Laurence A. Boxer

The phagocytes: neutrophils and monocytes

The production and deployment of phagocytes are central functions of the hematopoietic system. In the 1950s, radioisotopic studies demonstrated the high production rate and short lifespan of neutrophils and allowed researchers to follow the monocytes as they moved from the marrow through the blood to become tissue macrophages, histiocytes, and dendritic cells. Subsequently, the discovery of the colony-stimulating factors greatly improved understanding the regulation of phagocyte production. The discovery of the microbicidal myeloperoxidase-H2O2-halide system and the importance of NADPH oxidase to the generation of H2O2 also stimulated intense interest in phagocyte disorders. More recent research has focused on membrane receptors and the dynamics of the responses of phagocytes to external factors including immunoglobulins, complement proteins, cytokines, chemokines, integrins, and selectins. Phagocytes express toll-like receptors that aid in the clearance of a wide range of microbial pathogens and their products. Phagocytes are also important sources of pro- and anti-inflammatory cytokines, thus participating in host defenses through a variety of mechanisms. Over the last 50 years, many genetic and molecular disorders of phagocytes have been identified, leading to improved diagnosis and treatment of conditions which predispose patients to the risk of recurrent fevers and infectious diseases.

OXIDANT ACTIVITY IN EXPIRED BREATH OF PATIENTS WITH ADULT RESPIRATORY DISTRESS SYNDROME

Hydrogen peroxide levels were measured in the breath condensate of 43 patients receiving mechanical ventilation. In 16 patients the mean breath condensate peroxide level was 1.68 +/- 0.35 mumol/l on the day they met diagnostic criteria for adult respiratory distress syndrome (ARDS). The peak breath condensate peroxide level in the 27 patients in whom ARDS did not develop was significantly lower (0.34 +/- 0.08 mumol/l). Plasma lysozyme, a measure of in-vivo neutrophil turnover, was significantly higher in ARDS than in non-ARDS patients (9.2 +/- 2.2 U/ml v 3.4 +/- 1.1 U/ml). These findings support the hypothesis that neutrophil activation and oxidant production are involved in the pathogenesis of ARDS.

Correction of leukocyte function in Chediak-Higashi syndrome by ascorbate.

Because ascorbate potentiates chemotaxis of normal leukocytes, we examined the effect of ascorbate on polymorphonuclear leukocytes from a patient with the Chediak-Higashi syndrome. Chemotactic migration was 104+/-16 leukocytes per 10 fields (mean+/-S.D.) initially and 258+/-44 (P less than 0.001) after ascorbate, as compared to 182+/-10 in controls. There was no bactericidal activity by 40 minutes in the patients untreated leukocytes. After ascorbate bactericidal activity of patient and untreated control cells was the same. The addition of ascorbate reduced cAMP levels in the patients cells from a mean of 34.5 pmoles per 10(7) polymorphonuclear leukocytes to 5.9, as compared to a control value of 3.1+/-1.4. The association of elevated cAMP and impaired function in the polymorphonuclear leukocytes of patients with the Chediak-Higashi syndrome may be related to abnormal microtubular assembly.

Stable long-term risk of leukaemia in patients with severe congenital neutropenia maintained on G-CSF therapy

In severe congenital neutropenia (SCN), long‐term therapy with granulocyte colony‐stimulating factor (G‐CSF) has reduced mortality from sepsis, revealing an underlying predisposition to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). We have reported the early pattern of evolution to MDS/AML, but the long‐term risk remains uncertain. We updated a prospective study of 374 SCN patients on long‐term G‐CSF enrolled in the Severe Chronic Neutropenia International Registry. Long‐term, the annual risk of MDS/AML attained a plateau (2·3%/year after 10 years). This risk now appears similar to, rather than higher than, the risk of AML in Fanconi anaemia and dyskeratosis congenita.

Diminished polymorphonuclear leukocyte adherence: Function dependent on release of cyclic AMP by endothelial cells after stimulation of β-receptors by epinephrine

To investigate the biochemical and cellular basis for the rise in polymorphonuclear leukocyte (PMN) count during epinephrine administration, PMN from subjects receiving epinephrine were studied for their capacity to adhere to nylon wool fibers and endothelial cell monolayers. After administration of epinephrine, the PMN count increased by 80% at 5 min, and isolated PMN adherence to nylon fibers fell from a base line of 44+/-2-18+/-3%. In contrast, when subjects were infused with the beta-antagonist propanolol before receiving epinephrine, the PMN count failed to rise and PMN adherence was normal. Exposure of PMN endothelial cell monolayers to 0.1 muM epinephrine led to diminished PMN adherence that could be blocked by 10 muM propanolol but not by 10 muM phentolamine. Sera obtained from subjects 5 min after receiving epinephrine or from supernates derived from endothelial cell monolayers exposed to 90 nM epinephrine inhibited PMN adherence to nylon fibers. Addition of anticyclic AMP antisera but not anticyclic guanosine monophosphate antisera to the postepinephrine sera or to the postepinephrine supernate derived from the endothelial cell monolayers abolished their inhibitory effect of PMN adherence to nylon fibers. In contrast, direct exposure of PMN to epinephrine failed to affect their adherent properties. Because it has been previously shown that endothelial cells contain beta-receptors and respond to catecholamines by raising their intracellular concentrations of cyclic AMP, and that PMN adherence is attenuated by cyclic AMP, it would appear that diminished PMN adherence after epinephrine administration is mediated through endothelial cell beta-receptor activity, which in turn impairs PMN margination in vivo and could account for the rise in circulating PMN.

Protection of Granulocytes by Vitamin E in Glutathione Synthetase Deficiency

The administration of vitamin E (alpha-tocopherol) was found to improve polymorphonuclear leukocyte function in an infant with congenital deficiency of glutathione synthetase activity. Before therapy with vitamin E the abnormal leukocytes exposed to phagocytic challenge showed oxidant damage. They released 60 per cent more hydrogen peroxide than did normal leukocytes, iodinated only 20 to 25 per cent of the normal number of particles, and were unable to kill bacteria as effectively as normal leukocytes although the rates of phagocytosis were normal. These functional abnormalities disappeared when the patient was placed on 400 IU of alpha-tocopherol daily for three months. Associated with the functional improvement was a normalization of microtubule assembly during phagocytic challenge.

Purified human plasma kallikrein aggregates human blood neutrophils.

Exposure of human blood polymorphonuclear leukocytes (PMN) to purified active plasma kallikrein resulted in PMN aggregation when kallikrein was present at concentrations ranging from 0.4 to 0.6 U/ml (0.18-0.27 microM). Kallikrein-induced PMN aggregation was not mediated through C5-derived peptides, because identical responses were observed whether or not kallikrein had been preincubated with an antibody to C5. Moreover, kallikrein was specific for aggregating PMN, because no aggregation was observed with Factor XII active fragments (23 nM), Factor XIa (0.6 U/ml or 15nM), thrombin (1.6 microM), plasmin (2 microM), porcine pancreatic elastase (2 microM), bovine pancreatic chymotrypsin (2 microM), or bradykinin (1 microM). Bovine pancreatic trypsin (2 microM) aggregated PMN, but to a lesser extent than kallikrein (0.18 microM). Kallikrein was a potent aggregant agent for PMN because similar responses were observed with kallikrein (0.5 U/ml or 0.23 microM) and an optimal dose (0.2 microM) of N-formyl-methionyl-leucyl-phenylalanine. In addition, PMN incubation with kallikrein resulted in stimulation of their oxidative metabolism as assessed by an increased oxygen uptake. Neutropenia and leukostasis observed in diseases associated with activation of the contact phase system may be the result of PMN aggregation by plasma kallikrein.

Management of kostmann syndrome in the g-csf era

Kostmann (1956, 1975) described an inherited haematological disorder with severe neutropenia with an absolute neutrophil count (ANC) , 0 ́2 10/l and early onset of severe bacterial infections. Most children died because of these infections, despite antibiotic treatment. Different treatment strategies for congenital neutropenia (CN) included use of steroids and lithium (Barrett et al, 1977; Hraker et al, 1977), but these treatments did not show any long-term effect on neutrophil counts. Bone marrow transplantation (BMT) was the only curative treatment option for patients with human leucocyte antigen (HLA)compatible donors (Rappeport et al, 1980). Some patients who survived infections and treatment, however, underwent malignant transformation into acute myeloid leukaemia (AML) (Gilman et al, 1970; Rosen & Kang, 1979). The availability of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) in 1987 (Nagata et al, 1986; Souza et al, 1986) dramatically changed both the prognosis of CN and the quality of life for patients with CN (Bonilla et al, 1989; Welte et al, 1990). Since the establishment of the Severe Chronic Neutropenia International Registry (SCNIR) in 1994, data on 304 patients with CN have been collected to monitor the clinical course, treatment and disease outcomes in these patients. In clinical trials, . 90% of these patients responded to rHuG-CSF treatment with an increase in ANC . 1 ́0 10/l. Importantly, all responding patients required significantly fewer antibiotics and days of hospitalizations (Dale et al, 1993; Bonilla et al, 1994; Welte & Dale, 1996; Freedman, 1997; Welte & Boxer, 1997). Haematopoietic stem cell transplantation (HSCT) remains the only currently available treatment for those patients refractory to rHuG-CSF treatment that continue to have severe and life-threatening bacterial infections. Data from the SCNIR also demonstrate that for all CN patients, < 9% will develop leukaemia regardless of their treatment or response (Bonilla et al, 1994; Freedman, 1997; Welte & Boxer, 1997). The molecular and genetic basis for this disease is still largely unknown.

Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.

The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.

Neutrophil elastase mutations and risk of leukaemia in severe congenital neutropenia.

Severe congenital neutropenia (SCN) is a heterogeneous bone marrow failure syndrome predisposing to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). We studied 82 North American and Australian SCN patients enrolled in the Severe Chronic Neutropenia International Registry who were on long‐term treatment with granulocyte colony‐stimulating factor and for whom the neutrophil elastase (ELA2) gene was sequenced. There was no significant difference in the risk of MDS/AML in patients with mutant versus wild‐type ELA2: the respective cumulative incidences at 15 years were 36% and 25% (P = 0·96). Patients with either mutant or wild‐type ELA2 should be followed closely for leukaemic transformation.