Mycobacterium fortuitum by Human Granulocytes - Infection and ...

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PETER H. NIBBERING, ODElTE POS, ANNELIES STEVENHAGEN, AND RALPH VAN FURTH*. Department ofInfectious Diseases, University Hospital, 2300 RC ...
Vol. 61, No. 8

INFECrION AND IMMUNITY, Aug. 1993, p. 3111-3116 0019-9567/93/083111-06$02.00/0 Copyright © 1993, American Society for Microbiology

Interleukin-8 Enhances Nonoxidative Intracellular Killing of Mycobacterium fortuitum by Human Granulocytes PETER H. NIBBERING, ODElTE POS, ANNELIES STEVENHAGEN, AND RALPH VAN FURTH* Department of Infectious Diseases, University Hospital, 2300 RC Leiden, The Netherlands Received 16 September 1992/Accepted 28 April 1993

The results of this study show that recombinant interleukin-8 (IL-8) enhances the intracellular killing of Mycobacterium fortuitum by human granulocytes. This chemokine did not stimulate the phagocytosis of M. fortuitum by granulocytes at various bacterium-to-cell ratios. The killing process was not affected by the NADPH oxidase inhibitor diphenyleneiodonium bisulfate, which indicates that recombinant IL-8 stimulates oxygen-independent mycobactericidal mechanisms of granulocytes. IL-8 did not stimulate H202 production in granulocytes but primed the cells for enhanced H202 production upon stimulation with preopsonized M. fortuitum. In sum, the chemokine IL-8 not only is involved in the recruitment of granulocytes to the site of infection but also facilitates the elimination of microorganisms by increasing the efficiency of the bactericidal activity of granulocytes.

dissolved in pyrogen-free saline with 1% inactivated fetal calf serum and stored at -70°C. Maintenance of M. fortuitum. M. fortuitum (ATCC 12790; American Type Culture Collection, Rockville, Md.) was maintained as described previously (16) with minor modifications. The mycobacteria were passed bimonthly through specific-pathogen-free Swiss mice (IFFA-Credo, Saint-Germaine-sur-l'Abresle, France). For this purpose, 100 ,ul of a suspension of 106 mycobacteria per ml of phosphate-buffered saline (PBS) (pH 7.4) was injected intravenously into mice. After 5 days, the spleen was aseptically removed, homogenized, and plated onto blood agar plates. After incubation of the plates for 3 days at 37°C, colonies were transferred to culture medium, consisting of Bacto-Middlebrook 7H9 broth enriched with Bacto Middlebrook ADC (Difco Laboratories, Inc., Detroit, Mich.), supplemented with 0.05% Tween 80 and were cultured for 3 days at 37°C. Next, the mycobacteria were washed three times, frozen in PBS containing 50% (vol/vol) glycerol, and stored at -70°C. For experiments, a sample was thawed, washed, and resuspended in Hanks balanced salt solution (HBSS) (Oxoid Ltd., Basingstoke, United Kingdom) containing 0.01% gelatin and 0.01% Tween 80 (HBSS-gel-Tw). Clumps of bacteria were removed by repeated filtering of the mycobacterial suspension through cotton wool. The mycobacteria then were resuspended at a final concentration of 107/ml in HBSS-gelTw. Addition of Tween 80 to all media and the use of siliconized glassware in all experiments prevented clumping and adherence of the mycobacteria to the test tubes. Microscopic examination revealed no clumps of bacteria in these suspensions of M. fortuitum during a 3-h period of incubation. Isolation of human granulocytes. Buffy coat prepared from blood of healthy donors was diluted in PBS and then subjected to Ficoll-Hypaque density gradient centrifugation (p = 1.077; Pharmacia Inc., Uppsala, Sweden) at 440 x g and 18°C for 20 min (5). After resuspension of the pellets in PBS containing 0.5 U of heparin per ml (PBS-hep), the granulocytes were purified by dextran sedimentation (Plasmasteril; Fresenius A.G., Bad Homburg, Germany) at 1 x g and 37°C for 10 min. The remaining erythrocytes were removed by a single hypotonic lysis. Granulocytes were washed twice with PBS-hep at 160 x g for 10 min and

Interleukin-8 (IL-8), which is produced by a variety of cells upon appropriate stimulation (3, 23, 28, 37, 39), is a chemokine that stimulates the attachment of granulocytes and T lymphocytes to endothelial cells and the transendothelial migration of these leukocytes (1, 22, 35). Stimulation of granulocytes with this cytokine results in an increase in several activities, including chemotaxis (1, 31), phagocytosis of opsonized erythrocytes (10), inhibition of the proliferation of Candida albicans (12), the respiratory burst (1, 11, 38), synthesis of leukotrienes (3), release of enzymes from granules (1, 11, 38), and expression of several members of the 32-integrin family and complement receptor I (9, 10, 30). Mycobacterial infections are of renewed interest because of their prominence in patients suffering from AIDS (2, 17). These bacteria can multiply in resident macrophages, whereas activated macrophages are able to inhibit this intracellular proliferation (33, 36). Granulocytes also play a role in the defense against Mycobacteinum tuberculosis (4, 6, 21), although contrary results have been reported (8). A previous study showed that human granulocytes efficiently phagocytose Mycobacterium fortuitum, whereas subsequent killing of the ingested bacteria is limited (16). After activation of the granulocytes by recombinant gamma interferon, the intracellular killing of opsonized M. fortuitum was greatly enhanced (16). M. fortuitum, which is also pathogenic for humans (18, 40, 42), was chosen because this mycobacterium has a rather short doubling time and is less likely to form clumps when grown in suspension than Mycobacterium avium and M. tuberculosis are. Since IL-8 is involved in the stimulation of several antimicrobial functions of granulocytes and since the killing of ingested mycobacteria by these cells is not optimal, the present study was designed to investigate whether this cytokine also enhances the intracellular killing of M. fortuitum by human granulocytes.

was

MATERIALS AND METHODS rIL-8. Lyophilized recombinant human IL-8 (rIL-8), a gift from I. Lindley (Department of Immunostimulation, Sandoz Forschungsinstitut Gesellschaft M.B.H., Vienna, Austria), *

Corresponding author. 3111

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resuspended in the medium indicated for the assay in which they were studied. The viability of the granulocyte suspensions was more than 95%, as determined by trypan blue exclusion. Microbiological assay of phagocytosis and intracellular killing of M. fortuitum by granulocytes. Phagocytosis and killing of M. fortuitum by human granulocytes were determined simultaneously as described previously (16). Briefly, a suspension of 5 x 106 granulocytes and 5 X 106 M. fortuitum cells in 1 ml of HBSS-gel-Tw was incubated in the presence of 10% normal human serum (prepared from a donor with blood group AB and further referred to as serum) and various concentrations of rIL-8 at 37°C under slow rotation (4 rpm). At various intervals a sample was taken and centrifuged; the number of viable bacteria in the supernatant, which is a measure of the phagocytosis, was then determined. Another sample was used to assess the total number of viable bacteria after disruption of the cells, which is a measure of the killing of bacteria. The bacteria were grown on blood agar plates and counted as CFU. The intracellular killing of M. fortuitum by granulocytes was also measured after a short period of phagocytosis (26). For this purpose, a mixture of 5 x 106 granulocytes and 5 x 106 M. fortuitum cells was incubated in the presence of 10% serum at 37°C under slow rotation for 10 min. The supernatant containing extracellular bacteria was discarded, and the cells were washed; next, 5 x 106 granulocytes containing ingested bacteria per ml of HBSS-gel-Tw were incubated in the presence of 10% serum and various concentrations of rIL-8 at 37°C. At various intervals samples were taken, and after disruption of the granulocytes, the number of viable cell-associated bacteria was determined microbiologically. Microscopic assay to quantitate numbers of cell-adherent and intracellular M. fortuitum cells. The method to distinguish between cell-adherent and intracellular fluorescein isothiocyanate (FITC)-labeled M. fortuitum after phagocytosis by granulocytes has been described previously (13). M. fortuitum at a concentration of 108 bacteria per ml of buffer consisting of 50 mM NaHCO3 and 100 mM NaCl (pH 9.0) was incubated with 0.1 mg of FITC (Sigma Chemical Co., St. Louis, Mo.) per ml in the dark at room temperature for 20 min. After removal of free FITC, the bacteria were filtered through cotton wool and resuspended in HBSS-gel supplemented with 0.05% Tween to a concentration of 108 bacteria per ml. After incubation of 5 x 106 granulocytes per ml of HBSS-gel-Tw with various concentrations of FITC-labeled M. fortuitum at 37°C for 10 min and removal of the extracellular bacteria by three washings, the granulocytes were resuspended in HBSS-gel supplemented with 0.05% Tween. Next, a sample of this cell suspension was mixed with ethidium bromide (Sigma; final concentration, 25 ,ug/ml), and a preparation was made for microscopic analysis with a Leitz Orthoplan fluorescence microscope (Leitz, Wetzlar, Germany) at a magnification of x 1,000. The percentage of granulocytes associated with bacteria and the numbers of intracellular (i.e., green fluorescent) and cell-adherent (i.e., orange fluorescent) bacteria in granulocytes with one or more M. fortuitum cells were determined. Treatment of granulocytes with DPI. To investigate whether oxygen-dependent mechanisms are involved in the intracellular killing process, granulocytes were incubated with S ,uM diphenyleneiodonium bisulfate (DPI) (a generous gift of A. R. Cross, University of Bristol, Bristol, United Kingdom), which irreversibly binds to the flavoprotein of NADPH oxidase (14), at 37°C for 30 min before stimulation

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of the killing process. As a control, cells were incubated with 0.5% dimethyl sulfoxide, the diluant of DPI. Assay of hydrogen peroxide production by granulocytes. The hydrogen peroxide production by granulocytes at rest and upon stimulation was assessed by the horseradish peroxidase-mediated H202-dependent oxidation of homovanillic acid (34), and the results are expressed as nanomoles of H202(106 cells x 10 min). The following stimuli were used: serum-opsonized, heat-killed M. fortuitum; formylmethionylleucylphenylalanine (FMLP) (Sigma); and phorbol myristate acetate (PMA) (Sigma). For optimal responses to FMLP, granulocytes were incubated with 5 ,ug of cytochalasin B (Sigma) per ml at 37°C for 5 min. Calculations. The results are expressed as means and standard errors of the mean (SEM). Data were analyzed statistically by means of the Kruskal Wallis test or the Mann-Whitney U test where indicated. P < 0.05 was considered significant. Rate constants of intracellular killing (Kk per hour) were calculated as a measure of the intracellular killing of bacteria according to the following equation: Kk = [ln N(t = 1) - ln N(t = 2)]/[(t = 2) - (t = 1)], in which N(t = 1) is the number of viable cell-associated bacteria at time t = 1 and N(t = 2) is that at time t = 2 of the assay. The values given for Kk represent the mean and SEM of the rates determined during 1-h intervals. RESULTS Effect of rIL-8 on phagocytosis of M. fortuitum by granulocytes. After phagocytosis of FITC-labeled M. fortuitum by granulocytes at bacterium-to-phagocyte ratios of 5:1, 1:1, and 1:5, the percentages of phagocytes with one or more mycobacteria were 84 ± 6%, 42 + 10%, and 8 ± 2%, respectively (n = 3 or 4). Under these conditions the majority of the cell-associated mycobacteria were intracellularly localized; the respective values were 84 ± 9%, 71 + 5%, and 72 ± 9% (n = 3 or 4). At the various bacterium-togranulocyte ratios rIL-8 did not affect the percentage of granulocytes associated with M. fortuitum or the numbers of intracellular and cell-adherent mycobacteria (P > 0.5). Since at a bacterium-to-phagocyte ratio of 5:1 many extracellular mycobacteria were observed in the granulocyte preparation, in all further experiments an M. fortuitum-to-granulocyte ratio of 1:1 was used. The results of the phagocytosis experiments with the microbiological assay revealed also no effect of 1 to 27 nM rIL-8 on the ingestion of M. fortuitum by granulocytes, as illustrated for 27 nM rIL-8 in Fig. 1A. The growth of M. fortuitum in culture medium without granulocytes was not influenced by the chemokine (data not shown). Effect of rIL-8 on intracellular killing of M. fortuitum by granulocytes. When intracellular killing of the bacteria by granulocytes was assessed during continuous phagocytosis, approximately 30% of the M. fortuitum cells were killed by human granulocytes during a 2-h incubation period (Fig. 1A). In the presence of 3 to 27 nM rIL-8, the intracellular killing of opsonized M. fortuitum by granulocytes was significantly enhanced (Fig. 1); 1 nM rIL-8 did not affect (P > 0.1) the intracellular killing (Fig. 1B). The cell-free medium, obtained after incubation of 5 x 106 granulocytes per ml and 5 x 106 M. fortuitum cells per ml in HBSS-gel-Tw containing 10% serum with or without 27 nM rIL-8 for 2 h at 37°C, did not affect the growth of M. fortuitum (results not shown). The intracellular killing of M. fortuitum by granulocytes assessed after phagocytosis of the mycobacteria in the presence of serum was rather poor; the addition of 27 nM

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FIG. 2. Effect of rIL-8 and serum on intracellular killing of M. fortuitum by granulocytes. Granulocytes were allowed to ingest M. fortuitum in the presence of 10% serum for 10 min. After removal of the extracellular bacteria, granulocytes were reincubated in either HBSS (0), 10% serum (0), 27 nM rIL-8 (M), or 10% serum with 27 nM rIL-8 (@), and then the intracellular killing of M. fortuitum was assessed. Intracellular killing is represented by the percent decrease of viable intracellular bacteria. Data are means and SEM of three experiments. *, P < 0.05 for the difference between the value for granulocytes stimulated with serum plus IL-8 and the value for serum alone.

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120 min 60 0 FIG. 1. Effect of rIL-8 on antimycobacterial activities of granulocytes. (A) Effect of 27 nM rIL-8 on phagocytosis and intracellular killing of M. fortuitum by human granulocytes. Granulocytes and M. fortuitum in HBSS-gel-Tw supplemented with 10% serum were incubated at 37°C. At various intervals the numbers of extracellular bacteria and of total viable extracellular plus cell-associated bacteria were determined. Phagocytosis in the presence (U) or absence (0) of rIL-8 is represented by the percent decrease in the number of extracellular bacteria. Data are means and SEM of three experiments. Intracellular killing of M. fortuitum in the presence (0) or absence (0) of rIL-8 is represented by the percent decrease in the number of viable cell-associated plus extracellular bacteria. Data are means and SEM of nine experiments. (B) Effect of various concentrations of rIL-8 on intracellular killing of opsonized M. fortuitum by granulocytes. After phagocytosis of opsonized bacteria and removal of the extracellular bacteria, granulocytes were incubated with 10% (vol/vol) serum with or without rIL-8, and the intracellular killing of M. fortuitum by granulocytes was determined. Results in the presence of 1 (0), 3 (0), 9 (0), or 27 (U) nM rIL-8 are expressed as the percent increment in intracellular killing of bacteria by granulocytes relative to the percent killing in the absence of rIL-8. Data are means and SEM of nine experiments *, P < 0.05.

rIL-8 significantly enhanced the intracellular killing of this bacterium (Fig. 2), and similar results were found with 9 nM rIL-8 (results not shown). In the absence of serum, intracellular killing of M. fortuitum by granulocytes did not occur,

and addition of rIL-8 had no effect (Fig. 2). There was no decrease in the number of bacteria when M. fortuitum was incubated for 2 h in the cell-free supernatant obtained after phagocytosis of mycobacteria by granulocytes for 2 h (results not shown). Next, the killing assay was performed alternately with or without rIL-8. The rates of intracellular killing of M. fortuitum by granulocytes in the presence of rIL-8 were significantly higher than those in the absence of the cytokine (Table 1). When granulocytes containing M. fortuitum were incubated with serum and rIL-8 during the first hour and next only with serum, the rate of intracellular killing during the second hour decreased significantly. Addition of rIL-8 to cells that had been incubated during the first hour with serum resulted in a significant increase in the killing rate during the second hour (Table 1). Together these results demonstrate that IL-8 had to be continuously present for its stimulatory effect on the intracellular killing of M. fortuitum. The stimulatory effect of rIL-8 on the intracellular killing of M. fortuitum by granulocytes isolated with the use of heparin was the same as that by granulocytes isolated without this agent (results not shown), indicating that the stimulatory effect of the cytokine on granulocytes was not mediated by heparin. Effect of DPI on intracellular killing of M. fortuitum by granulocytes. DPI was used to investigate whether oxygendependent mechanisms are involved in the intracellular killing of M. fortuitum stimulated by serum or by serum plus IL-8. The rates of intracellular killing of M. fortuitum in DPI-incubated and control granulocytes stimulated with serum plus rIL-8 during the first hour of the assay amounted to 0.72 + 0.20/h and 0.74 + 0.22/h, respectively, and after stimulation with serum the respective rates were 0.51 +

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TABLE 1. Effect of IL-8 on rate of intracellular killing of M. fortuitum by granulocytesa Kk (h-') during h:

Stimulus during h: 1

2

Serum + rIL-8 Serum Serum + rIL-8 Serum + rIL-8 Serum Serum

1

2

0.97 ± 0.08b 0.47 + 0.13 0.73 0.43 0.93 0.37

Serum + rIL-8 Serum Serum + rIL-8 Serum

± 0.06c,d ± 0.16d ± 0.19c ± 0.06

a Granulocytes containing M. fortuitum were incubated with serum in the presence or absence of rIL-8 for 1 h, washed, and then reincubated with serum in the presence or absence of rIL-8 for 1 h longer. Intracellular killing was assessed, and the rates of intracellular killing (Kk) during the first and the second hours were calculated. Results are means and SEM of four experiments. Final concentrations were 10% (vol/vol) serum and 27 nM rIL-8. b Significantly different from value obtained with serum alone (P < 0.01). c Significantly different from value obtained with serum alone during h 2 (P < 0.01). d Not significantly different from value obtained with serum alone during h 1 (P < 0.2).

0.13/h and 0.45 ± 0.22/h (n = 4), demonstrating that DPI did not inhibit the killing process. The effect of this inhibitor on the H202 production by granulocytes after stimulation was determined to investigate whether DPI affected the oxidative metabolism of the cells. The respective values for DPIincubated granulocytes after stimulation with 100 ng of PMA per ml or i09 serum-opsonized killed Staphylococcus aureus cells per ml were 0.6 + 0.7 and 0.7 + 0.6 nmol of H202/(106 cells x 10 min) (n = 4), and the values for control granulocytes were 15 ± 2 and 4.3 ± 0.5 nmol of H2021(106 cells x 10 min) (n = 4), demonstrating that DPI completely blocked the oxidative metabolism of granulocytes. Effect of rIL-8 on hydrogen peroxide production by granulocytes. H202 production by resting granulocytes amounted to 0.1 ± 0.2 nmol of H20J(106 cells x 10 min) (n = 7); 9 and 27 nM rIL-8 did not stimulate H202 production in these cells (P > 0.1) (Table 2). Granulocytes incubated with 9 and 27 nM rIL-8 for 1 h and then stimulated with 5 x 108 opsonized M. fortuitum cells per ml, 50 nM FMLP, or 2.5 ng of PMA per ml produced slightly more H202 than did cells incubated with PBS (Table 2). Such an enhancement of the H202 production by rIL-8-incubated cells was not found when a higher concentration of FMLP (1 p,M) or PMA (100 ng/ml) was used as a stimulus (results not shown). TABLE 2. Effect of IL-8 on H202 production by granulocytes' rIL-8

(nM) 0 9 27

H202 production [nmol/(106 cells x 10 min)l by granulocytes stimulated with: FMLP Nothing Opsonized M. fortuitum

0.1 ± 0.2 0 ± 0.1 0.1 ± 0.2

0.5 ± 0.1b 0.9 ± 0.5b,c 1.0 ± 0. OX

0.8 ± 0.2 1. 3 ± 0o4b,c 1. 4 ± 0.2I'C

2.3 ± 1.1b 3.0 ± 0.7b,c 3.5 ± 1.3

a 106 granulocytes per ml were incubated with various concentrations of rIL-8 or PBS (control cells) for 1 h. Next, granulocytes were stimulated with 5 x 103 opsonized M. fortuitum cells per ml (n = 3), 50 nM FMLP (n = 3), or 2.5 ng of PMA per ml (n = 9) for 10 min, and the production of H202 by granulocytes (mean ± SEM) was determined (34). b P < 0.01 for the difference between values for cells incubated with and without a stimulus. c p < 0.01 for the difference between the values for rIL-8-incubated and control cells.

DISCUSSION The main conclusion that can be drawn from the present results is that IL-8 enhances the intracellular killing of M. fortuitum by human granulocytes without affecting the phagocytosis of bacteria by these cells. The concentrations of the cytokine required for this stimulatory effect are at a level shown to be chemotactic in vivo (7, 32). Since microbiological assessment of the decrease in the number of extracellular mycobacteria can be an inaccurate measure of the phagocytosis of M. fortuitum by granulocytes, ingestion of FITC-labeled bacteria was also determined microscopically. The results revealed that about 75% of the cell-associated bacteria were intracellularly localized. This demonstrates that quantitation of the phagocytosis of M. fortuitum with the microbiological assay is reliable. In the present study two methods for the assessment of the intracellular killing of M. fortuitum by granulocytes were used. The first method measured the killing of M. fortuitum during ongoing phagocytosis, a condition most closely resembling the process occurring in vivo. This assay has a few disadvantages. First, the rate of phagocytosis can affect the rate of intracellular killing. Second, the effects of changes in the conditions which affect the rate of phagocytosis or intracellular killing are difficult to assess independently. Third, the occurrence of extracellular killing of M. fortuitum during the assay cannot be controlled. To avoid these drawbacks, the intracellular killing of M. fortuitum by granulocytes was also investigated after a short period of phagocytosis (26). With this approach, it was found that serum is required for the intracellular killing of M. fortuitum by granulocytes. Previously, we had demonstrated that human granulocytes kill ingested S. aureus in the absence of serum, although serum factors, such as immunoglobulin G and C3b, enhance the process considerably (25, 27). It thus appears that the requirement for serum for the intracellular killing of bacteria by granulocytes depends on the bacterial species involved. Human monocytes always require serum stimulation for the intracellular killing of bacteria, since otherwise it does not occur (27). It has been suggested that clumping of mycobacteria during the killing assay could be misinterpreted as bacterial killing (8). This is not the case, however, since no decrease in CFU or formation of clumps of bacteria was observed when the bacteria were incubated in cell-free medium or with disrupted granulocytes for up to 2 h. Since stimulation of the intracellular killing of M. fortuitum by granulocytes by IL-8 was not affected by heparin, we concluded that this agent, which is routinely used during isolation of granulocytes, does not interfere with the interaction between IL-8 and these cells. Furthermore, heparin did not influence the binding of FITC-labeled IL-8 to gran-

ulocytes (unpublished results).

The mechanisms mediating the intracellular killing of mycobacteria by granulocytes are not clear. There is some evidence that reactive oxygen intermediates are involved (19, 20), although most authors now suggest that the killing process is dependent on nonoxidative mechanisms (21, 29, 33). These results led us to investigate the possible contribution of reactive oxygen intermediates to the killing of M. fortuitum by granulocytes. Since DPI did not affect the killing of M. fortuitum by granulocytes, we conclude that the intracellular killing of M. fortuitum by granulocytes is mediated by oxygen-independent killing mechanisms. Our observation that rIL-8 hardly affects H202 production by these cells supports this conclusion. Furthermore, we have found that defensins and lysozyme, constituents of lysosomes in

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granulocytes (15, 24), mediate the nonoxidative killing of M. fortuitum (16a). Since IL-8 induces the rapid polymerization of actin filaments (10, 41), it might be that this cytokine stimulates the intracellular killing of M. fortuitum by granulocytes by a more efficient phagosome-lysosome fusion. Both gamma interferon and IL-8 enhance the intracellular killing of M. fortuitum by granulocytes, but the mechanisms underlying these effects differ. Granulocytes have to be exposed to gamma interferon for 18 h before the enhancing effect of the cytokine can be demonstrated, but the presence of this cytokine is not required during the killing assay (16). The stimulatory effect of IL-8 is independent of preincubation, but the cytokine must be present during the assay. This indicates that gamma interferon primes granulocytes for an enhanced antimycobacterial activity, while IL-8 together with serum stimulates the intracellular killing of M. fortuitum. ACKNOWLEDGMENTS This study was supported by a grant for AIDS research from the Ministry of Welfare, Health and Culture of The Netherlands and by a grant from the AIDS fund. We thank A. C. Bezemer and T. P. L. Zomerdijk for excellent technical assistance. REFERENCES 1. Baggiolini, M., P. Imboden, and P. A. Detmers. 1992. Neutrophil activation and the effects of interleukin-8/neutrophil-activating peptide 1 (IL-8/NAP-1), p. 1-17. In M. Baggiolini and C. Sorg (ed.), Cytokines, vol. 4. Interleukin 8 (NAP-1) and related chemotactic cytokines. Karger, Basel. 2. Barnes, P. F., A. B. Bloch, P. T. Davidson, and D. E. Snider. 1991. Tuberculosis in patients with human immunodeficiency virus infection. N. Engl. J. Med. 324:1644-1650. 3. Bazzoni, F., M. A. Cassateila, F. Rossi, M. Ceska, B. Dewald, and M. Baggiolini. 1991. Phagocytozing neutrophils produce and release high amounts of neutrophil-activating peptide NAP-1/ IL-8. J. Exp. Med. 173:771-774. 4. Bloch, H. 1948. The relationship between phagocytic cells and human tubercle bacterium. Am. Rev. Tuberc. 58:662-670. 5. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest.

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