Clin. exp. Immunol. (1976) 26, 463-468.

Neutrophilic granulocytes in acute bacterial infection SEQUENTIAL STUDIES ON LYSOZYME, MYELOPEROXIDASE AND LACTOFERRIN N. E. HANSEN, H. KARLE, V. ANDERSEN, J. MALMQUIST & G. E. HOFF Department ofInfectious Diseases; Division ofHaematology A, Department ofMedicine TA, Department ofClinicalMicrobiology; Rigshospitalet, University of Copenhagen, Denmark and Department of Medicine, University of Lund, Malmo General Hospital, Malmi, Sweden

(Received 8 July 1976) SUMMARY

The changes in intraneutrophilic and plasma concentrations of the three antibacterial proteins lysozyme, lactoferrin, and myeloperoxidase were studied sequentially during acute bacterial infection in nine patients. Intraneutrophilic concentrations of the three proteins were decreased by more than 50% during the 1st week of infection, followed by a slow increase over the following 2 weeks. Nadir values coincided with maximal toxic granulation of the neutrophils. The data suggest that neutrophilic granulocytes are deficient during early bacterial infection, possibly because of deficient synthesis of antibacterial proteins in the bone marrow, and that neutrophil toxic granulation is the visual counterpart of this defect. The plasma concentrations of the three proteins showed considerable differences: whereas plasma lysozyme did not show any sequential changes, plasma myeloperoxidase was high at the start of infection and quickly decreased towards normal values, and plasma lactoferrin, high in the first samples, showed a secondary peak 1 week after onset of disease, before normalization was seen. These differences may result from differences in the signals governing the release of the proteins from neutrophilic granulocytes, and it is suggested that these signals are specific for the individual antibacterial protein and not for the different types of neutrophil granules.

INTRODUCTION Some of the most interesting and perplexing changes of neutrophil function are those associated with bacterial infection. These changes include alterations in neutrophil morphology (vacuolization, toxic granulation), reduced bactericidal capacity (Copeland et al., 1971, Soelberg & Hellum 1972, Messner et al., 1973; Koch 1974) and reduced levels of the bacteriolytic enzyme lysozyme (Hansen & Andersen 1973); besides, intraneutrophilic antibacterial proteins have been shown to be released from the cells during phagocytosis (Cohn & Hirsh, 1960). Hitherto, however, most studies have been conducted at one time only during the infectious process, or in in vitro systems, whereas few studies are available on the sequence of events during bacterial infection in vivo. In the present study we investigated, during the course of bacterial infection in man, neutrophil morphology and the intraneutrophilic and plasma levels of the antibacterial proteins lysozyme, lactoferrin and myeloperoxidase, which all originate in the neutrophil granules. Our studies on lymphocytes and antibodies are reported in the companion article (Andersen et al., 1976). Patients were elected for study when they had an acute onset of bacterial infection which quickly brought them to hospital; this permitted an accurate time estimate of the onset of the disease, sampling of blood early in the disease, and frequent sampling for several weeks. Correspondence: Dr N. E. Hansen, Department of Medicine C, Gentofte Hospital, 2900 Hellerup, Denmark.

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N. E. Hansen et al. MATERIALS AND METHODS

Patients. Nine adult patients were studied; the age of the patients ranged from 23-79 years. Eight patients had meningitis (five caused by N. meningitidis, two by S. pneumoniae, and one by E. coli) and one had pneumonia (S. pneumoniae). Two of the patients also had cirrhosis of the liver, one had maturity onset diabetes, and one had been splenectomized for congenital sphaerocytosis; however, none of the patients had histories ofrecurrent infections prior to the present admission. All patients recovered and were discharged in good health. Most patients were re-examined 1 year after discharge, and the parameters studied were found to be normal. The latter finding and the histories of the patients were taken as evidence that they had no primary neutrophil defects. On admission, prior to aetiological diagnosis, all patients with meningitis were treated with G-penicillin (2 million u twice a day), sulphonamide (sulphadiazine 2 g four times a day), and streptomycin (0 5 g four times a day); following aetiological diagnosis, streptomycin was discontinued. The patient with E. coli meningitis was treated with sulphonamide and ampicillin (0 5 g four times a day), which was later replaced by chloramphenicol (0-5 g four times a day). All patients with meningitis were furthermore treated with hydrocortisone (100 mg) on admission; after aetiological diagnosis steroids were discontinued in the patients with N. meningitidis and E. coli meningitis, whereas prednisone (10 mg four times a day) was given to the two patients with S. pneumoniae meningitis until fever subsided. The patient with pneumonia was treated with penicillin. The course of the infection was fairly similar in eight of the patients who recovered uneventfully, except for the development of arthritis in two of the patients. One patient had a stormy course; this patient had been splenectomized for congenital sphaerocytosis and was admitted with severe S. pneumoniae meningitis, which relapsed twice during his stay in hospital for nearly 3 months. He finally recovered and was discharged without sequelae. Sampling. The first blood samples were taken on admission, i.e. 24-48 hr after the first symptoms of the disease. Subsequent blood samples were taken three times a week for at least 3 weeks, longer in case of prolonged disease. Morphology of neutrophils. Vacuolization and toxic granulation of the neutrophils were estimated on blood smears (MayGrtlrwald-Giemsa) in the light microscope. By toxic granulation is understood the appearance of large, heavily stained granules in the neutrophils, which contrast sharply with the fine scatter of normal neutrophil granules. Toxic granulation was graded arbitrarily on a scale from 0-6 taking into account both the degree of toxic granulation in the individual cell and the percentage of cells showing toxic granulation. Lysozyme. Neutrophilic granulocytes were isolated from blood (EDTA anticoagulated) by Ficoll centrifugation and solubilized by n-butanol as previously described (Hansen & Andersen, 1973). Lysozyme in EDTA plasma and neutrophil extracts was measured with a turbidometric method measuring the lysis of Micrococcus lysodeikticus with human lysozyme as standard (Hansen & Andersen 1973). The normal values are (twenty-five normals): plasma lysozyme 2-4+ 06 cpgfml (s.d.), intraneutrophilic lysozyme 2-7± 0-8 pg/106 cells (s.d.). Measurements were carried out in duplicate. Lactoferrin and myeloperoxidase. Lactoferrin and myeloperoxidase were measured on EDTA plasma and neutrophil extracts with radioimmunoassays as previously described (Hansen, Malmquist & Thorell, 1975). The normal range is for plasma lactoferrin 0-13-042 ,ug/ml and for plasma myeloperoxidase 0-02-0-18 ,ug/ml as determined on forty-nine and fortythree normal persons respectively. Although no normal range for intraneutrophilic lactoferrin is available, intraneutrophilic lactoferrin has been shown to be of the same magnitude as intraneutrophilic lysozyme, i.e. around 3 pg/106 cells (Masson, Heremans & Schonne, 1969). Intraneutrophilic myeloperoxidase is for unknown reasons difficult to quantitate accurately; the intraneutrophil level, however, is similar to that of lysozyme and lactoferrin (Spitznagel et al., 1974). Lactoferrin and myeloperoxidase measurements were carried out in triplicate. Circulating bacterial antigen. Bacterial antigens in the blood were measured with a counterelectrophoresis technique (Hoff & Hoiby, 1974).

RESULTS

Figs 1 and 2 show the composite curves from the eight patients with relatively similar courses of disease, whereas Figs 3 and 4 show the developments in the patient with relapses of pneumococcal meningitis. It appears from Fig. 1 that the intraneutrophilic lysozyme activity, although low already in the first sample, decreased further to reach nadir values 5-8 days after onset of disease after which a slow normalization was seen over the following 2 weeks. The lowest intraneutrophilic lysozyme activity averaged 0-8 pg/106 cells, representing a 70% reduction from the normal mean (2-7 pg/106). Toxic neutrophil granulation rose slowly to reach a maximum on days 5-8 before a decrease was seen; as shown in Fig. 1 toxic granulation varied inversely with intraneutrophilic lysozyme activity (r = 043, n = 97, P0-1). Intraneutrophilic lactoferrin showed the lowest values in the first sample (mean: 0 73 jig/106) followed by a slow, more than two-fold increase with maximum 3 weeks after onset of disease (mean: 1-82 pg/106).

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FIG. 1. Changes in intraneutrophilic lysozyme, neutrophil toxic granulation, and blood neutrophil concentration during bacterial infection. Composite curves (mean+ s.e.m.) from eight patients. The hatched area shows the normal mean+ s.d. for intraneutrophilic lysozyme. (a) Neutrophilic granulocytes (x 103/pul); (b) toxic granulation (arbitrary scale); (c) intraneutrophilic lysozyme (,pg/106). FIG. 2. Changes in the plasma concentrations of lysozyme, lactoferrin, and myeloperoxidase during bacterial infection. Composite curves from eight patients (mean+ s.e.m.). The stippled areas indicate the normal ranges. (a) Plasma lysozyme (pug/ml); (b) plasma lactoferrin (,pg/ml); (c) plasma myeloperoxidase (,pg/ml).

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FIG. 3. Intraneutrophilic lysozyme and toxic granulation in a patient with meningitis (S. pneumoniae). The patients had two relapses of his meningitis as indicated by the arrows, which indicate the presence of pneumococcal antigens in the blood. The stippled area indicates the normal range. (a) Body temperature (0C); (b) toxic granulation (arbitrary scale); (c) intraneutrophilic lysozyme (/ug/106). FIG. 4. Plasma lysozyme, myeloperoxidase and lactoferrin and blood concentration ofneutrophilic granulocytes in the same patient as in FIG. 3. The hatched area indicates the normal range. (L), Plasma myeloperoxidase (gfg/ml); (o), plasma lactoferrin (pug/ml); (0), plasma lysozyme (ug/ml). (a) Neutrophilic granulocytes (x 103/pl).

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Intraneutrophilic myeloperoxidase activity showed a similar development although a more pronounced day-to-day variation was seen; in the first sample the mean concentration was 1-45 pg/106 followed by a slow, more than twofold increase with maximum 3 weeks after onset of disease (3-60 Pg/106). Fig. 2 shows the variations in the plasma concentrations of lysozyme, lactoferrin, and myeloperoxidase. Considerable differences are seen between these three antibacterial proteins: plasma lysozyme was within the normal range during the whole period of observation and showed no sequential changes at all; plasma myeloperoxidase showed high values in all patients in the first samples (range 0-22-1-08 pg/ml), followed by a fast decrease towards normalization; plasma lactoferrin was high in the first sample in four of the eight patients (range 0 43-4 9 pg/ml), after normalization in the next sample a secondary peak was seen in seven of the eight patients on days 6-11 (range 0-41-0-94 pg/ml) before normalization. Both plasma myeloperoxidase and lactoferrin correlated significantly with the blood neutrophil counts (plasma myeloperoxidase: r = 0 49, n = 98, P