The cell-free H₂O₂-MPO-Cl^‾-luminol system was designed to model the respiratory burst. The CL peaked within a few minutes, then tapered off for the next 23 minutes of recording (Figure 1a). Calprotectin increased (p < 0.05) CL (Figure 1b), whereas cytidine deaminase inhibited it already after one minute (p < 0.05).

In the four experiments depicted in Figure 1 MPO (0.1-0.3 µg/mL) and calprotectin (16 µg/mL) in combination had an additive effect on light emission (p < 0.05). A moderate increase (21%) was also observed for 8.0 µg/mL calprotectin (p < 0.05), provided that the data for only the first 10 minutes were used. For a total of nine experiments the effect was not only additive (p < 0.001), but also potentiated synergistic (p < 0.01); the average CL integrals being MPO 8.9, calprotectin 4.8 and MPO + calprotectin 19.1. Even though stimulation by calprotectin varied considerably (see below), it was not significantly affected by addition of, or preincubation with, calcium (1-2 mM). It is noteworthy that PMN may contain >10.000 µg/mL calprotectin [7].

Dose-response experiments showed that the CL was enhanced more than linearly with increased concentrations of MPO (0.1-0.4 μg/mL), and at high activity of MPO (not shown) the inhibitory effect of a constant cytidine deaminase concentration (3.2 µg/mL) was reduced (p < 0.01). On the other hand, with a constant MPO concentration, cytidine deaminase exerted increasing inhibition in the dose range 0.8-6.4 µg/mL (Figure 1b). The PMN concentration of cytidine deaminase has been measured to be 100-200 µg/mL [14].

In the same molar or mass concentration ranges used for cytidine deaminase and calprotectin, control human serum albumin (HSA) had no effect on MPO-CL, but inhibited the response at concentrations ≥100 µg/mL (p < 0.05). CL was not induced with MPO or calprotectin when luminol was replaced by lucigenin (0.1 mM), which is supposed to mostly reflect superoxide formation. The calprotectin had no detectable peroxidase activity in the ECL assay.

As demonstrated for the first time here, calprotectin itself can induce CL. However, the activity of calprotectin was lower than for MPO, and we wanted to test if it could be enhanced by modifying the experimental procedure. At first we tested pH changes and found that the response (AUC) to calprotectin (8 µg/mL) was increased from 1 to 19 (p < 0.001) and the MPO-dependent response decreased (AUC from 10 to 7.3, p < 0.01) along with elevation of pH from 7.0 to 7.9 (Figure 2a-d). In four new experiments these results were confirmed with another buffer (Dulbecco’s phosphate-buffered saline), and calprotectin luminescence could be markedly enhanced by a pH increase from 7.1 to 7.4. In additional tests we found that 2-4 µg/mL of calprotectin, with pH 7.9 and 60-500 µM H₂O₂ was appropriate for routine use.

Controls, relevant according to molar and mass concentrations, with HSA (up to 1000 µg/mL) or human immunoglobulin (20 µg/mL) yielded CL (AUC) of ~1 or less (not shown). A pH-dependent pattern was also observed for CL elicited by PMA-stimulated human granulocytes, rat granulocytes, and rat peritoneal cells (Figure 2f-h). This response (Figure 2i) resembled the pH dependency of the calprotectin-response more than that of the MPO-response (Figure 2d, Figure 2e).

This pattern was confirmed when H₂O₂ instead of PMA was used as stimulator of human PMN (Figure 3), thereby presumably bypassing the contribution by phagocyte NADPH oxidase. This H₂O₂ response was almost aborted by azide (1 mM) at pH 6.1, and the 12-minute response was consistently reduced at pH 6.9-7.8, probably reflecting an inhibitory effect of azide on the heme enzyme (MPO). However, as pH increased, the response in the azide groups was changed (Figure 3a-d), and the 60-minute CL integral (at pH 7.4 and 7.8) was increased above control without azide (Figure 3e, p < 0.01). With 20000 PMN/well (Figure 3f), we found that 16 µM of H₂O₂ was sufficient to yield a significant (p < 0.05) response.

We also wanted to compare responses of MPO and calprotectin to different reactants, some of them known to affect MPO. The responses of MPO and calprotectin to increasing concentrations of H₂O₂ were very different (Figure 4a), even though both agents showed H₂O₂-dependency. H₂O₂ (500 µM) + luminol, alone, yielded a slight response (AUC~0.9) only at high pH (7.8). MPO was much more sensitive than calprotectin to the inhibitory effect of azide (Figure 4b). In fact, there was a slight stimulation of calprotectin-H₂O₂-CL at 0.02-0.1 mM azide (p < 0.05). Calprotectin-H₂O₂-CL was independent of the presence of NaCl, whereas MPO-H₂O₂-CL required a high NaCl concentration (Figure 4c), presumably for HOCl production. The response pattern was similar for inhibition of MPO and calprotectin by added cysteine and melatonin - added as anti-oxidants [18]-with MPO the more sensitive (p < 0.01) (Figure 4d, Figure 4e).

The combination of calprotectin (4 µg/mL) and NaOCl (20 µM) strongly enhanced CL in the presence of H₂O₂ (Figure 5), possibly caused by oxidation of calprotectin by NaOCl [19]. Since HOCl is the end product in the MPO-system, this finding may explain the synergy observed by combining MPO and calprotectin (Figure 1). NaOCl itself may generate significant CL, but high background values are largely avoided at low concentrations of H₂O₂ (Figure 5). Noteworthy, it was necessary to incubate the mixture of calprotectin and NaOCl for 17-20 minutes at room temperature in these experiments, before the CL reaction could be initiated by adding luminol and H₂O₂ and enhancement demonstrated [19]. Dose-response experiments showed that a calprotectin concentration of 0.5-1.0 µg/mL was sufficient, when combined with NaOCl and H₂O₂ (>16 µM), to induce a significant CL response, whereas no response was observed in the absence of NaOCl.

A8 remained a monomer in solution while A9 formed a dimer as well (Figure 6), but to a different extent in different batches. Dimerization apparently enhanced the capacity to generate CL in the presence of H₂O₂ (data not shown).

The activity of A9 (Figure 7a, Figure 7b) clearly depended upon both protein concentration (5-40 µg/mL) and pH, as did A8, which produced less CL (not shown). Mercaptoethanol-treated A9 elicited no detectable CL. A9 with NaOCl + 32 μM H₂O₂ gave a marked pH-dependent CL-response (Figure 7c). An even stronger response was observed with 63 μM H₂O₂ (Figure 7d). However, increasing concentrations above 3.6 µg/mL led to a gradual decrease of the CL, in contrast to the set-up without NaOCl included (Figure 7a). Similarly, HSA + NaOCl evoked a slight increase of CL up to 25 µg/mL albumin, declining to no response at 200-1,000 µg/mL. HSA without NaOCl did not cause any CL (2.5 to 1,000 µg/mL). At pH 7.8 similar responses were observed for A8 and A9 when combined with NaOCl (not shown). The activity of mercaptoethanol-exposed A9 was also restored by NaOCl.

A molar ratio of 1.6-3.2 between H₂O₂ and NaOCl proved to be optimal for A9-induced CL. We assessed the procedure by keeping the ratio constant at 1.6 in the dose ranges of 4-64 µM for H₂O₂ and 2.5-40 µM for NaOCl and obtained a bell-shaped curve for A9-CL, with 1.2 µg A9 per mL (Figure 8). Low background levels of the NaOCl + H₂O₂ were maintained. We saw A9-CL significantly above background (p < 0.05) with only 5.0 µM NaOCl + 8 µM H₂O_2. These values of NaOCl and H₂O₂ are in the physiological range [4,20,21]. It is possible to trigger ROS formation even more by increasing the H₂O₂ concentration (with constant NaOCl), but this tends to elevate the NaOCl-induced background levels and leads to more variable responses. For routine use, 10 or 20 µM NaOCl and H₂O₂ in the 16-64 µM range is recommended.

4-HBA (2 mM), considered to be an agent trapping hydroxyl radicals [22,23,24], stimulated (p < 0.05) the luminol-H₂O₂- dependent (500 µM H₂O₂) MPO-response by 28% (five experiments). On the contrary, 4-HBA abrogated CL (p < 0.05) by A8 (500 µM H₂O₂, three experiments) and A9 (500 µM H₂O₂, two experiments). In five experiments 4-HBA did not affect CL elicited by NaOCl alone, but suppressed (see Figure 7d) the intense CL generated by NaOCl + A9 (1.2 µg/mL) + H₂O₂ (63 µM) by 65% (p < 0.001).

Histidine, assumed to be a singlet oxygen scavenger [25], strongly inhibited the NaOCl-increased CL induced by A9, whereas inhibition was weaker in the absence of NaOCl, and then similar to the effect on H₂O₂-induced MPO responses (Figure 9).

A temperature increase from 24 to 37°C consistently enhanced CL integrals induced by A8 and A9 in combination with H₂O₂, but only with NaOCl in the reaction mixture (p < 0.001; data not shown). With MPO, or A9, + H₂O₂, we observed no consistent effect of temperature change.

In the absence of NaOCl, iron ions (FeSO₄, 140 µM) enhanced luminol-H₂O₂-CL, and the effect was additive when the iron salt was combined with calprotectin. FeCl₃ had a similar effect. Lower concentrations (10-20 µM) stimulated only marginally. When NaOCl was included, there was no additional stimulatory effect of iron ions (0.1-100 µM). Deferoxamine, an iron chelator (200 µM), significantly (two experiments) reduced the CL-response (p < 0.01) elicited by A8, A9, A9 + NaOCl, NaOCl alone, and MPO. The strongest reduction (90%) was observed with A9 + NaOCl, whereas an approximately 50% decline was obtained for MPO and NaOCl set-ups. The interpretation of these results is difficult since deferoxamine not only chelates iron, but may also bind and inactivate OH-radicals and possibly other ROS [26]. Taken together, the possibility exists that trace amounts of iron or other metals can activate calprotectin.

Hydrogen peroxide (30%), 5-amino-2,3-dihydro-1,4-phtalazinedione (luminol), bis-N-methylacridinium nitrate (lucigenin), myeloperoxidase (MPO), phorbol 12-myristate 13-acetate (PMA), mercaptoethanol, deferoxamine mesylate, 4-hydroxybenzoic acid, sodium azide, melatonin, L-cysteine, L-histidine and human serum albumin (HSA, MW ~ 67 kDa) were purchased from Sigma (St. Louis, USA), and Hepes buffer from BioWhittaker (Walkersville, MD, USA). Dextran 500 (Pharmacia, Uppsala, Sweden) was dissolved in 0.9% NaCl and used as a 3 or 6% solution. Sodium hypochlorite (NaOCl, 0.5 M in 0.1 M NaOH), purchased from BDH (Dorset, England), was diluted with water. Luminol and PMA were dissolved in dimethylsulphoxide (Sigma) and further diluted in water or a buffered salt solution. Phosphate-buffers of different pH were made by mixing appropriate portions of 0.2 M NaH₂PO₄ and 0.2 M Na₂HPO₄. Peroxidase was measured with the enhanced chemiluminescence method (ECL, Amersham Pharmacia Biotech, Oslo). Calprotectin, purified from human neutrophils [46], was generously provided by Axis-Shield (Oslo, Norway) and dialysed against Hanks’ balanced salt solution (HBSS, Invitrogen, Norway) or phosphate-buffered saline before use. The two subunits (S100A8 and S100A9) preferentially combine as a heterodimer, and tend to remain associated during purification [47]. However, they may also combine as homodimers and even trimers or tetramers [47], so exact characterization by MW is not always possible. Cytidine deaminase (MW~52 kDa) was produced as recombinant protein [13] and dissolved in HBSS.

Competent E. coli BL21 CodonPlus(DE3)-RIL cells were transformed with pET28-S100A8 or pET28-S100A9 [48], grown in 2 L of LB medium with 25 μg/mL kanamycin to OD₆₀₀ of 1 and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma) for 4 h at 30 ºC. Extract was prepared by sonication of the cell pellet in 40 mM Na₂PO₄, pH 8.0, 300 mM NaCl (sonication buffer) and cleared with centrifugation. The cell extract was applied on a Ni-column equilibrated with sonication buffer. The column was eluted with a gradient of 50-300 mM imidazole in sonication buffer, and purification of A8 and A9 (with and without mercaptoethanol) was monitored by protein gel electrophoresis. The N-terminal 6xHis were removed by Thrombin (T-4648, Sigma) cleavage, which enhanced the CL-inducing activity of the dialysed proteins. The purified A8 migrated as a monomer of apparent molecular weight 10 kDa, while A9 migrated as a monomer and a dimer of apparent molecular weight 13 and 26 kDa (Figure 6), respectively. Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad), with BSA as a standard.

A cell-free system was used as model of the respiratory burst in PMN: H₂O₂ + MPO + chloride + luminol. In some experiments MPO was replaced by calprotectin. The luminol-enhanced CL was measured as relative light units in a luminometer (Luminoskan, Termo Labsystems, Helsinki, Finland), reading the samples in 96-well plates (White Cliniplate, Thermo Fisher Scientific, Vantaa-Finland). In some experiments (Figure 7, Figure 8) we used a FLx800 luminometer (Biotek Instruments, Inc. Winooski, Vermont, USA). In relative terms the results were similar with the two luminometers, but the observed values (arbitrary light units) were quite different. Unless otherwise indicated, the recording was done at 37 ^oC. Each well contained 25 µL H₂O₂ solution (final concentrations are indicated), 5-10 µL MPO, 50 µL luminol (0.1 mM), 10-20 µL test substance and buffer (140-170 µL) to yield a total volume of 250 µL. MPO (10-40 µg) was dissolved in 1 mL water and stored at 4 ^oC. The activity was stable for some months, but upon further dilution it was often strongly reduced after a couple of days. The chemiluminescence was recorded (2-4 wells per group) at 1-2 minute intervals for 25-30 (or 60 minutes, Figure 3) minutes, and the integral of the response curve (AUC, area under the curve) was calculated. MPO concentration of 0.05-0.3 µg/mL mostly yielded AUC in the 5-30 range, but substantially increased responses were sometimes observed with modified procedures. Significant responses with MPO, calprotectin or cells were observed with 8-16 µM H₂O₂ as stimulator. However, consistent results were best obtained at higher concentration (30-500 µM). A control group with buffer, but without luminol and H₂O₂ was always included. Tris-buffers should be avoided because it triggers substantial CL when combined with NaOCl.

Human blood from healthy colleagues was collected into vacutainers containing EDTA. The granulocytes were separated as described previously [49]. Male Fisher 344 rats (Møllegaard Breeding Centre, Ejby, Denmark) were also used. They were treated in accordance with institutional and national guidelines for animal research. Rat granulocytes were prepared as follows: The rats were anaesthetized with CO₂ and killed by decapitation, and the trunk blood was collected into tubes containing EDTA. One part (3-6 mL) of EDTA-blood was mixed with one part of dextran 3% to cause aggregation and sedimentation of erythrocytes. Then 3 mL of Lymphoprep (Axis-Shield, Oslo, Norway) was installed underneath leucocyte-rich dextran-plasma (4-8 mL) in 15 mL tubes and centrifuged for 15 min at 600 g at room temperature. The bottom fraction, comprising >95% granulocytes, was collected and washed once. The cell button was suspended in 4 mL lysis solution (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM EDTA) and incubated at room temperature for 7 minutes to lyse remaining red cells. If necessary, the procedure was repeated. Rat peritoneal cells were obtained by washing the peritoneum of dead animals with 25 mL phosphate-buffered saline. The cells were pelleted by centrifugation and resuspended in phosphate buffer or HBSS supplied with 20 mM Hepes buffer (pH 7.4) and 5 mM glucose.

The results are given as means with their standard error (SEM). An analysis of variance (ANOVA) procedure was used to test for dose-response of cytidine deaminase and calprotectin. Student’s t-test was used to assess the difference between two groups. Two-sided P values < 0.05 were considered statistically significant.