3 Neutrophil function tests A . P. H A Y N E S J. F L E T C H E R

INTRODUCTION The clinical relevance of neutrophil function is usually thought of in terms of host defence because a lack of neutrophils is associated with overwhelming bacterial and fungal infection. To fulfill this defensive role, circulating neutrophils must adhere to the endothelium of capillaries and venules adjacent to the inflammatory locus, migrate through the vessel wall to the area of inflammation, phagocytose opsonized bacteria, kill ingested organisms and, finally, inactivate their own toxic products to prevent damage to normal tissue (Figure 1). Different aspects of this complex process, such as chemotaxis or phagocytosis, can be separately defined and studied in vitro but they are clearly linked by common mechanisms. For example, margination, chemotaxis and phagocytosis all depend upon neutrophil adhesion. However, it may be misleading to depend upon a single test of neutrophil function for although a number of responses can be elicited by a single stimulus, they may be independently regulated with distinct intracellular transduction pathways. Which tests are chosen from the range available will depend upon the questions to be answered. In general it is more time-consuming to measure physiological functions such as chemotaxis, phagocytosis or microbial killing. It is often easier to isolate a single product of a given function, such as the respiratory burst or degranulation, and measure it. This review evaluates the various methods available for measuring neutrophil function in vitro and relates them to what is known of neutrophil function in vivo. For a detailed discussion of the laboratory techniques required for the various methods, see the excellent manual produced by Metcalf (Metcalf et al, 1986). CELL SEPARATION As soon as a needle is inserted into a vein, the behaviour of neutrophils is changed. In vitro artefacts cannot be avoided but, if possible, they should be recognized and minimized. Circulating neutrophils are resting cells expressing low numbers of low affinity receptors. Outside the circulation the Bailli&e"s Clinical Haematology--

Vol. 3, No. 4, October1990 ISBN0-7020-1475-3

871 Copyright© 1990,byBailli~reTindall All rights of reproductionin any formreserved

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[ CIRCULATINGPMN ]

~--ADHERENCE}

..

CHEMOTAXIS

.~ ~ BACTERIAL ~ / / J ' TISSUE FACTORS FACTORS / 4 7 ~ / " e.g. adenosine e.g. FMLP LI~?'~FIC_DERtVEDPRODUCTS e.g. PAl:, LT B4, NAF ( I L S ) Cells leave the vascular space along a gradient of chemotactic factors originating from the inflammatory locus

I'ACT,VATION3

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Mediators such as IL-1, TNF or lipopotysaccharide released at the inflammatory locus cause both PMN and endothelial cells to expressenhanced numbers of surface adhesion molecules leading to margination. [PHAGOCYTOSIS]

Complem^emreceptor e.g. L,r], L,r~

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r|mmunoglobulin receptors e.g. FcR II

Receptorson the PMN surface recognize particles opsonized with complement or immunoglobulin RESPIRATORYBURSTgenerates toxic oxygen species DEGRANULATION- both intra- and extraceltular release of antimicrobial agents and digestive enzymes Leads to microbial killing and digestion of debris

Figure 1. Stages in the neutrophil response to inflammation.

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number and affinity of receptors can be increased by simply incubating in artificial media or by changes in temperature (Fearon and Collins, 1983; Andersson et al, 1987; Tennenberg et al, 1988). Consequently, great care must be taken when obtaining a pure preparation of neutrophils if cells resembling those in the peripheral blood are to be obtained. Venesection should be performed using a wide-bore needle with a polypropylene syringe and the blood transferred to an anticoagulated polypropylene tube. If heparin is used as anticoagulant then it must be recognized that complement activation will still occur, generating C5a des Arg in the plasma. This may not matter if blood is kept on ice and separated immediately, but it is probably better to chelate calcium with citrate or EDTA to avoid complement activation of neutrophils. Neutrophils are purified from other cells by centrifugation through a density gradient. A reliable two-step procedure involves obtaining a buffy coat by gravity sedimentation of red cells with the addition of dextran, followed by separation of neutrophils by centrifugation through FicollHypaque (Boyum, 1968). This may be simplified to a one-step procedure using only Ficoll-Hypaque; this has the advantage of speed but the disadvantage of increased red cell contamination of the neutrophils (Bignold and Ferrante, 1987). Percoll can be substituted for Ficoll-Hypaque but has no particular advantage over it (Dooley et al, 1982). In laboratories that have the necessarY equipment, centrifugal elutriation provides a method for obtaining large numbers of pure neutrophils (Berkow and Baehner, 1985). Whichever method is chosen, a number of problems can be recognized. Separation media, particularly dextran and Percoll, are readily contaminated with endotoxin. Exposure of cells to endotoxin during the time required for separation will increase their subsequent response to other stimuli such as the complement component C5a (Haslett et al, 1985). The neutrophils described in many publications are in fact granulocytes for, whilst it is relatively easy to remove red cells and mononuclear cells, it remains difficult to remove eosinophils by the commonly used techniques. This is not important for most function tests but it should be noted. Red cell contamination can be removed by hypotonic lysis but this immediately introduces more artefacts. Hypotonic conditions, even for as little as 20 s, will influence the neutrophil membrane, and if ammonium chloride is used then both the pH gradients within subcellular compartments and calcium homeostasis will be disturbed (Styrt and Klempner, 1988). Isolated neutrophils should be washed in calcium-free buffer to remove traces of plasma proteins and separation media. For most tests, cells are then suspended in a buffer containing divalent cations and 0.1% albumin, to maintain the integrity of the cell membrane. The final preparation should be at least 95% pure and cell viability confirmed to be greater than 98% by the exclusion of trypan blue, a vital dye taken up only by non-viable cells. ADHERENCE

The first step in the neutrophil response to inflammation is adhesion to the

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A. P. HAYNES AND J. FLETCHER

endothelium of dilated capillaries. In vitro, it is easy to label neutrophils with fluorescent cytoplasmic markers or radiolabel them with chromium-51. These labelling techniques permit cell detection by simple microscopy or by scintillation counting after cell lysis. Using such labelled cells it is possible to measure adhesion to glass or plastic coated with an extracellular matrix protein such as fibronectin (Gallin et al, 1982). The mechanism of this adhesion cannot be easily defined and these techniques do not model for the contribution of the endothelial cell to adhesion. It is more physiological to study the adhesion of labelled neutrophils to a monolayer of endothelial cells (Charo et al, 1985). The latter can be grown from human umbilical vein endothelium or from the capillaries of mesenteric fat removed at laparotomy (Jaffe et al, 1973). With either of the cell detection systems it is important to wash away non-adherent cells, and this is particularly true of radiolabelling because spontaneous cell lysis can release label. Measurements made on monolayers have additional undesirable features, most do not account for flow, which is obviously relevant to adhesion in vivo, and differences may exist in the properties of endothelial cells obtained from different tissues. An alternative to these difficult techniques is to use monoclonal antibodies to measure the surface expression of the glycoprotein receptors involved in neutrophil adhesion (Todd and Freyer, 1988). The adhesion molecules expressed by neutrophils are members of the integrin family. The integrins are a large family of glycoproteins that mediate adhesion between cells in many tissues and have two non-covalently linked subunits forming a transmembrane complex (Hogg, 1989). The three integrins on neutrophils share a common [3 subunit, termed CD18, but each has a distinct o~subunit, C D l l a for LFA-1, C D l l b for Mac-1 and C D l l c for p150,95 (Shaw, 1987). Monoclonal antibodies may also be used to measure the expression of complimentary ligands, termed ICAM-1 and ELAM-1, on the endothelium of inflamed vessels (Dustin and Springer, 1988; Bevilacqua et al, 1989). These monoclonal antibodies have allowed the study of neutrophil adhesion in cell suspension/monolayer experiments and in histological preparations. However, adhesion may occur independently of integrin expression by mechanisms such as complement bridging (Marks et al, 1989) or by interaction with ligands that are not members of the integrin family (Vedder and Harlan, 1988). Abnormalities in neutrophil adhesion in disease states are therefore best examined by a combination of integrin expression and a monolayer technique. CHEMOTAXIS Chemotaxis is the directed migration of cells in a gradient of chemoattractant. Chemokinesis is the enhanced motility of cells in the presence of chemoattractant in comparison with the random motility of unstimulated cells. Directed migration of cells depends upon the expression of chemoattractant receptors, intact signal transduction pathways, normal adhesion and normal motility. Neutrophils adopt a characteristic morphology during

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chemotaxis, with a blunt leading edge and an attenuated body containing the nucleus and cytoplasmic structures (Singer and Kupfer, 1986). Degranulation occurs selectively at the leading edge of the cell. As a result the highest concentration of receptors is at the leading edge with a gradient across the cell. This interacts with the chemotactic gradient and results in directed migration (Falloon and Gallin, 1986). Bound receptors are probably internalized, the ligand digested and the receptor returned to the cell surface (Niedel el al, 1979). Chemoattractants stimulate both chemotaxis and chemokinesis, hence techniques measuring only the fastest-moving cells will fail to distinguish between directed and random migration. Differentiation betwen directed and random migration can be achieved by applying an elaborate chequerboard analysis as described by Wilkinson (1982). At present, the conditions used for in vitro tests of chemotaxis differ greatly from those which are relevant in vivo and, whilst they are valid in screening biological fluids for the presence or absence of chemotactic activity, it is difficult to relate in vitro tests to normal neutrophil physiology. In vitro assays are temperature-, pHand osmolarity-sensitive, with a rapid decline in neutrophil motility occurring outside the physiological range. As with all other in vitro assays of neutrophil function it is important to avoid endotoxin contamination of buffers and apparatus. The chequer-board analysis can be used to generate dose-response curves which, for most chemoattractants, are bell-shaped with decreasing motility seen above a critical concentration. The potency of different agents can be compared from the concentration required to give a 50% maximal response. Chemotaxis is usually assessed by measuring migration either under agarose or through cellulose nitrate filters. The agarose method consists of cutting three wells into a preformed agarose gel. Neutrophils suspended in buffer containing divalent cations are placed in the central well and buffer or chemoattractant placed in the two flanking wells. Chemotaxis is assessed by comparing the distance migrated towards the chemoattractant with distance migrated towards buffer, using simple microscropy or an optical imaging system (Nelson et al, 1975). In its simplest form the technique measures only the fastest moving cells. This has the advantage of ease but means that it examines only a subpopulation of cells. Filter techniques may provide a better model of in vivo chemotaxis than agarose techniques because they require cells to deform to pass through pores within the filter. In these techniques a filter is used to separate a suspension of cells from a solution of chemoattractant or buffer. Care must be taken to exclude air bubbles from underneath the filter during filling and the fluid levels on either side of the filter must be identical to exclude hydrostatic effects upon cell distribution. After incubation, cells within the filter are fixed and stained and the filter cleared so that the distance migrated by cells can be measured microscopically (Fulk et al, 1980). This technique has the advantage that cell morophology is usually sufficiently preserved to enable identification of neutrophils amongst a mixed cell population, but unfortunately it has a low sensitivity because cells may migrate through the filter completely and escape detection. Sensitivity can be improved by

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adding a second filter to 'catch' all cells passing through the first. Alternatively, cells can be radiolabelled with chromium 51 and the appearance of radioactivity in the lower chamber used to follow cell movement (Gallin et al, 1980) (however, radiolabelling is inadequate if a mixed population of cells is to be studied). A chequer-board analysis can be applied to distinguish between chemotaxis and chemokinesis. It should be noted the results generated by filter techniques will depend upon the physical characteristics of the filter, such as thickness, pore size and construction material. The technical difficulties associated with in vitro assays of chemotaxis can make the comparison of results generated in different laboratories particularly difficult. Unfortunately, in vivo assays give no more help. In vivo assays use the basic principle of the skin window technique described by Rebuck (see Dale and Wolff, 1971), and they measure several different aspects of the inflammatory response, including vasodilatation, adhesion and chemotaxis. Reproducible quantitative result are difficult to produce due to the difficulty of applying a standard stimulus. Nevertheless, in spite of these problems, in vivo techniques can occasionally provide useful data in disease states. PHAGOCYTOSIS Neutrophils recognize particles to be phagocytosed as a result of interaction between their surface receptors and opsonins on the surface of the particles, The most important opsonins are the Fc portion of immunoglobulin G, for which neutrophils express low-affinity receptors (FcRII and FcRIII) and, when activated, high-affinity receptors (FcRI) (Unkeless, 1989). Neutrophils also express receptors for the complement components C3b and C3bi (Fearon, 1980); receptors for C3bi are part of the Mac-1 complex and like FcRI are expressed on activated cells (Gallin, 1985). The interaction between receptor and opsonin leads to rapid ingestion of the particle and, having ingested one particle, a neutrophil is more likely to ingest others. Consequently any method for measuring phagocytosis must be able to detect the initial rate of uptake and not just the end-point after the process is complete, since the same end-point can be reached at different rates. As far as possible, the whole population should be tested with a high ratio of particles to cells, but not so high that cell rupture and death occur. Dilute suspensions of cells and particles reduce the chances of interaction occuring between the two, so both concentration and ratio must be optimized for a given assay. In practice, a ratio of 5 : 1 and a concentration of 1 x 106 to 2 x 106 neutrophils per millilitre are appropriate. Many methods of assessing phagocytosis have been described. The easiest is light microscopy, but this has many disadvantages. Firstly, it is very difficult to count the number of ingested particles, hence it is usual to count the number of cells containing particles. This will only be an accurate measure of phagocytosis if expressed as a rate, because even in the presence of grossly defective phagocytosis all neutrophils will take up some particles, especially with high target:ceU ratios. More importantly, it is difficult to

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distinguish particles inside the neutrophil from those that are attached to the surface but have not been ingested. Phagocytosed particles within a vacuole are surrounded by a clear halo, but even so it is difficult to accurately locate particles by simple microscopy. This problem also applies to methods that separate phagocytes and particles by differential centrifugation or that rely upon the uptake of radiolabelled particles. However, there are several methods which avoid these pitfalls. In a suspension of bacteria and neutrophils, uridine uptake occurs only by viable extracellular bacteria. If phagocytosis is stopped at intervals and tritiated uridine added then the rate of phagocytosis can be calculated from the declining levels of radioactivity in the extracellular fluid (Bridges et al, 1980). Alternatively, bacteria or inert particles labelled with a fluorescent dye can be used to measure uptake and a second dye, such as trypan blue, which is excluded from the neutrophils, used to quench the fluorescence of extracellular, attached particles (Hed et al, 1987). Fluorescence detection by flow cytometry allows rapid measurements on a large number of cells and the study of subpopulations of ceils.

DEGRANULATION The contents of neutrophil granules are listed in Table 1. They are discharged in vivo and in vitro when cells are stimulated with chemoattractants or opsonized particles. Granules contain a number of substances which are easy to measure biochemically: for example, myeloperoxidase (Worthing1. Contents of the major cytoplasmicgranules of neutrophils. The secondary granules form at a later stage of neutrophil maturation than the primary granules and outnumber them by approximately2 : 1.

Table

Function Microcidal agents

Serine proteases Metalloproteinases Acid hydrolases

Others

Primary granule

Secondary granule

Myeloperoxidase Lysozyme Defensins Cationic proteins Etastase Cathepsins B/D Proteinases CathepsinsB/D 13-Glucuronidase Glycerophosphatase N-acetylglucosamine c~-mannosidase

Lysozyme Lactoferrin

Tertiary granule

Collagenases

Gelatinase

B12 binding protein Cytochrome b245 C3bi receptor FMLP receptor Histaminase

C3bi receptor

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A. P. HAYNES AND J. FLETCHER

ton Enzyme Manual, 1978, Worthington Biochemical Corporaton, Freehold, New Jersey) or ~3-glucuronidase (Gallin et al, 1982) from primary granules can be measured spectrophotometricaUy; elastase from the primary granules (Neumann et al, 1984) or lactoferrin (Horl et al, 1986) can be measured by an ELISA; or vitamin B~2 binding protein which, together with lactoferrin, is a secondary granule protein, can be measured by a radioassay (Gallin et al, 1982). Unfortunately, when these techniques are applied to measuring these constituents either inside cells or released from cells, there are a number of technical problems. Myeloperoxidase is usually measured by its reaction with ortho-dianisidine and this is affected by haemoglobin, so contaminating red cells must be removed from the neutrophil population. Eosinophils also contain a potent peroxidase which can be distinguished from the neutrophil enzyme using aminotriazole (Cramer et al, 1984). For these reasons [3-glucuronidase is an easier marker to use for primary degranulation. Lysozyme is easy to measure by its ability to lyse micrococci (Gallin et al, 1982) but it is not a specific marker for either type of granule. The preferred marker for secondary granules is lactoferrin, but this highly charged protein does stick to glass and plastic making it difficult to extract it entirely from cells. Whichever indicator is used, the accurate interpretation of results requires an understanding of the normal behaviour of primary and secondary granules. Primary granules are lysosomes concerned with the killing and digestion of ingested particles. Their contents are discharged into the phagocytic vacuole and very little leaks outside the cell. Within the vacuole, primary granule enzymes are denatured by the action of hypochlorite (Weiss, 1989). Consequently, loss of primary granule enzymes, detected either by staining cells or extraction of the enzyme and spectrophotometric measurement, indicates previous activation. The extracellular release of primary granule markers can be increased by first treating the cells with the fungal product cytochalasin B which inhibits microtubule function and phagocytosis. However, it must be remembered that cells treated with cytochalasin B have been poisoned and display abnormal metabolism (Koenderman et al, 1989). In contrast, secondary granules fuse with both the vacuole and the cell surface. The latter results in the translocation of receptors to the cell surface and discharge of contents outside the cell. A reduction of secondary granule markers inside circulating cells indicates previous activation. These markers along with the primary granule enzyme elastase can also be measured in plasma. However, the plasma level will depend upon circulating cell numbers, cell turnover and plasma clearance rates, and it does not have a simple relationship with neutrophil activation. RESPIRATORY BURST

The respiratory burst is the term used to describe the increase in oxygen consumption and hexose monophosphate shunt pathway activity, which accompany neutrophil activation. Like granule discharge, the respiratory burst can be measured in a variety of ways and interpretation of the results

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requires an understanding of neutrophil physiology. Secondary granule discharge brings cytochrome b245 t o the cell surface (this is one of the components of an electron transport chain termed the NADPH oxidase, which catalyses the one electron reduction of oxygen to superoxide anions; Segal, 1989). Secondary granule discharge alone is insufficient to activate the enzyme, which requires the addition of cytosolic components to complete the electron transport chain (Figure 2). The superoxide anions produced spontaneously dismutate to form hydrogen peroxide so consuming protons. Oxidase activity can therefore be measured directly by oxygen consumption (Root and Stossel, 1974), superoxide or hydrogen peroxide production, or indirectly by the metabolism of glucose via the hexose monophosphate shunt (Stjernholm, 1980) which is the source of NADPH. In practice, it is easy to measure either superoxide or hydrogen peroxide production, although they will not necessarily give the same results as protons are needed for superoxide to dismutate spontaneously. The most accurate way of measuring superoxide is by spectrophotoscopy following the reduction of ferricytochrome c (Nauseef et al, 1983). This reaction is inhibited by superoxide dismutase (SOD), hence the reduction specific to superoxide can be defined as the SOD-inhibitable change in STIMULATION

S"UNT_l

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LPE"°x'°ASE 1

xl,l, Figure 2. The neutrophil respiratory burst. Phagocytosis activates the NADPH oxidase which transfers electrons onto oxygen, generating superoxide anions and consuming NADPH. Superoxide is used to generate reactive species important for microbial killing. Abbreviations: SOC, soluble oxidase component; MPO, myeloperoxidase; GSH and GSSG, respectively, reduced or oxidized glutathione,

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A. P. HAYNES AND J. FLETCHER

optical density. Care must be taken as hydrogen peroxide formed by dismutation of superoxide can oxidize the reduced ferricytochrome c resulting in an underestimate of superoxide production. Superoxide can also be measured specifically by lucigenin-enhanced chemiluminescence, which, like the ferricytochrome c reaction, can be verified by the inhibitory action of SOD (Gyllenhammar, 1987). The chemical basis of chemiluminescence is the release of photons by the interaction of free radicals with other compounds, and in the presence of lucigenin the light production is amplified and specific for superoxide anions. Light production can be measured continuously in a luminometer by an array of photomultiplier tubes. Unlike ferricytochrome c reduction, chemiluminescence cannot be calibrated in terms of the number of moles of superoxide produced. There are two commonly used methods for measuring the extracellular release of hydrogen peroxide. Scopoletin is fluorescent but in the presence of hydrogen peroxide and horseradish peroxidase it is oxidized to a nonfluorescent derivative (Boveris et al, 1977). This method is sensitive to other hydrogen donors such as serum proteins, which must be excluded. The other method depends upon the hydrogen peroxide-mediated oxidation of phenol red (Pick and Mizell, 1981). Colour changes are detected spectrophotometrically, but the range over which colour changes are linearly proportional to the concentration of hydrogen peroxide is narrow. Hydrogen peroxide production can also be measured by chemiluminescence using luminol as an enhancer and catalase to make the results specific (Edwards, 1987). Intracellular hydrogen peroxide production can be measured by loading cells with the derivative 2,7-dichlorofluorescein (Bass et al, 1983). Neutrophils are loaded with the lipid-soluble acetate ester, which is nonfluorescent. Inside the cell, the acetate groups are cleaved to yield a derivative that is unable to leak out of the cell and which is converted to a brightly fluorescent compound by the action of hydrogen peroxide. When combined with flow cytometry this method permits the analysis of subpopulations of cells. MICROBIAL KILLING Phagocytosis activates the neutrophil respiratory burst and degranulation. The products of the respiratory burst and substances released into the phagosome by degranulation have important antimicrobial actions (Lehrer et al, 1988). Superoxide anions generated by the NADPH oxidase spontaneously dismutate to form hydrogen peroxide, which is toxic to some micro-organisms. Hydrogen peroxide, in a reaction catalysed by the primary granule enzyme myeloperoxidase, interacts with chloride ions to form hypochlorous acid (Figure 3). The latter reacts with amines to generate chloramines which are powerful oxidizing agents and destroy micro-organisms Within the phagosome. Chloramines are probably the major toxic products of the respiratory burst. Primary degranulation releases a number of antimicrobial substances into the phagosome (see Table I and Figure 3). Defen-

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sins are arginine- and cysteine-rich peptides which represent over 5 % of the total protein content of neutrophils (Ganz et al, 1985); they have a broad spectrum of antimicrobial activity, killing fungi and Gram-negative and Gram-positive organisms. Other similar agents present in primary granules include cathepsin G and bactericidal/permeability-increasing factor. Based upon the observation that microbial killing still occurs under anaerobic conditions, killing mechanisms are usually divided into those which are oxygen-dependent and those which are oxygen-independent (Thomas et al, 1988). This division is to some extent artificial because oxygen-dependent mechanisms require the presence of myeloperoxidase, a primary granule enzyme, and the action of products released by degranulation requires a normal sequence of pH changes within the phagosome, generated by the activity of the NADPH oxidase (Mayer et al, 1989). The susceptibility to killing by a particular mechanism will also be influenced by microbial

NADP 4

NADPH

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, OXYGENSPECIES

° granule"

H\

\

G roteins

H(

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OXYGENDEPENDENT ]

I OXYGENINDEPENDENT]

Figure 3. The major killing mechanisms operating within the phagolysosome after ingestion of 0psonized particles Abbreviations: HOC1, hypohalous acid; MPO, myeloperoxidase.

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A. P. HAYNES AND J. FLETCHER

characteristics (Elsbach and Weiss, 1983). For most clinical purposes it is not necessary to define the mechanism of killing. In a mixture of neutrophils and micro-organisms, the number of organisms killed can be measured by simply disrupting neutrophils to release any ingested but living organisms and measuring the number which remain viable. Viability can be defined by colony growth on pour plates (Root et al, 1972), by changes in staining characteristics (Lehrer et al, 1969) or by the uptake of radiolabelled uridine (Yamamura et al, 1977). The number killed will depend upon the number phagocytosed and, unless the fate of ingested organisms can be measured separately, the data can only be interpreted if the rate of phagocytosis is normal. Intracellular killing as distinct from total killing can be measured in a number of ways. Extracellular staphylococci can be lysed with lysostaphin and the neutrophils disrupted to release ingested bacteria (Pruzenski et al, 1983). Similarly, lysozyme can be used to lyse extracellular micrococci (Grinstein and Furuya, 1988). A more generally applicable method is to use the uptake of radiolabelled uridine by living organisms to define both total and intracellular killing (Harvey et al, 1986). This technique involves arresting phagocytosis at intervals by chelation of calcium ions and cooling. Incubation with uridine then permits the measurement of extracellular organisms, and incubation with uridine after neutrophil disruption measures the total number of viable organisms, hence providing a measure of intracellular killing. All killing assays are sensitive to the conditions under which they are performed. Test organisms should be in growth phase and, since oxygen-independent mechanisms require metabolically active organisms, a suitable energy source should be supplied. Similarly, oxygenindependent killing mechanisms require interaction with the microbial surface, hence killing is sensitive to changes in divalent cations and pH. PRIMING

As already explained, activated neutrophils increase their expression of adhesion molecules and receptors for C3bi and immunoglobulin. In vitro this upregulation occurs at concentrations of chemoattractants or cytokines which are too low to directly activate the respiratory burst but which 'prime' the cells to increase their response to subsequent stimuli such as C5a des Arg or opsonized particles (Weisbart et al, 1985; Wirthmueller et al, 1989). Endotoxin from the cell walls of Gram-negative bacteria is a potent priming agent and it is for this reason that so much emphasis is put on avoiding bacterial contamination of buffers and equipment used in the study of neutrophil function (Guthrie et al, 1984). Priming is not only a source of artefact in vitro but must also be taken into account when interpreting the results of function tests in vivo. For example, a few hours after a surgical operation, circulating neutrophils show a reduced response to stimulation with C5a des Arg, but if the neutrophils are first incubated with tumoug necrosis factor e~, then their responses show no differences from normal cells primed by tumour necrosis factor eL(S. Crouch and J. Fletcher, unpublished

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observations). Another example is the behaviour of neutrophils from patients with renal failure who are receiving continuous ambulatory peritoneal dialysis. These cells show decreased phagocytosis of opsonized staphylococci, but when patients develop peritonitis, a common complication of this form of dialysis, the phagocytosis of circulating neutrophils becomes normal (Harvey et al, 1987). It is vital to realize that neutrophils are not static end-stage cells, their functional responses can be up- or downregulated, as appropriate, both in vitro and in vivo. CONCLUSION The vital role of the neutrophil in host defence is witnessed by the devastating effects of abnormalities in its function. It is therefore unfortunate that our understanding of the basic functions of this cell rely" heavily upon extrapolation of in vitro observations to events in vivo. It is remarkably easy to disturb normal neutrophil physiology by removing cells from the circulation and great care must be exercised to obtain results which are relevant to events in vivo. Bacterial, hence endotoxin, contamination must be excluded from all buffers and attention must be paid to physical parameters such as pH or osmolarity, which have non-specific effects upon neutrophil function. If all of these factors are given consideration, the neutrophil provides an excellent opportunity to study the biochemical regulation of a complex cell in health and disease. Clinical suspicion of impaired function is usually aroused by a marked susceptibility to infection. This can occur in unusual sites and be caused by atypical pathogens. In those patients susceptible to infection, abnormal neutrophil function is a less likely cause than neutropenia or abnormal immune function, such as opsonic deficiency. The screening of neutrophil function is only appropriate when these other factors have been shown to be normal. Neutrophil killing is the end-point of a number of integrated functions, and a killing defect may be the result of abnormalities in one or more of these. It is therefore essential when testing for abnormal neutrophil killing to test a range of functions. When investigating the role of the neutrophil in the pathogenesis of a given disease, it is important to carefully select the function tests that are appropriate to the questions to be answered. Much of the confusion in the literature surrounding acquired abnormalities in neutrophil function can be explained by a critical appraisal of the techniques employed, which are often inadequate and chosen inappropriately. The neutrophil is a cell of great scientific interest and clinical relevance, but quantitative tests of neutrophil function must be applied with consideration of and an understanding of the relevant technical problems. SUMMARY

The complexity of the neutrophil response to inflammation creates many

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difficulties for the study of neutrophil function in vitro. The environment in which a neutrophil is placed can have marked effects upon a variety of cellular functions. Quantitative tests of neutrophil function present problems not only with assay design but also in the isolation of cells from peripheral blood without disturbing their normal physiology. It is desirable to isolate neutrophils from other leukocytes because soluble factors released by other cells can influence neutrophil function, and other cells may interfere with functional assays; for example, monocytes will phagocytose opsonized particles and eosinophils contain a potent peroxidase. Attention to physical parameters such as temperature, pH or osmotarity, and rigorous exclusion of endotoxin, permits neutrophils to be isolated in a resting state. Subsequent function tests must be selected with an understanding of normal neutrophil physiology and applied with an awareness of any associated technical problems. The investigation of abnormal neutrophil responses may necessitate the screening of several tests of function; for example, defective neutrophil killing may be the result of abnormal chemotaxis, phagocytosis or degranulation. Which tests are appropriate will depend upon the questions to be answered and on the quantity of cells available for study. REFERENCES Andersson T, Dahlgren C, Lew PD et at (1987) Celt surface expression of receptors on human neutrophils. Journal of Clinical Investigation 79: 1226-1233. Bass DA, Parce JW, Dechatelet LR et al (1983) Flow cytometric studies of oxidative product formation by neutrophils. A graded response to membrane stimulation. Journal oflmmunotogy 130(4): 1910-1917. Berkow RL & Baehner RL (1985) Volume dependent human blood polymorphonuclear leucocyte heterogeneity demonstrated with counterflow centrifugal elutriation. Blood 65(1): 71-78. Bevitacqua MP, Stengelin S, Gimbrone MA et al (1989) Endothelial leucocyte adhesion molecule-l: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243(4895): 1160-1165. Bignold LP & Ferrante A (1987) Mechanism of separation of polymorphonuclear leucocytes from whole blood by the one step Ficoll-Hypaque method. Journal of Immunological Methods 96: 29-33. Boveris A, Martino E & Stopanni AM (1977) Evaluation of horseradish peroxidase scopoletin method for measurement of hydrogen peroxide formation in biological systems. Analytical Biochemistry 80: 145-158. Boyum A (1968) Isolation of mononuclear cells and granulocytes from human peripheral blood. Scandinavian Journal of Clinical and Laboratory Investigation 21(Suppl. 97): 77-89. Bridges CG, DaSilva GL, Yamamura M e t al (1980) A radiometric assay for the combined measurement of phagocytosis and intracellular killing of Candida albicans. Clinical and Experimental Immunology 42: 226-233. Charo IF, Yuen C & Goldstein IM (1985) Adherence of human neutrophils to endothelial cell monolayers. Effects of temperature, divalent cations and chemotactie factors on strength of adherence measured with a new centrifugation assay. Blood 65: 473--479, Cramer R, Saranzo MR, Dri Pet al (1984) A simple reliable assay for myeloperoxidase activity in mixed human neutrophil--eosinophiI suspensions: Application to detection of myeloperoxidase deficiency. Journal of Immunological Methods 70: 119-127. Dale DC & Wolff SM (1971) Skin window studies of the acute inflammatory responses of neutropenic patients. Blood 38: 136--142. Dooley DC, Simpson JF & Merryman HT (1982) Isolation of large numbers of fully viable

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