Acauired Abnormalities of Polymorphonuclear Neutrophil Function

J. Fletcher, A. P. Haynes, S. M. Crouch SUMMA R Y. Normal polymorphonuclear neutrophils (PMN) in the circulation are resting cells expressing small numbers of low af6nity receptors. During infiammation they are upregulated to increase expression of high af6nity receptors and discharge both primary and secondary granules. This is reflected by a pattern of changes which can be detected in PMN from the circtdation of patients with infection, trauma or burns. Different patterns of abnormality occur in patients with systemic disease and increased risk of infection such as diabetes and renal failure. Functional defects also occur in PMN from patients with acquired blood disorders. It is likely that PMN contribute to tissue damage in infhumuatory and vascular diseases so that drugs which modulate PMN function will he of future therapeutic benefit.

The title of this review poses the problem of defining abnormal function. If circulating polymorphonuclear neutrophils (PMN) from a patient are compared with those of a healthy individual, are the differences to be defined as abnormalities or are they simply physiological responses to disease? Unfortunately our understanding of PMN function is based almost entirely on in vitro manipulation of PMN from healthy volunteers and extrapolation of these results to what may happen in vivo. Consequently, when PMN are sampled from the circulation of patients the differences from normal may be physiological but for the purposes of this review will be defined as ‘abnormal’. Neutrophil function is a term which must be further qualified. PMN can be stimulated in vitro with both opsonised particles and with soluble stimuli such as the peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), derived from bacterial cell walls, or the complement component C5a des arg, generated in serum by incubation with zymosan. Different receptors and transduction pathways are involved and independently regulated. A single stimulus can elicit a J. Fletcher. A. P. Haynes, S. M. Crouch, Medical Research Centre, City Hospital, Nottingham, UK. Blood Reviews (1990) 4. 103-110 ‘P 1990 Longman Group UK LLd

number of different functions such as chemotaxis, degranulation and the respiratory burst. Consequently no single test will be an adequate screen for abnormal function. Furthermore, the technology is particularly critical when measuring chemotaxis, phagocytosis and microbial killing. Chemotaxis is often measured by a leading edge technique but this does not examine the whole PMN population, tests of phagocytosis should involve the whole population and should distinguish particles which are simply attached to the cell’s surface from those ingested within the cell and microbial killing should be measured by a technique which is independent of the rate of phagocytosis. 1 Because it is so difficult and time consuming to quantitate these ‘physiological’ PMN functions it is often simpler to isolate a single product of a given PMN function and measure this by a biochemical technique. The respiratory burst, which accompanies activation, generates superoxide and hydrogen peroxide which are both easily quantitated. The PMN cytoplasm contains at least 3 distinct types of granule and markers specific for each can be measured to assess degranulation: beta glucuronidase for primary granules, lactoferrin for secondary granules and gelatinase for tertiary granules. The granule 0268-96OX/9O/ONWO103

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ACQUIRED ABNORMALITIES TabIe

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Neutrophil Granule Contents

Function

Primary granule

Secondary granule

Microbicidal agents

Myeloperoxidase Lysozyme Defensins Cationic proteins Elastase Cathepsin G Proteinases N acetylglucosamine Cathepsins B/D Glucuronidase Glycerophosphatase Mannosidase

Lysozyme Lactoferrin

Serine proteases Metalloproteinases Acid hydrolases

Others

contents are listed in the Table. Whichever physiological function or biochemical product is chosen the results have to be placed in the context of our present understanding of normal neutrophil function. Normal PMN Function Circulating PMN are resting cells, physiologically inactive and expressing only limited numbers of low affinity receptors on their surface. During inflammation their responses are orchestrated by a variety of cytokines and chemotactic factors so that they attach to vascular endothelium, move through the vessel wall and release their antimicrobial arsenal at the appropriate site (Figure). The first step requires increased expression of adhesion molecules known as the LFA-1, Mac-l, ~150, 95 family which are contained within secondary and tertiary granules and are translocated to the cells surface.2*3 PMN in the dilated capillaries can then attach to complimentary receptors on the endothelium. Next, in response to chemotactic factors PMN migrate through the capillary wall and move toward the focus of inflammation. PMN adopt a particular morphology with a blunt leading front and more attentuated body containing the nucleus and cytoplasmic structures including granules. Release of granules appears to occur at the blunt front end resulting in the highest concentration of receptors and a gradient of receptors across the surface of the cell which is able to interact with the gradient of chemoattractant resulting in directed movement towards the site of inflammation.4 Bound receptors are probably internalised, the ligand digested and the receptor returned to the cell surface.5 During chemotaxis PMN are exposed to cytokines and low concentrations of chemotactic factors which cause secondary and tertiary granule discharge resulting in translocation of receptors to the cell surface but little or no stimulation of the respiratory burst. The cells are now ‘primed’ so that their responses to opsonised particles and higher concentrations of soluble stimuli are enhanced. Chemoattractant gradi-

Tertiary granule

Collagenases

Gelatinase

B12 Binding Protein Cytochrome bzd5 Histaminase FMLP receptor Mac- 1 complex

Mac- 1 complex

ents lead the PMN to particles opsonised by antibody and complement which are phagocytosed. The important opsonins are the complement components C3b and C3bi, for which PMN express receptors CR1 and CR3, and immunoglobulin for which PMN express receptors FCRI, FCRII and FCRIII. The CR3 receptor is part of the Mac-l complex and is expressed on the surface of ‘primed’ cells; similarly the high affinity FCRI receptor is only expressed by primed or activated cells. Following ingestion, the intracellular phase of PMN function begins as the components of the NADPH oxidase are assembled and begin pumping superoxide anions into the phagocytic vacuole where they dismutate into hydrogen peroxide. At the same time primary granules, which are lysozomes, discharge their enzymes into the vacuole (see Table). Myeloperoxidase catalyses the production of hypochlorite from hydrogen peroxide and chloride, hypochlorite further reacts with amines to produce chloramines6 The killing of microbes depends upon a number of mechanisms including hypochlorite, chloramines, cationic proteins and defensins. Organisms and other ingested particles are digested by proteinases and hydrolases.6 The final phase in the neutrophil response to inflammation is the control of activated PMN to prevent release of toxic products including superoxide and proteinases which might damage normal tissues. This is achieved by hypochlorite which in the absence of other substrates reacts with primary granule enzymes and the NADPH oxidase inactivating and denaturing them.6 Exudate fluids also contain proteinase inhibitors to limit the action of digestive enzymes released extracellularly. Finally, macrophages recognise and ingest ageing PMN so preventing their contents spilling into the tissues.’ Acquired abnormalities in PMN function may result either from a failure of the normal response to inflammation or from a breakdown in the mechanisms protecting normal tissue from damage. There are many points in the normal sequence of events at

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PM,Y 1 ADHERENCE

11

MEDIATORS EXUDATE FACTORS eg. C55a

_ _ G

:

TISSUE FACTORS eg. adenosine

BACTERIAL eg. FMLP

WBC DERIVED PRODUCTS eg. P.A.F, LTB,, NAF(IL8)

Mediators such as ILI, TNF or lipopolysaccharide released at the inflammatory locus cau88 both PMN and endothelial cells to express enhanced numbers of surface adhesion molecules leading to margination

Cells leave the vascular space along a gradient of chemotactic factors originating from the inflammatory locus

t

eg. CRl, CR3

lmmunoglobulin

Receptors on the PMN surface recognise particles opsonised with complement or immunoglobulin.

Respiratory Burs+generates toxic oxygen species. Degranulutiotioth intra and extracellular releases antimicrobial agents and digestive enzymes.

Leads to microbial killing and digestion of debris Figure

The neutrophil response to inflammation

receptors

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which defects may occur and it can be seen that susceptibility may result from isolated impairment of chemotaxis, degranulation, phagocytosis or the respiratory burst. This emphasises the importance of testing several functions when screening for PMN abnormalities. However, even if this is done, the results on circulating PMN are very difficult to interpret as activated PMN will home to sites of inflammation, pass out of the circulation and leave functionally immature or exhausted cells to provide only a confused mirror image of what is really happening. It is very difficult to obtain exudate neutrophils from an abscess or inflamed joint and impossible to distinguish between fresh cells entering from the circulation and older dying cells. Nevertheless it is only by examining ‘abnormal’ PMN from real patients that in vitro predictions can be verified and refined. PMN Function in Inflammation Infection

Morphological changes in circulating PMN in association with infection have been recognised for many years. Primary granules may fuse to form secondary lysozomes which are recognised as toxic granulation and the secondary lysozomes may then fuse to produce Dhole bodies. Specific enzyme staining shows a pattern with reduction of myeloperoxidase and increase in alkaline phosphatase. Myeloperoxidase is contained within primary granules and loss of this enzyme suggests that the primary granules of circulating cells have been discharged and their enzymes partially inactivated. Alkaline phosphatase is a cell membrane marker in human PMN and indicates the age of the cells since younger PMN contain more enzyme. Resting levels of superoxide and hydrogen peroxide are not increased but, at least in a subpopulation of the cells, the respiratory burst is primed to increase the response to phagocytosis of opsonised particles.8 Circulating PMN also show enhanced phagocytosis associated with increased expression of the high affinity immunoglobulin receptor FCRl .9 Thus in the circulation of patients with infection there are increased numbers of young PMN, at least some of which are upregulated as shown by increased expression of FCRI and increased respiratory burst activity, but there is an overall reduction in their primary granule enzyme content particularly myeloperoxidase. The net effect of these changes is difficult to predict but microbicidal activity is usually reduced on in vitro testing and the cells may therefore be less effective than normal in combating infection.** Trauma

Part of the difficulty in interpreting results of function in patients with infection is the great tion shown by PMN from different normal viduals although the responses of cells from a

PMN variaindisingle

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individual are usually consistent. Surgical trauma has important effects upon PMN but is also a situation where it is possible to study the effects of inflammation as the patients can act as their own controls. A study of women undergoing elective hysterectomy for non-malignant conditions shows a similar pattern of changes to those reported during infection. There is a rapid fall in the level of the primary granule enzyme myeloperoxidase and the secondary granule protein transcobalamin III accompanied by a rise in the level of these proteins in the plasma, indicating that PMN have been stimulated to discharge both primary and secondary granules. 11v12 These changes in granule content are accompanied by an overall reduction in intracellular microbicidal ability without any impairment in phagocytic ability. These changes are not explained by influx into the circulation of young cells as they do not correlate with the numbers of circulating cells nor with changes in alkaline phosphatase.” The reduction in granule proteins develops within 5 h of surgery and persists for several days. When the cells are stimulated with C5a des arg they show reduced responses as measured by the respiratory burst but their responses can be increased normally if they are first primed by incubation with tumour necrosis factor alpha. The explanation may be that trauma activates complement to generate C5a which rapidly binds to its receptor and is then internalised so that the cells become unresponsive until fresh receptors are expressed. The significance of these changes for postoperative complications is not clear. Activated PMN in the circulation are both more rigid and more adherent than normal and are consequently more likely to become trapped in small blood vesels and so may contribute to postoperative thrombosis. The reduction in intracellular microbicidal ability of individual cells may be compensated by the increase in the number of cells in the circulation and probably does not reflect the functional capability of cells migrating into the site of inflammation. Very severe trauma is sometimes complicated by the adult respiratory distress syndrome, ARDS, the hallmark of which is the accumulation of protein rich fluid in the lungs. One hypothesis is that release of cytokines leads to massive activation of complement within the circulation which in turn activates PMN so that they adhere in the pulmonary circulation where they release oxygen radicals and enzymes which damage the vascular endothelium.13 Vascular permeability is certainly affected by PMN and ARDS in experimental animals does not occur in the absence of PMN.14*15 However, since ARDS can occur in neutropenic patients other mechanisms must also be important. Burns

Severe burns cause the same changes in circulating PMN as infection and surgical trauma with degranu-

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lation of both primary and secondary granules.16 However, unlike surgical trauma, the changes are at their maximum 3-5 days after the injury and are associated with loss of opsonic and chemotactic activity in the serum due to depletion of complement components. i’ Many severely burned patients require parenteral nutrition and this may contribute to impairment of PMN chemotaxis and microbial killing. It is perhaps not surprising that infection is the major complication of burns.

that before elastase can damage connective tissue it is necessary to postulate that the antiproteinases are inactivated in the same way as lactoferrin may be inactivated in rheumatoid joints. No specific mechanisms have been postulated in the other diseases in which neutrophil mediated tissue damage is suggested and it appears to be assumed that simply the presence of neutrophils is enough.

Exudate PMN

It has been suggested that PMN may contribute to tissue damage surrounding infarcts.‘l The evidence comes from experimental myocardial infarction in animals where the extent of muscle damage is reduced by neutropenia or drugs which block PMN activation. The reperfusion injury following clot lysis may also be related to PMN as monoclonal antibodies against surface adhesion molecules limit the extent of the injury. ” It is suggested that activated PMN are stiff and adherent, so plug small blood vessels and release toxic products. There is no direct evidence for this in humans although drugs known to be of benefit following myocardial infarction such as beta-adrenergic blockers and calcium channel antagonists also reduce PMN responses. PMN may contribute to the varicose ulceration of patients with venous hypertension.23 It has been shown that PMN pool in the legs of such patients and block capillary loops in the skin so contributing to tissue hypoxia. The hypothesis is that hydrostatic pressure causes haemoconcentration within the skin capillaries resulting in anoxia and release of inflammatory mediators which in turn activate PMN to adhere to the capillary endothelium generating a vicious cycle of increasing tissue hypoxia.

PMN in inflammatory exudates have been acted upon by cytokines and chemoattractants which should result in expression of adhesion proteins and up regulation of responses to stimuli such as CSa des arg and FMLP. In experimental models such as ellicited cells in the guinea pig peritoneum or suction blisters in human skin these in vitro predictions are confirmed.” However, in uncontrolled human pathology they are much more difficult to demonstrate. PMN from rheumatoid joints actually show reduced responses to C5a des arg probably due to internalisation of bound receptors. 5 Whenever an infiltrate or exudate of PMN accumulates there is the potential for tissue damage by release of toxic oxygen radicals and proteinases. This mechanism has been suggested in a large number of diseases including chronic bronchitis and emphysema, gout, rheumatoid arthritis, immune vasculitis, glomerulonephritis, psoriasis and ulcerative colitis. This is an attractive hypothesis but there is actually very little evidence to implicate PMN in causing tissue damage in humans as distinct from animal models. The presence of PMN is not enough as the fact of rapid wound healing following neutrophil infiltration indicates that there are potent mechanisms to limit tissue damage. Joint damage in rheumatoid arthritis is blamed on oxygen radicals from inflammatory cells. It is suggested that superoxide can mobilise iron from ferritin and that the iron would then be available to catalyse the Haber Weiss reaction which produces very reactive and toxic hydroxyl radicals. ’ 9 However, PMN also release a very potent iron binding protein termed lactoferrin, which will mop up free iron. It is therefore also necessary to postulate that the lactoferrin is denatured by oxygen radicals and does not function. This is an interesting hypothesis but there is very little evidence to support its relevance in human disease. In lung injury the emphasis has been on release of proteinases, particularly elastase which can cause alveolar damage in experimental animals.6 In humans, neutrophil elastase complexed with antiproteinase is detectable in the plasma and the levels certainly increase when PMN are activated.” However, in vitro, very little elastase is released by activated neutrophils and there are potent antiproteinases in plasma so

Haemostasis

Acquired PMN Abnormalities in Systemic Disease

Diabetes Meilitus

Diabetic patients are susceptible to infections and this has lead to much investigation of PMN function. Most of the data in the literature is conflicting due to the use of inadequate and poorly understood techniques. Abnormalities have been described in adherence, chemotaxis and microbicidal activity but most can be explained by ketoacidosis and hyperosmolar states which are not relevant to the majority of diabetics. However, if the initial rate of phagocytosis rather than end point is measured, then phagocytosis is defective in insulin dependent diabetics attending a routine diabetic clinic.24 The mechanism for this effect is not known but may involve the glycosylation of receptors for immunoglobulin expressed on resting cells. Glycosylation may lead to further protein damage as the reaction between glucose and a protein backbone releases oxygen radicals. Claims that microbicidal activity is defective can only be justified

108 ACQUIRED ABNORMALITIES OF POLYMORPHONUCLEAR if microbial killing is measured measured independently of phagocytosis and this has not been done. Nevertheless an interesting hypothesis suggests that high plasma glucose levels stimulate conversion of glucose to its alcohol, sorbitol, by the aldose reductase pathway consuming NADPH and depriving the respiratory burst of this substrate.25 As yet there is no convincing evidence of defective intracellular microbial killing or that NADPH is critically depleted in diabetic PMN. End Stage Renal Failure

Patients with both acute and chronic renal failure die of infections. Furthermore treatment by continuous ambulatory peritoneal dialysis (CAPD) is often complicated by peritonitis. The reasons are complex. There is certainly a mild but measurable defect in opsonic activity in uraemic plasma and there is a lack of opsonic proteins in peritoneal dialysis fluid even after a 4-hour intraperitoneal dwell.26 Circulating PMN from patients treated either with haemodialysis or peritoneal dialysis show reduced responses to C5a des arg due to a reduction in the expression of surface receptors for this ligand. The mechanism is thought to be activation of complement within the circulation of these patients so that PMN are exposed to relatively high concentrations of C5a leading to internalisation of bound receptors. *’ Complement activation on the haemodialysis membrane produces sequestration of activated PMN in the lungs with consequent neutropenia and hypoxia during dialysis. In spite of these changes in complement, when normal PMN are suspended in uraemic plasma or used peritoneal dialysis fluid they phagocytose preopsonised organisms normally but fail to kill the ingested bacteria or fungi.** Both uraemic plasma and used peritoneal dialysis fluid contain a low molecular weight factor which interferes with intracellular killing by inhibiting discharge of primary granule enzymes into the phagocytic vacuole. Pregnancy The fetus is antigenically foreign to the mother and can be regarded as a foreign body. It is tolerated by the mothers immune system, of which PMN are a part, and therefore it is not surprising that pregnancy affects PMN function. As early as the twelfth week of gestation, circulating PMN show a significant reduction in myeloperoxidase associated with defective fungicidal ability.*’ As yet changes in secondary granule proteins and surface receptors have not been investigated. Throughout pregnancy there is a progressive rise in neutrophil alkaline phosphatase reflecting rising levels of oestrogen and progesterone. After delivery these changes disappear rapidly and PMN sampled from the circulation 4 weeks later are normal. The functional significance of these changes is completely unknown.

NEUTROPHIL

FUNCTION

Primary Blood Diseases Myeloproliferative Disorders

The function of PMN is linked to the number in the circulation. If demand is increased as occurs in infection then the number increases and as the infection is overcome so the number declines. By the same argument if there is a primary defect of PMN function then the number in the circulation will increase. How number and function are linked is not known but in the chronic myeloproliferative disorders, chronic myeloid leukaemia (CML) and myeloid metaplasia (MM), there are certainly abnormalities of function. Chronic Myeloid Leukaemia (CML) . CML is characterised by the Philadelphia chromosome, a translocation between the long arms of chromosomes 9 and 22 which brings together the c-abl oncogene from 9 and the bcr region of 22. The result is an abnormal mRMA which translates a protein with tyrosine kinase activity. 3o How this protein results in disease is not known but it is usually assumed that there will be an abnormality of early myeloid progenitor cells although there is no evidence for this. All the evidence points to ‘discordant maturation’ with accumulation of a population of PMN which are morphologically relatively mature but functionally immature.31 Buffy coat leukocytes, with no attempt to separate more mature cells, show only a slight (lo-12%) reduction in phagocytosis while at the same time ability to kill organisms is severely reduced.32 Discharge of secondary granules and superoxide production in response to phagocytosis of opsonised particles is normal but there is a failure of primary granules to fuse with the phagocytic vacuole so that normal myeloperoxidase catalysed production of hydrogen peroxide does not occur.33 However, even this is a relative defect as it is possible to isolate a population of PMN with almost normal function suggesting that the problem is not primarily within the PMN but is secondary to the environment in which they mature. The CML PMN also show low levels of alkaline phosphatase which probably reflects their longevity within the circulation and in the splenic pool. The alkaline phosphatase score improves following treatment to reduce the total number of circulating PMN and also improves following splenectomy or with intercurrent infection. An interesting in vitro observation is that the alkaline phosphatase score improves when PMN are incubated with products of mononuclear cells and in particular with recombinant G-CSF. 34 This data may be interpreted as indicating that in the right environment CGL neutrophils can mature normally but it is more likely that incubation over several days with cytokines selects for differentiation of a residual normal population of progenitor cells. Myeloid Metaplasia. Like CML this is a clonal pro-

liferation of myeloid cells and the marrow fibrosis is

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secondary and not clonal. No chromosomal abnormality has been identified and the evidence for clonality is based upon glucose-6-phosphatase dehydrogenase isoenzyme patterns. PMN functions have some features similar to CML as phagocytosis is normal while microbicidal activity is impaired. 3s Unlike CML myeloperoxidase in circulating PMN is deficient and there is a correlation between levels of this enzyme and ability to kill Candida. Alkaline phosphatase is either normal or increased. The pattern of these changes is similar to those seen in response to inflammation. To date there is no data about receptor expression, secondary granule proteins or the development of PMN function as the disease progresses. Myelodysplasia

This increasingly common diagnosis is a description of morphological changes in the bone marrow of patients who show a variety of abnormality in circulating red cells, platelets and PMN. There is considerable morbidity and mortality due to’infection which is certainly related to the number of circulating PMN and may also be related to abnormal function. Defective chemotaxis, phagocytosis and microbial killing have been described as has partial or complete lack of granule enzymes and alkaline phosphatase.36 However it has not been possible to correlate particular functional defects with either the FAB classification of myelodysplasia or the presence of particular chromosomal abnormalities. Since a number of patients progress to acute myeloid leukaemia the disease appears to be an abnormality of early progenitor cells which produces a variety of phenotypic abnormalities in circulating cells. Paroxysmal Nocturnal Haemoglobinuria

(PNH)

This disease is another acquired clonal disorder of marrow stem cells resulting in defective expression of receptors which are anchored in the cell membrane by a phosphoinositol (PI) glycan link. The PMN immunoglobulin receptor FCRIII is PI linked and its expression in PNH is reduced to less than 10% of normal. The result is decreased binding of dimeric immunoglobulin G but normal phagocytosis and superoxide production.37 Drug Effects on PMN Function The implication of the suggestion that neutrophils are important in causing tissue damage is that drugs which interfere with neutrophil function should be protective. Both steroids and non-steroidal antiinflammatory drugs do indeed interfere with neutrophi1 function in vitro but only at high concentrations. The mechanism appears to be a blockade of receptors on the cell surface as the effect can be removed by washing. 38 The clinical relevance of this observation

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is doubtful since the concentration of non steroidal agents required is higher than can be achieved in the plasma because of the high protein binding of these drugs. Massive doses of steroids are given intravenously to patients with renal graft rejection or vasculitis and have been given to patients with ARDS. The half-life of steroid in the circulation is a matter of minutes and therefore circulating neutrophils will only be exposed to effective doses for a relatively short time. Whether or not this is important for the effect of intravenous steroids is not clear. There are a number of other drugs which, at least in theory, should effect function as PMN express receptors for catecholamines, histamine and adenosine. In each case receptor binding leads to a reduction in responsiveness of PMN to chemoattractants including FMLP and C5a des arg. The intracellular transduction pathways involve adenyl cyclase and cyclic AMP although in other respects they almost certainly differ. Histamine reduces responses to chemoattractants at concentrations which are achieved in vivo and acts via an H2 receptor. 39 The PMN responsiveness is restored by treatment with H2 antagonists both in vitro and in vivo. Adenosine acts via A2 receptors. In vitro methylxanthines such as theophylline and pentoxifylline compete for this receptor so that at low concentrations they increase the responsiveness of PMN to chemoattractants. At higher concentrations they inhibit phosphodiesterase leading to an increase in intracellular cyclic AMP and impairment of PMN responses. This biphasic effect occurs in vivo so that immediately following oral ingestion of pentoxifylline there is down regulation of neutrophil responses followed by an increase in responsiveness before a return to normal.40 Catecholamines and calcium channel antagonists also down regulate neutrophil responses in vitro but their effects in vivo are unknown in spite of their very widespread use. Conclusions This review reveals that while a great deal is known concerning PMN function in vitro, its application to the understanding of human pathology is only just beginning. The fact that it is possible to use in vitro data to interpret acquired defects of PMN function in disease goes some way to validate the model of PMN function suggested by in vitro results. Greater understanding of PMN function in disease and the use of drugs, including cytokines, to manipulate PMN function will be important developments within the next few years. References 1. Harvey D M, Sheppard K, Fletcher J 1986 A method for measuring rate of neutrophil phagocytosis of Staphyloccocus epidermidis or Candida guilliermondii using uptake of tritiated uridine. Journal of Immunological Methods 93: 259-264 2. Bainton D F, Miller L J, Kishimoto T K, Springer T A 1987

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6. I.

8.

9.

10. 11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

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Leukocyte adhesion receptors are stored in peroxidasenegative granules of human neutrophils. Journal of Experimental Medicine 166: 1641-1653 Tonnesen M G, Andersen D C, Springer T A et al 1989 Adherence of neutrophils to cultured human micro vascular endothelial cells. Stimulation of chemotactic peptides and lipid mediators and dependence upon the Mac-l, LFA-1, p 150, 95 glycoprotein family. Journal of Clinical Investigation 83: 637-646 Nunoi H, Endo F, Chikazawa S, Matsuda I 1985 Regulation of receptors and digestive activity towards synthesised formylchemotactic peptide in human polymorphonuclear leucocytes. Blood 66: 106-114 Niedel J E, Kahane I, Cuatrecasas P 1979 Receptor mediated internalisation of fluorescent chemotactic peptide by human neutrophils. Science 2%: 1412-1414 Weiss S J 1989 Tissue destruction by neutrophils. New England Journal of Medicine 320: 365-376 Savill J S, Wyllie A H, Henson J E et al 1989 Macrophage phagocytosis of ageing neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. Journal of Clinical Investigation 83: 865-875 Bass D A, Olbrantz P, Sjejda P, Seed M C, McCall C E 1986 Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection. Journal of Immunology, 1%: 860-866 Simms H H, Frank M M, Quinn T C, Holland S, Gaither T A 1989 Studies on phagocytosis in patients with acute bacterial infections. Journal of Clinical Investigation 83: 252-260 Solberg C 0, Hellum K B 1972 Neutrophil granulocyte function in bacterial infections. Lancet ii:727-730 El-Maallem H, Fletcher J 1981 Effects of surgery on neutrophil granulocyte function. Infection and Immunity 32: 38-41 Davies J M, Sheppard K, Fletcher J 1983 The effect of surgery on the activity of neutrophil granule proteins. British Journal of Haematology 53: 5-13 Hammerschmidt D E, Weaver L J, Hudson L D, Craddock P R, Jacob H S 1980 Association of complement activation and elevated plasma C5a with adult respiratory distress syndrome. Lancet i: 941-949 Williamson L M, Sheppard K, Davies J M, Fletcher J 1986 Neutrophils are involved in the increased vascular permeability produced by activated complement in man. British Journal of Haematology 64: 375-384 Till G 0, Johnson K J, Kunkel R, Ward P A 1982 Intravascular activation of complement and acute lung injury. Dependency on neutrophils and toxic oxygen metabolites. Journal of Clinical Investigation 69: 1126-1135 Davis J M, Dineen P, Gallin J I 1980 Neutrophil degranulation and abnormal chemotaxis after thermal injury. Journal of Immunology 124: 1467-1471 Dobke M K, Pearson G, Roberts C et al 1983 Effect of circulating fibronectin on stimulation of leucocyte oxygen consumption and serum opsonising function in burned patients. Journal of Trauma 23: 882-890 Patv P B. Graeff R W. Waldman F M. Hunt T K. Mathes S J 1988 Biologic priming of neutrophils in subcutaneous wounds. Archives of Surgery 123: 1509-1513 Blake D R, Hall N D, Bacon P A et al 1981. The importance of iron in rheumatoid disease. Lancet ii: 1142-l 143 Weissman G, Smolen J E, Korchak H M 1980 Release of inflammatory mediators from stimulated neutrophils. New England Journal of Medicine 303: 27-34

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21. S&mid-Schonbein G W, Engler R L 1986 Granulocytes as active participants in acute myocardial ischaemia and infarction. Journal of Cardivascular Pathology i: 15-30 22. Simpson P J, Todd R F, Fantone J C et al 1988. Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti CD1 lb) that inhibits leucocyte adhesion. Journal of Clinical Investigation sr: 624-629 23. Thomas P R S, Nash G B, Dormandy J A 1988 White cell accumulation in dependent legs of patients with venous hypertension: a possible mechanism for trophic changes in the skin. British Medical Journal 2%: 1693-1695 24. Davidson N J, Sowden J M, Fletcher J 1984 Defective phagocytosis in insulin controlled diabetics: evidence for a react&m between glucose and opsonising proteins. Journal of Clinical Pathology 37: 783-786 25. Wilson R M, Reeves W G 1986 Neutrophil phagocytosis and killing. Clinical and Experimental Immunology 63: 478-484 26. Harvey D M, Sheppard K J, Morgan A G, Fletcher J 1987 The effect of dialysate fluids on phagocytosis and killing by normal neutrophils. Journal of Clinical Microbiology 25: 1424-1427 27. Lewis S L, Van Epps D E, Chenoweth D E 1986 C5a receptor modulation on neutrophils and monocytes from chronic haemodialysis and peritoneal dialysis patients. Clinical Nephrology 26: 37-44 28. Harvey D M, Sheppard K J, Morgan A G, Fletcher J 1987 Neutrophil function in patients on continuous ambulatory peritoneal dialysis. British Journal of Haematology 68: 273-278 29. El-Maallem H, Fletcher J 1980 Impaired neutrophil function and myloperoxidase deficiency in pregnancy. British Journal of Haematology 44: 375-381 30. Shtivelman E, Lifschitz B, Gale R P, Canaani E 1985 Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 315: 550-554 31. Strife A, Clarkson B 1988 Biology of chronic myelogenous leukaemia: Is discordant maturation the primary defect? Seminars in Haematology 25: l-19 32. El-Maallem H, Fletcher J 1976 Defective neutrophil function in chronic granulocytic leukaemia. British Journal of Haematology 34 95-103 33. El-Maallem H, Fletcher J 1979 Defective hydrogen peroxide production in chronic granulocytic leukaemia neutrophils. British Journal of Haematology 41: 49-55 34. Yuo A, Kitagawa S, Okabe T et al 1987 Recombinant GCSF and repair in myelodysplasia and chronic granulocytic leukaemia. Blood 70: 404-4i 1 35. El-Maallem H. Fletcher J 1977 Imoaired neutroohil function and myeloperdxidase deficiency in myeloid metaplasia. British Journal of Haematology 37: 323-329 36. Boogaerts M A, Nelissen V, Roelant C, Goossens W 1983 Blood neutrophil function in primary myelodysplastic syndromes. British Journal of Haematology 55: 217-227 31. Huizinga T W J, Schoot C E, Jost C et al 1988 The PIlinked receptor FcR III is released on stimulation of neutrophils. Nature 333: 667-669 38. Davies J M. Sheooard K. Fletcher J 1984 Inhibition of human neutroph;i secondary granule discharge by antiinflammatory agents. Inflammation 8: 343-352 39. Seligmann B E, Fletcher M P, Gallin J I 1983 Histamine modulation of human neutrophil oxidative metabolism, locomotion degranulation and membrane potential changes. Journal of Immunology 130: 1902-1902 40. Slater K, Wiseman M S, Shale D J, Fletcher J 1988 The effect of pentoxifylline on neutrophil function in vitro and ex vivo in human volunteers. Pentoxifylline and leucocyte function 1: 115-123