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Originally published online as doi:10.1189/jlb.0605338 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2006;79:87-94.)
© 2006 by Society for Leukocyte Biology

Common methodology is inadequate for studies on the microbicidal activity of neutrophils

Eva Decleva, Renzo Menegazzi, Sara Busetto, Pierluigi Patriarca and Pietro Dri1

Department of Physiology and Pathology, University of Trieste, Italy

1 Correspondence: Department of Physiology and Pathology, Via A. Fleming, 22, 34127 Trieste, Italy. E-mail: dri{at}units.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was ~60% and ~70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated.

Key Words: microbial killing • diphenyleneiodonium • CGD • MPO deficiency


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Protection against infection by killing of invading microorganisms is the most important function of polymorphonuclear leukocytes. This is clearly documented by chronic granulomatous disease (CGD), a primary immunodeficiency syndrome characterized by defective reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. CGD patients suffer from recurrent and severe infections as a result of the inability of their neutrophils to respond with a respiratory burst during phagocytosis and kill various microorganisms [1 , 2 ]. In spite of the rather consistent clinical features (life-threatening infections and granuloma formation), the common genetic defect observed in the most frequent form of the disease (abnormality of the gp91phox component of NADPH oxidase, X-linked inheritance), and the typical lack of a respiratory burst, published studies about the microbicidal activity of CGD neutrophils have given widely variable results with microbial killing ranging from severely compromised to nearly normal (Table 1 and refs. therein). As shown in Table 1 , a similar variability in the extent of inhibition of microbial killing has been found using normal neutrophils treated with DPI, a potent inhibitor of NADPH oxidase [10 , 19 ].


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  		Table 1. Summary of Literature Data on the Staphylocidal Activity of CGD Neutrophils and DPI-Treated Normal Neutrophils

 
Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water (refs. [20 21 22 ] and refs. in Table 1 ). While studying the effect of DPI on the staphylocidal activity of neutrophils, we obtained various degrees of inhibition in some experiments and no effect in others. Although comparable with literature data showing variability in the effect of DPI, we were bothered by the inconsistency of the results obtained and eventually found that the cell lysis step, i.e., dilution in distilled water or pyrogen-free water for clinical use (hereafter, referred to as water), was the source of error. Light microscopic examination of cell suspensions diluted in water revealed, in fact, that lysis of neutrophils was incomplete. Bacteria were not dispersed in the medium but remained associated with cell ghosts. All cell ghost-associated viable bacteria are expected to generate only one colony-forming unit (CFU), thus causing an overestimate of the microbicidal activity. We overcame the problem using water brought to pH 11, a highly effective way to lyse neutrophils [23 ], and found that staphylocidal activity was inhibited completely and reproducibly by DPI. In this paper, we have compared the effect of cell lysis in water and water at pH 11 on the evaluation of microbicidal activity against Staphylococcus aureus, Escherichia coli, and Candida albicans of normal neutrophils, DPI-treated neutrophils, and neutrophils from CGD and from myeloperoxidase (MPO)-deficient subjects. We show that incomplete cell lysis, such as that occurring using distilled water or pyrogen-free water for clinical use (pH 4.6–5.6), leads to an overestimate of microbial killing. It follows that in using this method, partial defects may pass unnoticed, and total defects are detected as partial. As most literature studies about the killing activity of neutrophils have been performed using distilled water as a lytic agent, our findings suggest that previous data about microbicidal defects and the effects of agents having the potential to modulate neutrophil microbicidal activity need reassessment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Reagents
DPI, cytochrome c (type VI, from horse heart), phorbol 12-myristate 13-acetate (PMA), and Sabouraud dextrose broth were obtained from Sigma Chemical Co. (St. Louis, MO). Becton Dickinson (Sparcks, MD) provided Luria-Bertani (LB) broth, Miller, dehydrated, and BactoTM agar. Percoll was obtained from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Saponin was from Merck (Darmstadt, Germany). All other reagents and chemicals were of the highest purity grade available. All solutions were made in endotoxin-free water for clinical use. DPI (10 mM stock solution) was dissolved in dimethyl sulfoxide (DMSO) at 50°C, and aliquots were kept at –20°C until use.

Microorganisms
S. aureus strain 502A (ATCC 27217) was a generous gift of Dr. John J. Iandolo (Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City). E. coli ATCC 25922, a serum-resistant strain, was kindly provided by Dr. Cristina Lagatolla (Dipartimento di Scienze Biomediche, University of Trieste, Italy). C. albicans was a clinical isolate kindly provided by Dr. Edoardo Crevatin (Unità Clinico Operativa di Igiene e Medicina Preventiva, University of Trieste).

Neutrophil isolation
Neutrophils were isolated from peripheral blood by density gradient centrifugation over Percoll, as described previously [24 ] and suspended in Ca 2+- and Mg2+-free HEPES-buffered saline (HBS) solution containing bovine serum albumin (BSA), 140 mM NaCl, 5 mM KCl, 5 mM glucose, 5 mM HEPES, pH 7.4, and 0.2% BSA. Immediately before use, cell suspensions were supplemented with 1 mM CaCl2 and 1 mM MgCl2.

Preparation of bacteria/fungi
S. aureus and E. coli were picked from single colonies grown on LB-agar plates, inoculated into LB broth, and grown for 18 h at 37°C. Frozen aliquots of C. albicans blastospores were diluted in Sabouraud broth and grown overnight at 30°C. Microorganisms were pelleted by centrifugation at 2000 g for 5 min, transferred into a microtube, washed once in 0.9% NaCl solution by centrifugation at 12,000 g for 10 s, and suspended in 1.5 ml 0.9% NaCl solution. Bacterial concentration was determined by measurement of turbidity at 500 nm. Blastospore concentration was determined by microscopic counting in a cell-counting chamber. Suspensions were diluted to 1 x 108 cells/ml (S. aureus and E. coli) or to 6 x 107 cells/ml (C. albicans) in HBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 (Ca2+/Mg2+-HBS) and opsonized with 10% pooled human serum for 30 min at 37°C in a shaking water bath. Opsonized bacteria/fungi were kept on ice until use.

Killing assay
Neutrophils (5x106/ml in Ca2+/Mg2+-HBS) were prewarmed for 10 min at 37°C in a shaking water bath in the presence or absence of 5 µM DPI (diluted in Ca2+/Mg2+-HBS from a 10 mM stock solution in DMSO). Opsonized microorganisms were added at a ratio of three to five bacteria/neutrophil or two to three blastospores/neutrophil, with a final neutrophil concentration of 4 x 106 cells/ml. Microorganisms were also incubated in the absence of neutrophils to account for growth during the assay. The tubes were incubated at 37°C in a shaking water bath. At the desired times, 50 µL samples were diluted in 2.5 ml pyrogen-free distilled water (pH 4.5–6.5), 2.5 ml 1% saponin solution in water, or 2.5 ml water brought to pH 11.00 with NaOH just before use; all the samples were then inverted twice. After standing for 5 min at room temperature and vortexing vigorously for ~5 s, 50 µL of the samples was diluted in 0.9% NaCl solution to give a bacterial or fungal concentration of 2 x 103/ml. Duplicates of 100 µL aliquots of each dilution were added to 10 ml molten (42°C) 1% agar in LB/Sabouroud broth, rapidly mixed, and plated on Petri dishes. The CFU were counted after an overnight incubation at 37°C (S. aureus, E. coli) or at 30°C (C. albicans). The percent killing was calculated as follows: 100 – [(CFU at time t/CFU at time 0)x100].

Flow cytometric analysis and light microscopy
Neutrophils (4x106/ml in Ca2+/Mg2+-HBS) were incubated with opsonized S. aureus for 30 min at 37°C. Cell suspensions were then centrifuged, and pellets were resuspended in PBS, distilled water, or water brought to pH 11.00 with NaOH. After 5 min at room temperature, isotonicity was restored with a 10x PBS solution, and the samples were analyzed with a FACSCaliburTM (Becton Dickinson). Forward (FSC)- and side-scatter (SSC) analysis was performed after a constant acquisition time of 35 s. Cytospins were also prepared and stained with DIP-Quick (Dyaset, Ferrara, Italy). Photographs were taken with a Nikon Eclipse E600 microscope equipped with a Nikon Coolpix995 digital camera.

Assay of superoxide (O2–) production
O2– production was measured by the O2–dismutase-inhibitable cytochrome c reduction assay [25 ]. Briefly, neutrophils were suspended at 1.5 x 106 cells/ml in Ca2+/Mg2+-HBS and incubated with or without 0.1–10 µM DPI for 10 min at 37°C in a shaking water bath. Aliquots (50 µL) were then added to flat-bottomed microtiter plate wells (F16 MaxiSorp Loose Nunc-Immuno Modules, Nunc, Roskilde, Denmark) containing 0.1 ml of the same medium, supplemented with 0.18 mM cytochrome c (final concentration, 0.12 mM), 15 ng/ml PMA (final concentration, 10 ng/ml), and DPI at the required final concentration. After incubation for 30 min at 37°C, the plate was read at 550 nm and 540 nm. The amount of reduced cytochrome c was calculated from the absorbance difference between 550 nm and 540 nm, using as a standard an absorbance difference of 0.037 O.D. units for 1 nmol reduced cytochrome c.

Statistical analysis
Statistical significance was tested by Student’s t-test calculated using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). Values of P < 0.05 were considered statistically significant.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
While studying the effect of DPI on killing of S. aureus by human neutrophils, we observed that in cell populations exhibiting high killing activity (Fig. 1A , group 1), the number of viable bacteria, expressed as CFU, found after lysis of DPI-treated cells, was higher than that released from control cells. This indicated inhibition of killing. On the contrary, no significant change in the number of CFU was detected using cell populations with low killing activity (Fig. 1A , group 2), indicating that DPI had no effect. In these experiments, cells were lysed by a 5-min treatment in water or in a 1% saponin solution in water, although similar results were obtained using water at 4°C, a 1% saponin solution in PBS, or when the treatment time was prolonged to 60 min (data not shown).



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  		Figure 1. Effect of DPI on the killing of S. aureus and on PMA-induced O2–production by neutrophils (4x106/ml in Ca2+/Mg2+-HBS), which were treated for 10 min at 37°C in the absence ({square}) or presence ({blacksquare}) of 5 µM DPI. After incubation with opsonized S. aureus at a bacteria-to-cell ratio of 3:1, neutrophils were lysed for 5 min in water (H2O) in a 1% saponin solution in water (saponin) or in water at pH 11 (H2O pH 11). Lysed samples were diluted immediately in 0.9% NaCl solution (H2O and H2O pH 11) or brought to pH 11 for a further 5 min prior to dilution in physiologic saline (H2O->H2O pH 11). Suspensions were then processed for colony counting as detailed in Materials and Methods. Data are expressed as number of colonies of S. aureus (CFU) after 30 min phagocytosis (CFU/Petri dish at time 0 of phagocytosis{cong}200). (A) Effect of DPI on the staphylocidal activity of neutrophil populations with high (group 1) or low (group 2) killing activity, determined after cell lysis in water or in saponin. Data are mean ± SEM of 14 (H2O) and six (saponin) experiments for group 1 and seven (H2O) and four (saponin) experiments for group 2. DPI-treated cells compared with control: ***, P < 0.001; **, P < 0.01. Inset: Dose-response curve of the effect of DPI on PMA-induced O2–production. For experimental details, see Materials and Methods. The data are from one experiment representative of three, performed in duplicate, and are expressed as percent inhibition of O2–production in comparison with untreated cells (154.6 nmoles O2–/106 neutrophils/30 min). (B) Effect of DPI on the staphylocidal activity of neutrophils determined after lysis in water (H2O), water at pH 11 (H2O pH 11), or water following adjustment to pH 11 (H2O->H2O pH 11). Data are mean ± SEM of six experiments. *, Control cells: H2O pH 11 versus H2O, P = 0.0152; ***, DPI-treated cells: H2O pH 11 versus H2O, P < 0.001.

 
The concentration of DPI used in these experiments (5 µM) completely inhibited PMA-induced O2–production (Fig. 1 , inset), thus excluding that the observed variability of the effect of DPI on killing was a result of incomplete inhibition of NADPH oxidase. As it is difficult to understand how DPI, at concentrations that totally block NADPH oxidase activation, might inhibit the microbicidal activity of some cell populations but not of others, we hypothesized that the observed discrepancies could be attributed to the failure of the lysis methods to disrupt neutrophils and free intracellular bacteria. As a consequence, the bacteria surviving within each neutrophil and not dispersed in the medium after the cell lysis step would give rise to and be counted as a single colony, thus leading to an overestimate of the bactericidal activity. This would be particularly relevant when killing is defective, as in DPI-treated neutrophils, and a large number of microorganisms remain viable inside each cell. To explore this possibility, we used water brought to pH 11 to dilute neutrophils after phagocytosis, a treatment that has been shown to effectively lyse these cells and release phagocytosed bacteria [23 ].

The results shown in Figure 1B confirm our hypothesis. In fact, lysis of neutrophils in water at pH 11 led to an increase in the number of CFU with respect to the number of CFU obtained after dilution in distilled water. Similar effects were observed in control and DPI-treated neutrophils. It is worth noting that in DPI-treated cells, the number of CFU after lysis in water at pH 11 roughly corresponded to the CFU expected ({cong}200) if no killing had occurred, indicating that killing is totally inhibited by this compound. Figure 1B also shows that the number of CFU obtained after lysis of neutrophils with water increased to values comparable with those observed after immediate lysis in water at pH 11, if the pH was subsequently brought to 11, thus excluding a direct effect of water (pH range, 4.5–6.5) on bacterial viability. These observations suggest that lysis of neutrophils in distilled water is not sufficient to free all intracellular bacteria, but this can be achieved in water at pH 11. To verify this hypothesis more directly, we carried out experiments in which phagocytosing neutrophils were suspended in physiologic saline, water, and water at pH 11 and analyzed by flow cytometry and light microscopy.

Figure 2A 2B 2C 2D 2E 2F , shows a dot-plot distribution of FSC versus SSC (left panels) and morphology (right panels) of neutrophils after 30 min of phagocytosis of S. aureus and subsequent dilution in physiologic saline (A and B), water (C and D), and water at pH 11 (E and F). In left panels, the total number of events measured after an acquisition time of 35 s, which gives an estimate of the relative number of particles per unit volume, is also reported. After treatment in water, the volume and complexity of neutrophils are reduced, with only a 20% decrease in the number of total events, compatible with the effect of the hypotonic shock, which is expected to alter the physicochemical properties of the cells. The situation is dramatically different after lysis in water at pH 11, when neutrophils have virtually disappeared, as indicated by the 97% decrease in the number of total events. The panels on the right side show light microscopy images of the same neutrophil-S. aureus suspensions used in flow cytometry and confirm what was seen in the panels on the left side of the figure. In fact, after treatment in water (D), homogeneously stained cell bodies were present, clearly identifiable as swollen nuclei at higher magnification (inset). It is worth noting that the peripheral areas of nuclei presented a faint rim of what appears to be cytoplasmic remnants still containing bacteria. After lysis in water at pH 11 (F), only faintly stained, ghost-like debris were observed. Figure 2B and 2D , also shows that the number of cell-associated bacteria is variable, indicating cell heterogeneity in phagocytosis. This may be an additional factor that could contribute to overestimation of killing when lysis is not performed correctly.



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  		Figure 2. Fluorescein-activated cell sorter analysis and morphology of neutrophils lysed in distilled water and water at pH 11. Neutrophils (4x106/ml in Ca2+/Mg2+-HBS) were incubated for 30 min with S. aureus at a bacteria-to-neutrophil ratio of 5:1. The phagocytosis mixture was then centrifuged and resuspended for 5 min in physiologic saline (A and B), distilled water (C and D), or water at pH 11 (E and F) and processed as described in Materials and Methods. Left panels show the FSC and SSC analysis of the samples. Right panels show the morphology of the same samples, as observed by light microscopy. Original magnifications: x20 (backgrounds) and x100 (insets).

 
Figure 3 shows the effect of DPI on killing of S. aureus by neutrophil populations obtained from different donors and exhibiting high (>70%) or low (<50%) killing activity, as determined after lysis in water and water at pH 11. As expected, DPI strongly inhibited the staphylocidal activity of both neutrophil populations when lysis was carried out in water at pH 11. In contrast, after treatment in water, DPI was found to inhibit killing of neutrophils with high bactericidal activity (42.0%±3.7) but had no effect on neutrophils with low bactericidal activity. In addition, Figure 3 shows that lysis in distilled water, as compared with lysis in water at pH 11, leads to an erroneously high estimate of killing also in control neutrophils with low killing activity (+22.8%±5.1; P<0.05), in agreement with the observations reported by Gargan et al. [23 ].



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  		Figure 3. Effect of DPI on the killing of S. aureus by neutrophil populations with high or low bactericidal activity as assessed after lysis in water and in water at pH 11. For experimental details, see the legend to Figure 1. Values are expressed as percent of staphylococci killed after 30 min of phagocytosis, with respect to the initial inoculum. Data are mean ± SEM of six (upper panel) and eight (lower panel) experiments. DPI-treated neutrophils ({blacksquare}) versus control neutrophils ({square}): ***, P < 0.001; NS, not significant.

 
The effect of lysis in water and water at pH 11 on the measurement of staphylocidal activity of control and DPI-treated neutrophils at various bacteria-to-cell ratios is shown in Figure 4 . As expected, after lysis in water at pH 11, a near complete inhibition by DPI was observed at all bacteria-to-cell ratios. On the contrary, lysis in water gave results comparable with those observed in water at pH 11, only at low bacteria-to-cell ratios (0.2:1). By increasing the number of bacteria per cell, killing in the presence of DPI, after lysis in water, appeared progressively less inhibited and was not different from that of control cells at a bacteria-to-cell ratio of 10:1. Thus, lysis in water provides reliable results only at low bacteria-to-cell ratios, when the likelihood of phagocytosis of more that one microorganism/cell is extremely low. Use of a high ratio (e.g., 10:1) may even obscure detection of a complete defect in microbial killing.



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  		Figure 4. Influence of the bacteria-to-neutrophil ratio on the assessment of S. aureus killing by DPI-treated cells after cell lysis in water and water at pH 11. Neutrophils (4x106 cells/ml in Ca2+/Mg2+-HBS) were incubated with S. aureus at the indicated ratios for 30 min at 37°C. Cells were then lysed in water (upper panel) or in water at pH 11 (lower panel) as described in Materials and Methods. DPI-treated neutrophils ({blacksquare}) versus control neutrophils ({square}): NS, not significant; for all other conditions, P < 0.01. Bars indicate SEM n = 4.

 
Figure 5A shows that the staphylocidal activity of control and DPI-treated neutrophils after lysis in water appears higher than that after lysis in water at pH 11 at all incubation times. In control cells, such a difference is no longer significant after 45 min of incubation when killing is high (87.7%±3.1 and 82.5%±5.3 in water and water at pH 11, respectively), and therefore, no more than one viable microorganism is expected to remain within each cell. Lysis in water at pH 11 shows that DPI completely inhibits bacterial killing throughout the 45-min incubation period, whereas after lysis in water, only a partial inhibition is observed. Figure 5B shows the dose-response curves of the effect of DPI on the staphylocidal activity of neutrophils after lysis in water and in water at pH 11. In both conditions, optimal inhibition is obtained at 0.5 µM DPI. However, a maximum inhibition of 39.3% ± 4.4 becomes apparent using water, whereas with water at pH 11, inhibition of killing is virtually complete. As expected, inhibitory concentration of 50% values for DPI were similar in the two lysis conditions (0.11 µM after lysis in water; 0.15 µM after lysis in water at pH 11). It is worth noting that at the two lower DPI concentrations used (0.05 µM and 0.1 µM), killing was still significantly inhibited after lysis in water at pH 11 (26.8%±3.3 and 42.3%±7.0, respectively), whereas no significant inhibition could be observed after lysis in water (9.0%±4.4 and 13.0%±6.6, respectively). This latter observation indicates that lysis in water may not allow detection of partial killing defects.



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  		Figure 5. Time-course and dose-response of the effect of DPI on killing of S. aureus after lysis in water and water at pH 11. Open symbols: Control PMN; solid symbols: DPI-treated PMN; circles: lysis in water; triangles: lysis in water at pH 11. Results are mean ± SEM of five (A) and four (B) experiments. (A) Time-course of staphylocidal activity of control and DPI-treated neutrophils. Control cells, water at pH 11 versus water at 45 min: NS, not significant; for all other incubation times, P < 0.05. (B) Dose-response curve of the effect of DPI on killing of S. aureus. Significance of DPI effect (one-sample Student’s t-test): *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
We next determined whether conditions for cell lysis are critical for the measurement of killing of microorganisms other than S. aureus. Figure 6 shows the effect of DPI on killing of E. coli and the fungus C. albicans. The results obtained with these microorganisms clearly emphasize the importance of efficient cell lysis (i.e., dilution in water at pH 11) for correct assessment of microbicidal activity. In fact, lysis in water at pH 11 compared with lysis in water shows that killing of C. albicans is also totally inhibited by DPI and demonstrates that killing of E. coli, which does not appear to be affected by DPI after cell lysis in water, is inhibited to a significant extent.



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  		Figure 6. Effect of DPI on killing of E. coli and C. albicans after lysis in distilled water and water at pH 11. Neutrophils (4x106/ml in Ca2+/Mg2+-HBS) were incubated for 30 min with E. coli at a bacteria-to-cell ratio of 5:1 and for 60 min with C. albicans at a ratio of 3:1. Results are mean ± SEM of five (E. coli) and four (C. albicans) experiments. DPI-treated neutrophils ({blacksquare}) versus control neutrophils ({square}): **, P < 0.01; ***, P < 0.001.

 
Figure 7 summarizes the results of studies with neutrophils from patients with conditions associated with defective microbicidal activity, i.e., CGD and MPO deficiency. As revealed by lysis in water at pH 11, CGD neutrophils (Fig. 7A) are almost totally incapable of killing S. aureus and C. albicans and moreover, show a small but significant defect in the killing of E. coli. These results are in agreement with those using DPI, indicating that DPI-treated, normal neutrophils represent a good in vitro model of CGD neutrophils. Different results are seen after lysis in water, where killing of E. coli appears normal, and killing of S. aureus and C. albicans appears to be only partially defective. It is noteworthy that the results obtained using water to lyse the cells are similar to those reported in the literature [3 4 5 6 7 8 9 , 11 , 13 , 16 , 18 , 26 , 27 ], which suggests that previously published results are inaccurate as a result of the lack of efficacy of lysis in distilled water. With MPO-deficient neutrophils (Fig. 7B) , no defective killing of S. aureus and only a partially reduced killing of C. albicans were observed after lysis in water, whereas after lysis in water at pH 11, killing of C. albicans was strongly defective, and a clear-cut deficiency in killing of S. aureus could also be demonstrated. Killing of E. coli was normal. The marked defect of candidacidal activity, revealed after lysis in water at pH 11, is in agreement with previous reports in which killing has been measured by vital staining techniques [28 , 29 ] but differs from the partial defect shown by others who used distilled water for cell lysis [30 ]. Killing of S. aureus by MPO-deficient neutrophils has been shown to be normal, delayed, or defective [28 , 29 , 31 32 33 ]. The basis for these inconsistencies might well lay in the unreliability of the microbicidal assays used, based on lysis in distilled water. Our results provide the possibility to explain these discrepancies.



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  		Figure 7. Effect of lysis in distilled water and water at pH 11 on the assessment of microbicidal activity of CGD (A) and MPO-deficient (B) neutrophils. Normal (open bars), CGD (solid bars), and MPO-deficient (shaded bars) neutrophils were incubated with opsonized microorganisms at a bacteria-to-cell ratio of 5:1 and a fungi-to-cell ratio of 3:1. Percentages of killing refer to 45 min (S. aureus), 30 min (E. coli), and 60 min (C. albicans) of phagocytosis. (A) Mean ± SEM of three to six experiments with four CGD patients. (B) Mean ± SEM of four experiments with two MPO-deficient individuals for S. aureus and C. albicans and data from one duplicate experiment with E. coli. Differences from normal neutrophils: *, P < 0.05; **, P < 0.01.

 
It has been proposed recently that killing of phagocytosed microorganisms depends on the release of cationic proteins, including proteolytic enzymes, from the proteoglycan matrix of the granules and that NADPH oxidase is responsible for such release via an increased flux of K+ in the phagocytic vacuole [18 ]. Our results about microbicidal activity of CGD neutrophils and MPO-deficient neutrophils cast doubts on the reliability of this hypothesis. In fact, killing of E. coli is only partially defective in CGD neutrophils and DPI-treated, normal neutrophils, indicating that activation of NADPH oxidase is not needed for an effective killing of this microorganism. Also, MPO activity does not seem to be needed for killing of E. coli, as MPO-deficient neutrophils killed this microorganism normally. Thus, killing of E. coli appears to be mediated almost exclusively by oxygen-independent microbicidal mechanisms, as previously suggested by others [26 , 34 ]. Furthermore, MPO-deficient neutrophils were found markedly defective in killing of S. aureus and C. albicans. MPO-deficient neutrophils are known to exhibit a normal-to-increased respiratory burst and degranulation (ref. [35 ] and refs. therein) and are consequently expected to release microbicidal proteins from the granules normally. Therefore, our results suggest that release of granule proteins is not sufficient to kill S. aureus and C. albicans effectively and that the oxygen-dependent and MPO-dependent microbicidal mechanisms are likely to play a major role in the killing of these microorganisms. It would therefore seem likely that the inability of CGD neutrophils and DPI-treated, normal neutrophils to kill these microorganisms is mostly a result of the lack of activation of the MPO-dependent microbicidal mechanisms.

Table 1 compares literature data about staphylocidal activity of CGD neutrophils and DPI-treated, normal neutrophils with the results of this study. It can be readily observed that CGD neutrophils and DPI-treated, normal neutrophils exhibit a considerable killing activity in various reports, although with highly variable results. This is in striking contrast with our data, showing that CGD neutrophils and normal neutrophils treated with DPI are almost completely unable to kill staphylococci.

In conclusion, we demonstrate that a critical methodological shortcoming, i.e., use of distilled water for cell lysis, is a major source of error in assessment of microbicidal activity, particularly when it is defective. The present data should induce investigators to reassess the microbicidal activity of neutrophils in conditions where defects are suspected and to re-examine the effects of treatments (including drugs, pH, ions), which might influence such an activity. Finally, our data suggest that the re-evaluation of microbicidal activity of other phagocytic cells, such as eosinophils, monocytes, and macrophages, is warranted.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Italian Telethon Foundation, Contract Grant Number EC0533, and by Fondo Commissario del Governo nella Regione Friuli-Venezia Giulia.

Received June 23, 2005; revised August 18, 2005; accepted August 30, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Johnston, R. B., Newman, S. L. (1977) Chronic granulomatous disease Pediatr. Clin. North Am. 24,365-376[Medline]
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