of the separation of functions lhas proved useful. GRANULE CONTENTS AND FUNCTIONS The two main neutrophil granules, specific and azurophilic, are separated on the basis of differences in content, morphology, density, and appearance at different stages of neutrophil maturation (Fig. 1). During myelopoiesis, the first granules become visible at about the promyelocyte stage and stain azurophilic in the bone marrow; these are the primary or azurophilic granules. Later on during maturation, synthesis of these granules ceases, and they are distributed to daughter cells during cell division, thus decreasing From the Bacterial Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda. Received for publication Nov. 21, 1985. Accepted for pu,blication Jan. 9, 1986. Reprint requests: Judith Falloon, M.D., Bacterial Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bldg. 10, Rm. llN112, Bethesda, MD 20892.
~1 FMLP:
CGD:
N-formylmethionylleucylphenylanine Chronic granulomatous
disease
the azurophilic granule content of the cells.” The granules produced second during neutrophil maturation, called secondary or specific granules, then come to predominate, comprising about two thirds of the granules of the mature ce11.5The differences between these granules have permitted the development of subcellular fractionation techniques leading to the separation of disrupted cells into granule-enriched fractions that have been used to probe further into granule content and function. The azurophilic granules sediment heterogeneously on sucrose gradient9 and contain microbicidal enzymes, proteases, acid hydrolases, and bactericidal cationic proteins (Table I).6-” These contents correlate with the postulated lysosomal function of azurophilic granules. The specific granule, a less dense granule that stains heavily for glycoprotein, contains lysozyme, collagenase, lactoferrin, vitamin B,,-binding protein, and possibly histaminase and sialidase.6-‘3 Recently, the specific granule-enriched fraction of neutrophils has been demonstrated to contain a unique b-type cytochrome’4. ” and possibly a flavoprotein’6 believed to be of importance in the formation of the complete NADPH oxidase enzyme responsible for the respiratory burst that produces the toxic oxygen products important in neutrophil function, including killing.17, ‘* Organelles that contain a preformed intracellular pool of receptors important in the response of the phagocyte to chemoattractants and interaction with serum complement proteins also cosediment with specific granules in gradients.“, ‘O A third granule type, a gelatinase containing tertiary granule, has been described as a separate entity with a density slightly less than that of specific granules on sucrose gradients.” Further knowledge of this granule is scanty; it most likely sediments with the specific granule-enriched fraction in most gradients and is potentially the site 653
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J. ALLERGY CLIN. IMMUNOL. MAY 1986
FIG. 1. A human neutrophil stained for peroxidase. Five nuclear lobes (1 to 5/ are visible in the cytoplasm. Small, unstained specific granules (SJ are present throughout the cytoplasm. The larger, darkly stained structures are azurophilic granules that stain positively for peroxidase. irregularly shaped channels (at arrow] represent invaginations of the cell surface and are continuous with the external environment. Fixation in aldehydes and osmium. Scale bar = 0.5 pm. (Micrograph courtesy of Dr. Marc Friedman, Georgetown University.)
of certain contents currently attributed to specific granules. Neutrophil degranulation occurs in the midst of many other events during the complex response of the cell to inflammatory stimuli. As a neutrophil is exposed to chemoattractants such as N-formylated peptides released by bacteria,22 complement proteins, or leukotriene products, it develops a ruffled membrane and changes from a round to a polarized cell with anterior broad lammellipodium and posterior-narrow uropod. Cytoskeletal microtubules and microfilaments reorganize, and there is anterior movement of granules while the nucleus moves toward the rear of the ce11.23Cells aggregate and adhere to endothelial surfaces. There is subsequent disaggregation, and cells become increasingly motile (chemokinetic) with directed movement (chemotaxis) toward the more concentrated source of chemoattractant. These events are accompanied by ion fluxes, changes in membrane potential and surface charge, increased glycolysis, alterations in phospholipid and arachidonic acid metabolism, protein phosphorylation, and production of toxic oxygen radicals.24. 25 In this way these cells are directed to tissue sites where they can alter and destroy bacteria and other target cells. At sites of inflammation, both phagocytosis and internal digestion of microorganisms or other particles occur, and the finely tuned exocytosis of granules, prompted also by chemoattractants, occurs. In order to understand further this eomplex neutrophil response, the role
of granule contents in inflammatory and plasma membrane events has been the focus of recent investigations . In keeping with their different functions, the extracellular release of specific and azurophilic granules is under separate control. In vitro, for example, chemoattractants and other substances can be used to cause specific granule secretion under conditions whereby azurophilic granule enzymes will not be released.3. 4. lo. 26 Migration through filters in response to chemotactic stimuli results in preferential specific granule discharge,27 as does migration through tissues in vivo, as demonstrated in exudate cells harvested from human dermal suction blisters. The strongest stimuli for azurophilic granule degranulation also stimulate concomitant specific granule exocytosis.3 Specific granules, then, and probably the tertiary granules2’ appear readily released during cell stimulation so that their contents are available to modify the response of the cell to stimuli as well as to effect function. Investigations into the functions of granule contents have proceeded through isolation and characterization of granule proteins and through observations made on the neutrophils of patients with abnormal granules. In the specific granule, lactoferrin is an iron-chelating glycoprotein that probably has an antimicrobial function via iron chelation or by direct bactericidal effect2* and may function in neutrophil adhesiveness,*’ hydroxyl radical formation,30 and in control of myelo-
VOLUME 77 NUMBER !j
TABLE
Neutrophil
I. Neutrophil Class
granule
0f constituent
Neutral serine proteases
Metalloproteinases Acid hydrolases
Other
references
in health
and
disease
655
contents*
Microbicidal enzymes
*From
granules
Azurophilic
granules
Myeloperoxidase Lysozyme Elastase Cathepsin G Proteinase3
Specific
granules
Lysozyme
Collagenaset
N-acetyl-P-glucosaminidase Cathepsin B Cathepsin D P-Glucuronidase P-Glycerophosphatase a-Mannosidase Bactericidal cationic proteins$
Lactoferrin Vitamin B,? binding proteins Cytochrome b Histaminase FMLP receptors5 C3bi receptors (CR3)S
3 and 10 with modifications.
TReleased asa latentenzyme. $Note added in proof: defensins, antimicrobial peptides in azurophilic granules, have recently Szklorek D, et al: J Clin Invest 76:1427, 1985). 9Present in a subcellular fraction cosedimenting with specific granules in sucrose gradients.
poiesis by inhibition of the production of granulocytemacrophage colony-stimulating factor.” Lysozyme,
present in both granules, digests glycopeptide debris in the cell,32 and hydrolyzes cell wall components of some bacteria.33 It may also modulate inflammation by suppressi:ng neutrophil chemotaxis and oxidative metabolism.‘4 The vitamin B,, binding protein of the specific granule remains enigmatic; its function is unknown. Cytochrome b, probably a specific granule membrane component, is believed to be a constituent of the membrane-associated electron transport chain comprising the NADPH oxidase responsible for the enhanced oxygen consumption and hydrogen peroxide generation of the respiratory burst of the neutrophil. I73I8 Unstimulated cells contain an inactive oxidase, which may consist of several subunits requiring assembly and then activation for activity. One subunit appears to be a cytochrome b, and one appears to be a flavoprotein, both believed to be partially located in the specific granule.14-16.35 Fusion of specific granule membrane containing cytochrome b w:ith the plasma membrane may be a key step in the formation of a complete oxidase enzyme,36 since the complete enzyme complex appears to be plasma membrane associated. Other specific granule functions include amplification of the inflammatory response by activation of the complement cascade, generation of the chemoattractant C5a,37 and release
been described
(Gang
T, Selsted
ME,
of a monocyte chemoattractant.3, ‘* Specific granule contents also promote neutrophil adherence to endothelium. It has recently been postulated that the specific granule membrane contains receptors important in neutrophi1 function and that fusion of granule membrane
with plasma membrane during exocytosis may provide membrane and receptors to the cell surface.3, 4o Since membrane turnover may be important in chemotaxis4’ and new membrane synthesis appears unnecessary, the specific granule membrane pool may be a requirement for membrane replacement during chemotaxis. By correlating specific granule degranulation with increased surface expression of receptors and through binding assays to subcellular fractions enriched for specific granules, some plasma membrane receptors have been demonstrated to exist in a preformed pool that may be on the specific granule membrane. These receptors include the chemotactic peptide receptor, often called the FMLP receptor for its identification by the binding of this synthetic peptide, and a complement receptor called CR3 that binds the C3b hydrolysis product called C3bi. 19,*O,42.43Activated neutrophils *have an asymmetric distribution of FMLP receptors on their cell surfaceti that could in part be caused by the discharging of specific granules at the leading edge of the cell during chemotaxis.3 This asymmetry, along with receptor turnover, may be cru-
666
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Falloon and Gallin
TABLE II. Models
of neutrophil Neutrophil
specific
granule
deficiency Congenital-specific granule deficiency
function
Aggregation Disaggregation C3bi receptors Increased expression Chemotaxis FMLP receptors Increased expression Chemoattractant generation from serum by secretedproducts Phagocytosis Bactericidal activity NL
= normal;
NT
= not tested;
& = decreased.
Reproduced
from reference
cial for optimal sensing of a gradient of chemoattractant. The specific granule membrane thus may play a central role in the responsiveness of the human neutrophil to inflammatory stimuli. Azurophilic granule proteins have predominantly digestive and bactericidal functions. Elastase along with specific granule collagenase may permit penetration through tissues,45 and other enzymes serve to degrade phagocytosed material. Lysozyme and cationic proteins have bactericidal activity.46*47 MPO, a heme protein, has cytotoxic and antimicrobial activity and can inactivate humoral agents.48 It forms a part of the MPO-halide-hydrogen peroxide bactericidal system whereby the MPO-catalyzed oxidation of a halide by hydrogen peroxide results in the production of a number of potent oxidants. These provide the ability to injure or kill via oxidation or halogenation of important mo1ecules.48 MPO also appears to contribute to regulation of the respiratory burst enzyme activity.49 In addition, it and other azurophilic granule proteins may modulate the inflammatory response by inactivating or digesting chemoattractants and lysosomal enzymes.50 For example, an azurophilic granule product is able to destroy C5a activity,27 and the MPO system has been demonstrated to oxidize chemoattractant peptides and neutrophil secretory products.5’ The ability of the cell to digest chemoattractants may be important in chemotaxis.52 Azurophilic granules, then, serve lysosomal functions and are important in target cell death and in modulation of the inflammatory response. A novel role of neutrophil granules in the regulation of intracellular calcium has recently been proposed.53 Stimulation of the neutrophil by chemoattract&t stimuli results in rapid rises in cytosolic free calcium believed to represent release from nonmitochondrial in-
Neutrophil
NL Absent NL
NL
Absent NL Absent 1 NL Absent Absent NL 3 with
cytoplasts
J NL
Absent NT NL
modifications.
tracellular stores.54 These calcium rises are important in regulating functional responses to stimuli. Since both specific and azurophilic granule-enriched subcellular fractions contain a magnesium-adenosine triphosphate-dependent calcium pump promoting calcium uptake, these granules may be an intracellular calcium storage site, thus linking granules with cell activation in yet another way. MODELS OF GRANULE Cytoplasts
DEFICIENCY
Our understanding of the role of neutrophil granules in the inflammatory response has been strengthened by experimental models of granule-deficient cells and by patients whose cells are lacking in granule contents (Table II). The model cell, the enucleated cytoplast, is a plasma membrane-bound bag of cytoplasm pinched off from a neutrophil and contains less than 10% of the original granules of the ce11.55,56 It has various functional abnormalities including poor chemotaxis, slower bactericidal activity, abnormal shape change in response to chemoattractants, and abnormal disaggregation. The cytoplast preparation, unlike the neutrophil, is unable to increase the plasma membrane expression of FMLP or C3bi receptors in response to degranulating stimuli. *O*56 Some of these abnormalities may relate to the absence of specific granule membrane to provide for the membrane turnover and receptor replenishment needed for cellular responses such as chemotaxis, shape change, and disaggregation. Congenital
specific granule
deficiency
The model of specific granule deficiency, patients with recurrent bacterial infections (Table III) and a severe deficiency in the specific granule markers vitamin B,2 binding protein and lactoferrin, has been
VOLUME 77 NUMBER 5
TABLE
Neutrophil
III. Clinical
Granule
type
Specific granules
presentations
of patients
B, Baggiolini
Screening
Early onset recurrent infections with catalase-positive bacteria and fungi, granulomatous lesions Delayed separation of umbilical stump, leukocytosis, recurrent bacteria1 infections, periodontal disease Bacterial infections, infectious mortality (relates to extent of burn) Generally none; some patients with other host defense defects such as diabetes have bacterial or fungal infection (particularly Candida albicans) Recurrent bacterial infections, partial oculocutaneous albinism, progression to peripheral neuropathy, accelerated phase (organomegaly, coaguloplymphoma-like athy, neutropenia, syndrome)
Chediak-Higashi syndrome
described.57 The cells of these patients are morphologically abnonnal with misshapen nuclei. No granules are visible on Wright’s stain, but myeloperoxidase staining reveals azurophilic granules. The presence of empty specific granules has been suggested by electron microscopy studies5* Other abnormalities such as a reduction in the plasma membrane marker alkaline phosphatase, altered azurophilic granule complex carbohydrate staining by electron microscopy, and abnormal granule sedimentation in subcellular fractionation studies57-59are present. In vitro, these neutrophils demonstrate abnormal chemotaxis and bacterial killing. Like cytoplasts, these cells do not increase receptors after stimulation.“. 6o In vivo, one patient has demonstrated deficient neutrophil and monocyte recruitment to skin windows, although monocyte chemotaxis was not deficient in vitro. In addition, secretory products from the cells of this patient were unable to generate chemoattractants from serum.@’ Disaggregation was also abnormal.@’ These neutrophils were demonstrated to be lacking in gelatinase activity, suggesting absence of tertiary granules as well, * Although the underlying defect in this disorder is complex and not well understood, the abnormalities documented point to a role of the specific
*Dewald
manifestations
CGD (cytochrome deficient)
b
M, Gallin
J: Unpublished
observation.
in health and disease
657
abnormalities
Recurrent severe bacterial infections
MPG deficiency
and
granule
Specific granule deficiency
Thermal injury
Azurophilic specific granules
neutrophil
Clinical
Disorder
C3bi receptor (CR3) deficiency
Azurophilic granules
with
granules
test
Peripheral blood smear (misshapen nucleus, no granules on Wright’s stain) and enzyme contents of neutrophils Nitroblue tetrazoleum reduction test
Deficient binding of monoclonal antibodies against the C3bi receptor to neutrophils
MPG stain of neutrophils
Peripheral blood smear (characteristic large lysosomal granules in white cells)
granule and possibly the tertiary granule in chemotaxis, receptor upregulation, chemoattractant generation, disaggregation, and control of bacterial infections. Specific granule thermal injury
deficiency
secondary
to
Another model for specific granule-deficient cells is that of thermal injury. In bum patients a decrease in neutrophil lysozyme with increase in blood lactoferrin and lysozyme has been reported with neutrophil P-glucuronidase being normal, suggesting preferential specific granule degranulation.6’.62 Davis et a1.62correlated this decrease in neutrophil lysozyme with abnormalities of chemotaxis in bum patients. These abnormalities have, in turn, been correlated with increased mortality.63 After a bum, preferential specific granule exocytosis may interfere with chemotaxis, perhaps by lack of membrane and preformed receptors for tumover and, along with defects in oxidative metabolism,bl may contribute to compromised host defenses. Neonatal
neutrophils
Neonatal neutrophils comprise another system with decreased neutrophil lactoferrin but normal neutrophil MPO content.65 This suggests a relative specific granule deficiency in comparison with adult cells. In addition, neonatal neutrophils manifest a host of com-
668
Falloon and Gallin
plicated abnormalities when they are compared to adult cells. These include abnormalities of chemotaxis,@ adherence, 66. 67 disaggregation,68 membrane potential,69 phagocytosis and microbicidal activity,” and oxidative metabolism.@ In particular, hydroxyl radical production appears diminished, which has been attributed to lactoferrin deficiency.65 The specific granule deficiency in these cells may be in part responsible for the increased susceptibility of neonates to infection. C3bi receptor
(CR3) deficiency
Other patients have been described who appear to be deficient in a group of leukocyte membrane glycoproteins including the C3bi receptor,” a receptor normally found in neutrophil plasma membrane and specific granule-enriched cell fractions. Patients with this abnormality present with delayed separation of the umbilical stump, recurrent and severe bacterial and fungal infections, impaired wound healing, diminished pus formation, severe periodontal disease, and persistent leukocytosis. The severity of the clinical manifestations appears to be related to the degree of deficiency of the membrane glycoproteins.” In vitro, slightly differing abnormalities have been described, generally involving abnormalities of neutrophil aggregation, adherence, spreading, chemotaxis, and defective phagocytosis of some opsonized particles. Bactericidal activity is generally normal.” This defect involves deficiencies in a family of multimeric highmolecular-weight glycoprotein surface antigens with a common beta subunit and different alpha chains. The C3bi receptor is one of these molecules.74 Work in which the in vitro abnormalities of this disease are mimicked by adding a monoclonal antibody against the antigen to normal cells75,76suggests that the functional abnormalities are related to the missing glycoprotein. Studies of these patients suggest that the C3bi receptor functions in neutrophil adherence, aggregation, and chemotaxis and that its absence from the granules and plasma membrane of the neutrophil is responsible for severe human disease. CGD
Another patient group with abnormal granules is a subset of patients with CGD. CGD is a group of related disorders characterized by recurrent infections and granulomatous lesions.77-79The infections are due to an inability of the neutrophil to kill most bacteria and fungi because of an abnormality in oxidative metabolism with inability to produce toxic oxygen metabolites, including hydrogen peroxide. This abnormality is believed to result from various genetically absent or defective constituents of the membrane-associated
J. ALLERGY CLIN. IMMUNOL. MAY 1996
oxidase that normally triggers the respiratory burst or from defective activation of this oxidase. In one subset with X-linked inheritance, the patients’ specific granule-enriched fractions appear deficient in cytochrome b, ‘O,” and another subset may be deficient in a flavoprotein. 3s In a subset of patients with autosomal recessive inheritance, there is an absence of stimulusinduced phosphorylation of a protein distributed in plasma membrane and granule fractions that may interact with cytochrome b.82 In this way, abnormal specific granules may lead to an inability to mount a normal respiratory burst so that one of the important antibacterial mechanisms of the neutrophil is crippled. Abnormalities of azurophilic Myeloperoxidase deficiency
granules:
No patients lacking azurophilic granules have been described, but the inherited or acquired absence of azurophilic granule MPO is a relatively common neutrophil abnormality occurring in about 1 in 2000 persons.83 It is interesting that the lack of this protein, believed to play a key role in bacterial killing by normal neutrophils, does not usually result in illness, although some patients have been described with Candida albicans infections. The patients with infections generally have another illness such as diabetes mellitus contributing to compromised host defenses. In vitro, MPO-deficient cells undergo an augmented respiratory burst,49 and although bacterial killing is delayed, it is eventually normal, perhaps the result of the compensatory role of unusually vigorous production of toxic oxygen metabolites. The benign nature of MPO deficiency points out the complexity of neutrophil bacterial killing and the importance of killing mechanisms other than the MPO-halide-hydrogen peroxide system such as oxidative killing by other products of oxygen and nonoxidative killing by exposure of bacteria to the interior of the phagolysosome and to granule contents.&, 47,84,” The Chbdiak-Higashi
syndrome
The Chediak-Higashi syndrome is an inherited abnormality characterized by morphologically abnormal granules in neutrophils and other granule-containing cells.86 The giant granules of the cells are believed to be products of abnormal granule fusion,*’ perhaps related to abnormal microtubule metabolism88~89 or to altered membrane properties.” The neutrophils are functionally abnormal with decreased chemotaxis, abnormal lysosomal degranulation, and delayed and deficient bactericidal capacity.‘lT 92The patients have increased susceptibility to bacterial infection as well as variable complicated systemic manifestations, including neutropenia, blood coagulation abnormalities,
VOLUME 77 NUMBER 5
Neutrophil
neuropathy, partial albinism, adenopathy, hepatosplenomegaly, and an increased incidence of lymphoproliferative disorders.86 Since the underlying defect in this syndrome is unknown, the role of the abnormal granules in the neutrophil defect is unclear. Granule deficiency collection
secondary
to leukocyte
Granule exocytosis may be involved in the diminished functional capacity of neutrophils intended for transfusion that were collected by filtration leukapheresis.“’ In this method cells are collected by reversible adhesion to nylon fibers. Neutrophils become partially degranulated during this process with loss of specific granules being greater than that of azurophilic granules. These cells are functionally abnormal with abnormal posttransfusion kinetics, chemotaxis, phagocytosis, and bactericidal activity. Because of these defects, as well as increased recipient side effects with filtration leukapheresis cells, alternative collection methods are now prefemed.94 POSSIBLE ROLE OF NEUTROPHIL GRANULES IN SYSTEMIC DISEASE There is growing evidence that neutrophils and their granules contribute to a variety of systemic diseases through local release of damaging granule enzymes or toxic oxygen products. In particular, the neutrophil is believed important in the pathogenesis of both acute and chronic lung disease. In an acute lung disease, the adult respiratory distress syndrome, it is believed that activation of neutrophils by blood stream complement results in neutrophil trapping in the lung. The local release of toxic neutrophil products such as oxygen metabolites, proteolytic enzymes, and arachidonic acid products results in damaged alveolar endothelium and plasma exudation. 95-97In some animal models lung damage is dependent on production of reactive oxidants and can be diminished by inactivators of these oxidants, particularly by scavengers of hydrogen peroxide or hydroxyl radical.98, 99 In a chronic lung disease, emphysema, it is believed that lung irritants induce alveolar macrophages to release neutrophil chemoattractants. Neutrophils arriving in the lung degranulate, releasing enzymes such as elastase that contribute to the disruption of alveolar elastic fibers that results in emphysema. ‘O”Other systemic illnesses potentially related to neutrophil granule products include rheumatoid and crystalline arthritis, serum sickness, immune vasculitis and glomerulonephritis, myocardial infarction, and even malignancies at sites of chronic inflammation. ‘01-‘04The neutrophil and its granule components are thus potentially involved in many human disease states involving inflammation.
granules
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SUMMARY The granules of the neutrophil, in addition to contributing to its distinctive morphologic appearance, are critical to its unique functions. Specific granules appear necessary for neutrophil recruitment to sites of inflammation, for upregulation of receptors important in the control of chemotaxis and the respiratory burst, for disaggregation, for bactericidal activity, and for chemoattractant generation. The azurophilic granules supply enzymes for digestive and bactericidal functions and supply MPO to the MPO-halide-hydrogen peroxide bactericidal system. Azurophilic granule contents also regulate inflammation by degrading inflammatory products. Both granules may play a role in intracellular calcium regulation. In addition to these activities that protect the host from infection, granules also, under certain circumstances, contribute to disease processes. For these reasons, greater knowledge about granule contents, control of degranulation, inactivation of toxic granule contents and products, and the role of granules in neutrophil membrane events and function has widespread implications for treatment of patients with neutrophil dysfunction syndromes and patients with multiple other systemic diseases.
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