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Published ahead of print on October 25, 2007, doi:10.1164/rccm.200603-311OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 132-141, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200603-311OC

Original Article

Optimized Dialysis and Protease Inhibition of Sputum Dithiothreitol Supernatants

Edward M. Erin1, Gavin R. Jenkins1, Onn Min Kon2, Angela S. Zacharasiewicz3, Grant C. Nicholson1, Helen Neighbour1, Rachel C. Tennant1, Andrew J. Tan1, Brian R. Leaker4, Andrew Bush3, Peter J. Jose5, Peter J. Barnes6 and Trevor T. Hansel1

1 Clinical Studies Unit, National Heart and Lung Institute, Imperial College, London, United Kingdom; 2 Department of Respiratory Medicine, St. Mary's Hospital, London, United Kingdom; 3 Department of Respiratory Paediatrics, and 4 Department of Nephrology, Royal Free Hospital, London, United Kingdom; and5 Department of Leukocyte Biology, and 6 Department of Thoracic Medicine, Imperial College, London, United Kingdom

Correspondence and requests for reprints should be addressed to Trevor T. Hansel, M.D., Ph.D., NHLI Clinical Studies Unit, Royal Brompton Hospital, Fulham Road, London SW3 6HP, UK. E-mail: t.hansel{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Dithiothreitol (DTT) is commonly used to liquefy induced sputum samples before assessment of cytology, but causes reduction of disulfide bonds and denaturation of proteins.

Objectives: To process sputum supernatants containing DTT to enable quantification of cytokines and chemokines.

Methods: A standard solution of 22 pooled chemokines and cytokines was incubated with DTT at the concentrations used during sputum liquefaction and then dialyzed under 20 different denaturant and redox conditions.

Measurements and Main Results: After incubation of the standard solution with DTT there was loss of detectable protein mediators on immunoassay, but optimized dialysis permitted recovery of chemokines to 96 ± 4% and cytokines to 91 ± 6%. Optimized dialysis of DTT supernatants from subjects with asthma covering a range of severities (n = 35) was performed in the presence of a cocktail of protease inhibitors and demonstrated significantly elevated levels of the chemokine CXCL10 (IFN-{gamma}–inducible protein-10), CXCL8 (IL-8), and CCL3 (macrophage inflammatory protein-1{alpha}); with lower but significantly elevated levels of CCL2 (monocyte chemotactic protein-1), CCL11 (eotaxin), and CCL5 (regulated on activation, normal T-cell expressed and secreted) in severe asthma. In sputum from subjects with severe asthma there were also significantly elevated levels of IL-4, IL-5, IL-13, tumor necrosis factor-{alpha}, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-12(p40).

Conclusions: The technique of optimized dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines. The detection of elevated levels of particular sputum chemokines and cytokines in individual patients may provide a rationale for specific therapies.

Key Words: sputum cytokines • dithiothreitol • dialysis • protease inhibitors



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Analysis of chemokines and cytokines in sputum is a controversial and technically difficult area. Dithiothreitol is used to liquefy conventionally processed sputum and its effects on analysis of sputum mediators require assessment and correction.

What This Study Adds to the Field
The technique of optimized dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines.

 
Inhalation of hypertonic saline is a relatively noninvasive method to induce sputum expectoration, and processing has permitted the assessment of inflammation in a range of respiratory diseases (1–3). In response to the interest in sputum analysis, a European Respiratory Society task force has published standardized sputum methodology (4, 5). Despite being derived from different compartments, inflammatory cell counts in asthma are related in sputum, bronchoalveolar lavage, and bronchial mucosal biopsies (6, 7). Furthermore, in clinical practice a treatment strategy aimed at normalization of the induced sputum eosinophil count has been shown to reduce asthma exacerbations without the need for additional antiinflammatory therapy (8, 9).

Dithiothreitol (DTT) is effective for liquefaction of sputum, releasing cells and permitting cytospin preparations to determine the cell differential. However, the reducing and denaturant effects of DTT diminish the detectable levels of mediators (10, 11), and a comprehensive table summarizing the effects of DTT on inflammatory proteins has been published (12). In addition, elegant work on the validity of fluid-phase detection of sputum IL-5 has demonstrated that there is a problem with proteases in causing decreased detectable levels (13), and that IL-5 levels can be significantly increased by adding protease inhibitors (PIs) (14). A number of groups have reported elevated levels of CXCL8 in induced sputum from subjects with asthma (15–17); while tumor necrosis factor (TNF)-{alpha}, CXCL1, CCL2, and CXCL8 are elevated in chronic obstructive pulmonary disease (15, 18, 19) and CXCL8 is elevated in cystic fibrosis (20).

Removal of DTT by dialysis against phosphate-buffered saline (PBS) has been shown to cause an increase in detectable levels of IL-13 in induced sputum supernatants from subjects with mild asthma (21), and our group has previously used dialysis against PBS to partially recover activity of CXCL8 in sputum supernatants (22). Dialysis removes low-molecular-weight DTT (MW 154), and this facilitates the reforming of disulfide bonds in sputum proteins, resulting in the partial recovery of immunoreactivity of some proteinaceous mediators.

Alternative methods of sputum processing have included homogenization (23), ultracentrifugation (15, 24), and ultrasonication (25) before analysis of levels of soluble mediators, including cytokines and chemokines. However, these physical methods involve cell lysis, release intracellular proteases, and probably provide a signal different from that of extracellular supernatant obtained after liquefaction of sputum with DTT. In addition, comparison of sputum samples solubilized with PBS, DTT, and dornase-{alpha} has demonstrated differences in detectable levels of mediators (20).

In the present study, we assess the effects of DTT on cytokine standards, and then examined various dialysis conditions to determine which favor denaturation and refolding of proteins to restore detectable levels of these standards (26). Bovine serum albumin (BSA) was added to minimize nonspecific binding of sputum supernatant proteins and a cocktail of PIs was used to minimize proteolysis of cytokines and chemokines. We used the Luminex bead array immunodetection system (Luminex Corporation, Austin, TX), which has the advantages of being semiautomated and being able to detect a panel of 22 cytokines/chemokines in small volumes of fluid. After performing "optimized dialysis" to remove DTT from sputum supernatants, we then measured levels of chemokines and cytokines in patients with asthma of differing severity.


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Adult subjects with asthma and healthy control subjects were recruited for this study from the Clinical Studies Unit, Royal Brompton Hospital (London, UK). Subjects with asthma were defined by the terms of the Global Initiative for Asthma (27), had features of episodic bronchospasm, and had demonstrable reversibility after inhalation of a β-adrenergic agonist. Subjects with mild asthma had an FEV1 greater than 70% and were not taking inhaled steroids, subjects with moderate asthma had an FEV1 of 60–70% and were taking inhaled corticosteroids, and subjects with severe asthma had an FEV1 less than 60% and were generally taking higher doses of inhaled corticosteroids but not oral corticosteroids. The study was approved by the Royal Brompton Hospital Ethics Committee and all subjects gave informed, written consent for sputum inductions, with the entire clinical investigation conducted according to the principles expressed in the Declaration of Helsinki.

For the cross-sectional study individual sputum samples were collected from 6 normal subjects (mean FEV1, 110.2% of predicted); 16 subjects with mild asthma (mean FEV1, 95.3% of predicted), not taking inhaled corticosteroids; 8 subjects with moderately severe asthma, taking inhaled corticosteroids (mean FEV1, 66.8% of predicted); and 11 subjects with severe asthma, taking inhaled corticosteroids (mean FEV1, 52.3% of predicted) (Table 1). To assess intrasubject variability induced sputum samples from 20 subjects with moderate or severe asthma (mean FEV1, 58.4% of predicted) were collected. Each subject attended for sputum induction on four visits on Day 1, Day 8, Day 50, and Day 77.


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  		TABLE 1. PATIENT CHARACTERISTICS AND SPUTUM CYTOLOGY

 
Effects of DTT
Standards.
A single standard solution of pooled chemokines and cytokines was reconstituted in assay buffer (Beadlyte human multi-cytokine standards 4 and 6; Upstate Cell Signaling Solutions/Millipore, Billerica, MA), such that IL-1{alpha} and IL-1β were at 800 pg/ml. This standard solution (1 ml) was incubated with DTT (Sigma-Aldrich, St. Louis, MO) in Dulbecco's PBS (Sigma-Aldrich) (4 ml of 6.5 mM DTT) to give a final DTT concentration of 5.2 mM, and compared with standard solution (1 ml) added to PBS (4 ml). Incubation of the standard with and without DTT was performed for 15 minutes at room temperature, as in the situation when sputum was liquefied with DTT solution, when 4 ml of 6.5 mM DTT (0.1%) was added to 1 g (1 ml) of sputum to obtain a 5.2-mM solution. PBS (4 ml) was then added to the standard solution in 5.2 mM DTT (5 ml), giving the standard in 2.9 mM DTT. This mimicks the liquefaction of sputum (1 g) in 4 ml of 6.5 mM DTT solution in PBS, followed by 4 ml of PBS to give a final concentration of 2.9 mM DTT. During the overnight incubation in the Luminex assay system, assay beads were exposed to standard solution containing DTT at a concentration of 1.92 mM; because standard in 2.9 mM DTT (50 µl) was mixed with bead solution (25 µl).

Conjugated beads.
Beads were incubated with DTT (1.92 mM at 4°C for 2 and 12 h) (see Table E1 in the online supplement), because this is the DTT concentration present during the Luminex assay. After incubation, DTT was removed by applying a vacuum manifold pump to the bottom of a filter plate before pooled standard solution was added.

Unconjugated beads.
Unconjugated beads lacking specific capture antibodies (donation from Luminex Corporation) were incubated with DTT (1.92 mM at 4°C for 2 and 12 h). DTT was removed by applying a vacuum manifold pump to the bottom of a filter plate before the pooled standard solution and pooled secondary antibodies were added.

Range of Dialysis Conditions
A range of 20 different dialysis conditions was set up, using a 5 x 4 fractional matrix system to provide an efficient screen for denaturation and refolding conditions (Pro-Matrix protein refolding kit; Pierce Biotechnology, Rockford, IL). Five denaturant chaotrope conditions and four redox environments were included in this matrix (Table 2; see Table E2 in the online supplement for complete data). The pH was maintained at 8.2 with 0.055 M TRIS buffer, and the redox environment was stabilized with EDTA. This basic screening protocol for protein refolding involved incubation with a strong denaturant, guanidine hydrochloride (550 mM or 1.1 M), or a weak denaturant, L-arginine (440 or 880 mM), to destabilize protein interactions. Concurrent use of redox environments was assessed using previously optimized levels and ratios of oxidized to reduced glutathione (final concentrations: 0.2 mM:2 mM, 0.4 mM:2 mM, and 1 mM:1 mM). A second dialysis was performed with PBS at pH 7.4 (12 h).


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  		TABLE 2. RECOVERY OF CHEMOKINES AND CYTOKINES AFTER TREATMENT OF POOLED STANDARD SOLUTION WITH DITHIOTHREITOL UNDER VARIOUS DIALYSIS CONDITIONS

 
Sputum Induction and Initial Processing
All sputum samples were collected and processed in a standardized manner (28). Briefly, after pretreatment of the patient with inhaled albuterol (200 µg), sputum was induced with nebulized hypertonic saline at 3.5%, for three periods of 5 minutes. Induced sputum was selected away from saliva and liquefied with 4 volumes of 6.5 mM DTT in PBS (Figure 1). The samples was incubated on a roller at room temperature for 15 minutes, and then 4 volumes of PBS was added and the samples were filtered through 48-µm nylon gauze. The sputum supernatant was collected after centrifugation at 400 x g for 10 minutes and stored at –70°C.


Figure 1
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  		Figure 1. Sputum processing methodology. A schematic is presented to illustrate the steps in sputum processing. These include dithiothreitol liquefaction followed by filtration, dilution, protease inhibition, and a two-step dialysis procedure. Chemokines and cytokines are then measured in the sputum extracts by bead array immunoassay (Luminex). EDTA = ethylenediaminetetraacetic acid; PBS = phosphate-buffered saline.

 
Protease Inhibition and Bovine Serum Albumin
PI cocktail.
A liquid cocktail of six broad-spectrum PIs was used (cat. no. P8340; Sigma-Aldrich), being added at 50 µl/ml of sputum DTT-containing supernatant. This gave the following final concentrations of PIs in the supernatant: 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF; 5 mM), aprotinin (400 µM), leupeptin (100 µM), bestatin (200 µM), pepstatin A (75 µM), and E-64 (70 µM). AEBSF is a broad-spectrum serine PI; aprotinin also inhibits serine proteases; leupeptin inhibits both serine and cysteine proteases; bestatin inhibits aminopeptidases; pepstatin A inhibits acid proteases; and E-64 inhibits cysteine proteases.

Ethylenediaminetetraacetic acid.
In addition, the disodium salt of ethylenediaminetetraacetic acid (EDTA; 0.5 M) (cat. no. 15575-020; Invitrogen Ltd, Paisley, UK) was added at 2 µl/ml of sputum DTT-containing supernatant, to give a final concentration of 1 mM. EDTA inhibits some metalloproteases.

BSA (20%).
BSA (20%) (cat. no. A3803; Sigma-Aldrich) was then added to the DTT-containing supernatant to give a final concentration of 1% (50 µl of BSA [20%] to 1,000 µl of supernatant). BSA was added at this step to reduce the nonspecific binding of the chemokines and cytokines to the dialysis sack and to adjust the final BSA to 1%, which is the same as the BSA concentration in the Luminex assay buffer.

Optimized Dialysis
Preparation of dialysis tubing.
Dialysis tubing (pore size, 3.5 kD; diameter, 16 mm) (cat. no. 3500/1; Medicell International Ltd, London, UK) was cut into 20-cm lengths and heated in a fume cupboard at 80°C for 45 minutes in Milli-Q water (4 L; Millipore, Billerica, MA) containing EDTA (0.5 mM) and sodium bicarbonate (0.2%). The process was repeated for a second 45 minutes in a fresh 4-L volume of Milli-Q water with EDTA (0.5 mM) and sodium bicarbonate (0.2%), to remove the preservative coating from the tubing. The tubing was then washed and flushed with Milli-Q water, using a Pasteur pipette. The tubing was knotted at one end, and sputum supernatant (containing DTT, PI cocktail, EDTA, and BSA) was added, before tying the other end to form the dialysis sack.

Dialysis buffers.
The first buffer for optimized denaturation and refolding was prepared by adding the following constituents to 4 L of Milli-Q water (Figure 1; and see condition F in Table 2 and condition 12 in Table E2): TRIS buffer (33.31 g, later giving 0.055 M in 5 L) (cat. no. 271195Y; VWR International, Lutterworth, UK), sodium chloride (6.136 g, giving 21 mM in 5 L), potassium chloride (0.328 g, giving 0.88 mM in 5 L), L-arginine (383.24 g, giving 440 mM in 5 L) (cat no. A5006; Sigma-Aldrich), reduced glutathione (3.07 g, giving 2 mM in 5 L) (cat. no. G4251; Sigma-Aldrich), oxidized glutathione (1.22 g, 0.4 mM in 5 L) (cat. no. G4376; Sigma-Aldrich), and EDTA (10 ml of 0.5 M, giving 1 mM in 5 L) (cat. no. 15575-020; Invitrogen). The pH was then adjusted by cautious dropwise addition of hydrochloric acid (concentration, 1.0 M; with mechanical stirring in a fume cupboard). The volume was then made up to 5 L with Milli-Q water, and the pH was adjusted to 8.2.

The second dialysis buffer was Dulbecco's PBS buffer, pH 7.4 (cat. no. P4417; Sigma-Aldrich) with 10 ml of EDTA (final concentration, 1 mM).

Dialysis procedure.
See Figure 1 for details.

First dialysis: After tying both ends of the dialysis tubing, the sack was floated with a floater/identity tag in the first prechilled dialysis buffer (4-L volume in a 5-L flask) and rotated with stirrers (4°C for 12 h). There were up to 50 sacks in a 5-L flask to maintain at least a 100:1 ratio of buffer to total supernatant volumes.
Second dialysis: Samples within Visking tubing (Medicell International Ltd) were then transferred to a second flask for dialysis (4°C for 12 h) in stirred prechilled PBS (4-L volume in a 5-L flask). Last, dialysis sacks were removed, the tubing was cut, and a pipette was used to recover the remaining fluid before placing it in a cryotube for storage at –80°C until analysis. All the chemokines and cytokines studied were too large to exit through the 3.5-kD molecular mass cutoff dialysis membrane.

Multiplex Bead Array Immunoanalysis
The levels of human IL-1{alpha}, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, CXCL8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, eotaxin, CCL5, CCL2, CCL3, CXCL10, granulocyte-macrophage colony–stimulating factor (GM-CSF), IFN-{gamma}, and TNF-{alpha} were analyzed in a multiplexed bead array system (Millepore Biosciences, Dundee, UK). Samples were read with a commercially available Luminex 100 IS instrument. This assay is designed for analysis of multiple quantitative measurements of supernatants. Using this 96-well microtiter plate–formatted assay it is possible to profile the level of multiple chemokines and cytokines in a single well, using as little as 50 µl of sample. However, to increase sensitivity, the gain of the RP1 reporter channel was increased from a value of 3,885 to 9,000 and a final late wash step was added to reduce the associated increase in background. The lower limit of detection obtained for cytokines and chemokines was not more than 1.9 pg/ml, except for GM-CSF, IL-4, IL-5, and IFN-{gamma}, for which it was 2.3 pg/ml. The lower limit of detection is the extrapolated value (in picograms per milliliter) on the linear part of the standard curve that is 2 standard deviations above the mean fluorescence intensity (MFI) of the zero standard replicates.

Dilution Factors
The values expressed are not corrected for the dilution of sputum liquefaction in DTT (1 volume of sample to 4 volumes of 6.5 mM DTT in PBS and then 4 volumes of PBS), and also are not corrected for the addition of PI cocktail, BSA, and EDTA (0.102 ml/ml of DTT supernatant). Thus, the overall dilution is 1 in 9.102 and the unprocessed sputum levels may be estimated by using a multiplication factor of 8.102.

Statistical Analysis
We have used summary statistics depending on whether our data are normally distributed (parametric) or not normally distributed (nonparametric). Normally distributed data are expressed as means with SEM (or confidence interval), and this was appropriate for the effects of DTT on pooled standards. However, when measuring detectable levels of cytokines and chemokines in sputum samples, this was nonnormally distributed data, because many levels were below detection limits. Hence, for sputum samples we have used summary statistics for nonparametric data, including medians. The sputum cell differential and FEV1 data have been correlated with the chemokine and cytokine data, using nonparametric Spearman correlation coefficients.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of DTT
Standards.
Incubation of the standard solution with DTT resulted in decreased detectable levels of 6 chemokines (21 ± 11%, mean ± SEM) and 16 cytokines (41 ± 7%) (Table 2). The remaining chemokine standard immunoreactivity was only 3–16% for CCL2, CCL3, CCL5, CCL11, and CXCL10, whereas CXCL8 had a greater remaining immunoreactivity of 74%. IL-4, IL-7, IL-12(p70), IL-13, and TNF-{alpha} had less than 10% remaining immunoreactivity. The effects of DTT on chemokines and cytokines at a range of concentrations were also studied (for results, see Figure E1).

ANTIBODY-CONJUGATED BEADS. The ability of antibody-conjugated beads to detect corresponding chemokines and cytokines in the standards was affected to a variable extent by incubation of the beads with DTT. IL-12(p40) remaining at 30% at 2 and 12 hours; and IL-1{alpha}, IL-1β, IL-3, CCL2, and CCL11 were less than 80% at 12 hours (see Table E1).

Unconjugated beads.
Nonspecific binding of the secondary antibodies to the unconjugated beads did not occur after DTT incubation with the beads (data not shown). This was at the concentration of DTT (1.92 mM) that beads would be exposed to in the assay of DTT-containing sputum supernatants.

Optimized Dialysis Conditions
Removal of DTT by dialysis against PBS across a 3.5-kD molecular mass cutoff pore size dialysis membrane with a buffer change facilitated reoxidation of disulfide bonds. This resulted in the recovery of immunoreactivity of most, but not all, of the mediators tested (see Figure E1 and Table 2).

After using optimized dialysis under condition 12, recovery of the 6 chemokines was 96 ± 4% (mean ± SEM) and that of the 16 cytokines was 91 ± 6%. A 100% recovery level was obtained for all cytokines with dialysis buffer condition 12 except for IL-4 (94%), IL-7 (66%), IL-10 (83%), IL-12(p40) (91%), and IL-12(p70) (3%) (Table 2). This buffer system was selected as the optimized dialysis system to be used in further experiments. Pooled standard solution of chemokines and cytokines not exposed to DTT were also dialyzed under the 20 different buffer systems, and no significant loss of immunoreactivity was found for any of the assessed proteins (data not shown).

Protease Inhibition
Effects of PI cocktail on standards.
The PI cocktail consists of rapidly acting irreversible antagonists of proteases, so that short-term incubation can be performed to detect effects. After incubation of the 7-PI cocktail (including EDTA) for 10 minutes with the pooled Luminex standard there were no significant changes in detectable levels of any of the 6 chemokines and 16 cytokines, compared with an assay buffer control (data not shown).

Effects of PI cocktail on standards when spiked in sputum.
A known amount of pooled standard (500 pg/ml) was added to a pooled DTT-derived sputum supernatant from six subjects with asthma, and then optimized dialysis was undertaken with and without the PI cocktail. There were increases in detectable levels of most chemokines and cytokines, but lower detection of IL-8, possibly due to chelation by EDTA of divalent cations that are required for IL-8 refolding (Table 3).


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  		TABLE 3. EFFECTS OF PROTEASE INHIBITOR COCKTAIL ON DETECTABLE LEVELS OF POOLED STANDARD SOLUTION WHEN SPIKED INTO POOLED DITHIOTHREITOL-CONTAINING SUPERNATANT

 
Effects of optimized dialysis on PIs.
The PI cocktail was added to sputum supernatants containing DTT before dialysis under optimized conditions of pH, denaturant conditions, and redox environment. The PI cocktail has immediate and irreversible effects on proteases, because they are noncompetitive inhibitors with low Ki values. Therefore, optimized dialysis is unlikely to affect protease inhibition.

Variability
Technical variability.
A pooled sample obtained from six subjects with mild asthma (FEV1 > 70% of predicted) was analyzed, using the optimized dialysis system 12 with protease inhibition. The pooled sample (6 ml) was divided into 6 (1-ml) samples and each was dialyzed separately under identical conditions. Coefficients of variation were less than 10% (see Table E3).

Intrasubject variability.
Individual samples from 20 subjects with moderately severe asthma were collected. Each subject attended for sputum induction on four visits. A coefficient of variation for each mediator was calculated for each patient on 4 visits, and a mean (SEM) coefficient of variation was then derived for the group of 20 subjects (see Table E3). Samples that were not dialyzed showed greater variability (coefficients of variation up to 178%) compared with samples that were processed with optimized dialysis (coefficients of variation up to 19.7%). A further slight reduction in variability was seen if dialysis was performed in the presence of the PI cocktail. This compared favorably with the variability of sputum cell differential data concurrently processed during the same four visits. A mean (SEM) coefficient of variation for absolute eosinophil cell counts (cells per milliliter) was 39.8 (5.6), for absolute neutrophil cell counts (cells per milliliter) it was 21.1 (7.8), for absolute macrophage cell counts (cells per milliliter) it was 14.2 (7.4), and for absolute lymphocyte cell counts (cells per milliliter) it was 11.4 (6.5). Interestingly, it was seen that this group of patients with moderate and severe asthma had remained clinically stable when assessed at the four visits, as the mean (SEM) coefficient of variation for FEV1 was 9.1 (1.1).

Levels of Chemokines and Cytokines in Patients with Asthma
Optimized dialysis and protease inhibition increased the levels of cytokines and chemokines in induced sputum from patients with asthma, and this allowed for superior discrimination between patients with asthma of differing severity and a control group of healthy subjects (Figures 2, 3, and 4). In particular, statistically significant differences were observed for all six chemokines between the severe asthma group and the healthy subject group for CCL2 (P < 0.01), CCL3 (P < 0.001), CCL11 (P < 0.001), CXCL10 (P < 0.001), CXCL8 (P < 0.05), and CCL5 (P < 0.01). In addition, statistically significant differences were also observed for the cytokines TNF-{alpha} (P < 0.01), IL-4 (P < 0.01), IL-5 (P < 0.01), IL-6 (P < 0.001), IL-12(p40) (P < 0.01), IL-13 (P < 0.01), and GM-CSF (P < 0.001) between the severe asthma group and healthy control group. Addition of the PI cocktail to sputum samples generally caused an increase in detectable levels of chemokines and cytokines, when employed without optimized dialysis (data not shown). However, in all cases the increase was less than that shown with optimized dialysis. The effects of optimized dialysis plus protease inhibition are shown in Figures 2, 3, and 4.


Figure 2
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  		Figure 2. Chemokines in sputum supernatants from patients with asthma. Levels of chemokines were determined in sputum supernatants from patients with asthma of different severity. Lower limit of detection obtained for all chemokines shown: <=1.9 pg/ml. D = dialysis; IP-10 = IFN-{gamma}–inducible protein-10; MCP-1 = monocyte chemotactic protein-1; MIP-1{alpha} = macrophage inflammatory protein-1{alpha}; Nil = no treatment; ns = nonsignificant; PI = protease inhibition with a cocktail of inhibitors; RANTES = regulated on activation, normal T-cell expressed and secreted. *P < 0.05; **P < 0.01; ***P < 0.001 (see Table E4 for a complete set of P values). Medians are shown as horizontal bars.

 

Figure 3
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  		Figure 3. Cytokines in sputum supernatants from patients with asthma. Levels of cytokines were determined in sputum supernatants from patients with asthma of different severity. Lower limit of detection obtained for cytokines shown: <=1.9 pg/ml (except for granulocyte-macrophage colony–stimulating factor [GM-CSF] and IFN-{gamma}, for which it was 2.3 pg/ml). Dashed line indicates limit of detection for GM-CSF. ND = Not detectable; TNF-{alpha} = tumor necrosis factor-{alpha}; for other abbreviations see Figure 2. *P < 0.05; **P < 0.01; ***P < 0.001 (see Table E4 for a complete set of P values). Medians are shown as horizontal bars.

 

Figure 4
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  		Figure 4. (A) IL-4, IL-5, and IL-13 in sputum supernatants from patients with asthma. Levels of cytokines were determined in sputum supernatants from patients with asthma of different severity. Lower limit of detection obtained for IL-4 and IL-5 was 2.3 pg/ml (dotted line indicates limit of detection) and for IL-13 it was 1.9 pg/ml. For abbreviations, see Figures 2 and 3. *P < 0.05; **P < 0.01; ***P < 0.001 (see Table E4 for a complete set of P values). Medians are shown as horizontal bars. (B) Eosinophils, IL-4, IL-5, and IL-13 in sputum supernatants from allergic and nonallergic patients with severe asthma. Subjects with severe asthma were classified according to allergic status: allergic asthma, serum total IgE levels > 150 IU/ml; nonallergic asthma, serum total IgE levels < 150 IU/ml. Levels of interleukins are those after dialysis and protease inhibition (D+PI), in the same subjects as in A. (C) IL-1β, TNF-{alpha}, IL-6, GM-CSF, IFN-{gamma}, and IL-12(p40) in sputum supernatants from allergic and nonallergic patients with severe asthma. Severe asthma subjects were classified according to allergic status: allergic asthma, serum total IgE levels > 150 IU/ml; nonallergic asthma, serum total IgE levels < 150 IU/ml. Levels of interleukins are those after dialysis and protease inhibition (D+PI), in the same subjects as in A.

 
Values quoted are not corrected for dilution with DTT, PBS, and PIs. Hence true physiological levels of chemokines and cytokines within sputum would be expected to be higher than the levels described.

A key question concerns whether optimized dialysis and protease inhibition of DTT-containing supernatants has advantages, other than broadly increasing detectable levels of chemokines and cytokines, and causing less variability in detectable levels. Table E4 shows comparisons of the significance when comparing patients with asthma of varying severity with a normal population in terms of detectable cytokine and chemokine levels, either with or without optimized dialysis and protease inhibition. After optimized dialysis and protease inhibition, differences in the levels of CCL2, CCL3, CCL5, CCL-11, IL-4, IL-5, IL-6, IL-13, and GM-CSF became significant, whereas previously they had not been significant.

Correlation of Sputum Cell Differential Counts and Mediators after Optimized Dialysis
Sputum neutrophils (percentage) were found to correlate (Spearman correlation coefficient) with levels of the following mediators after optimized dialysis in the presence of PIs: MIP-1{alpha} (r = 0.39; P < 0.05), eotaxin (r = 0.39; P < 0.01), IL-8 (r = 0.4; P < 0.01), TNF-{alpha} (r = 0.42; P < 0.01), GM-CSF (r = 0.47; P < 0.001), IL-6 (r = 0.42; P < 0.01), IL-7 (r = 0.3; P < 0.01), and IL-12(p40) (r = 0.47; P < 0.01). These correlations were seen only when the mediators were processed using optimized dialysis in the presence of PIs. Sputum neutrophils (percentage) correlated significantly with asthma severity in terms of FEV1 expressed as a percentage of the predicted value (r = –0.57; P < 0.05).

Sputum eosinophils (percentage) were found to correlate with eotaxin (r = 0.3; P < 0.01), MCP-1 (r = 0.37; P < 0.01), MIP-1{alpha} (r = 0.55; P < 0.001), IL-4 (r = 0.38; P < 0.01), IL-5 (r = 0.37; P < 0.01), IL-6 (r = 0.33; P < 0.05), and IL-13 (r = 0.35; P < 0.01). These correlations were again seen only after optimized dialysis. Sputum eosinophils (percentage) also correlated with FEV1 expressed as a percentage of the predicted value.

Asthma severity, measured as FEV1 expressed as a percentage of the predicted value, correlated significantly with the following mediators after optimized dialysis in the presence of PIs: MIP-1{alpha} (r = –0.44; P < 0.01), MIP-1{alpha} (r = –0.45; P < 0.01), eotaxin (r = –0.47; P < 0.001), IP-10 (r = –0.46; P < 0.01), IL-8 (r = –0.31; P < 0.05), and RANTES (r = –0.37; p < 0.01) (see Figures 3 and 4), IL-1β (r = –0.45; P < 0.01), TNF-{alpha} (r = –0.47; P < 0.01), IL-4 (r = –0.58; P < 0.001), IL-5 (r = –0.59; P < 0.001), IL-6 (r = –0.58; P < 0.001), IL-7 (r = –0.3; P < 0.01), GM-CSF (r = –0.57; P < 0.001), IL-12(p40) (r = –0.39; P < 0.01) (see Figures 3 and 4), and IL-13 (r = –0.3; P < 0.01). IFN-{gamma} was not correlated with disease severity.

Among the patients with severe asthma, it was noted that those with allergic asthma (IgE > 150 IU/ml) had higher levels of eosinophils, IL-4, IL-5, and IL-13. Conversely, those with nonallergic asthma (IgE < 150 IU/ml) had higher levels of IFN-{gamma} and IL-12(p40) (see Figures 4B and 4C).


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
After protease inhibition and optimized dialysis of sputum supernatants containing DTT, we showed that there is an increase in some chemokines and cytokines as detected with a multiarray system. Using this technique, we noted that elevated levels of some chemokines and cytokines were found on comparing levels between normal subjects and subjects with asthma of varying severities. This method of processing enables the characterization of particular sputum cytokines and chemokines in patients with asthma, and may prove helpful to predict those patients with severe asthma who may respond to specific therapies, for instance, therapy directed against TNF-{alpha} (29).

Levels of IL-4, IL-5, IL-13, and eosinophils were all found to be elevated in the same patients with severe allergic asthma. This finding is in agreement with bronchoalveolar lavage data (30), although IgE synthesis occurs in the bronchial mucosa in both allergic and nonallergic asthma (31). Levels of these cytokines increase in late-phase nasal secretions in some patients with allergic rhinitis after nasal challenge with timothy grass pollen (30, 31) and IL-13 has been reported in sputum from subjects with asthma after use of dialysis (32). Detection of IL-5 in sputum remains problematic according to other workers, even when PIs are used to elevate levels (14). However, IL-5 and IL-4 may not be important pathogenic inflammatory mediators, because a monoclonal antibody against IL-5 was largely clinically ineffective in severe persistent asthma (33), and a soluble IL-4 receptor construct was discontinued in clinical trials in asthma because of lack of efficacy (34). Cytokines responsible for IgE production may be pathogenic in some patients with severe allergic asthma, because systemic monoclonal antibody directed against IgE is clinically effective (35), but the factors responsible for the elevated IgE production may be present in other compartments than sputum.

Our study found significantly elevated levels of TNF-{alpha}, IL-6, GM-CSF, and IL-12(p40) in sputum from some subjects with severe asthma, treated with inhaled but not oral corticosteroids. This picture is more of a neutrophil- and macrophage-related disease, and is suggestive of helper T-cell type 1 involvement. Indeed, a number of separate groups have noted neutrophilic as opposed to eosinophilic inflammation in severe asthma (36–38). In particular, our results are consistent with reports of the clinical success of therapy directed against TNF-{alpha} in severe refractory asthma (39, 40). Furthermore, patients with refractory severe asthma had increased expression of membrane-bound TNF-{alpha}, TNF-{alpha} receptor-1, and TNF-{alpha}–converting enzyme by peripheral blood monocytes (39).

The most prominent chemokines found in the sputum of subjects with severe asthma receiving inhaled but not oral corticosteroids were CXCL10, CXCL8, and CCL3. CXCL10 is a widely expressed chemokine that can cause nonspecific activation of self-reactive lymphocytes in autoimmune disease via CXCR3 on helper T type 1 cells (41). CXCL8 is associated with neutrophilic responses. In severe asthma, there were also significantly elevated but lower levels of sputum CCL11 and CCL5 (42), providing a rationale for the use of chemokine receptor antagonists in severe asthma. Chemokine receptors are G protein–coupled receptors that represent tractable targets for new drugs (43, 44).

The effect of DTT on recovery of immunoreactivity of proteins may be partly related to the number and location of disulfide bonds. IL-4, IL-7, IL-10, and IL-12 are cytokines with more complex structures that are vulnerable to denaturation by DTT. For example, IL-12(p70) is composed of a subunit with one disulfide bond and a subunit with five disulfide bonds, and these subunits are linked by a further two disulfide bonds. IL-12(p70) is the cytokine that is most affected by DTT and has the least recovered immunoreactivity.

The immunoreactivity of IL-5 was one of the least affected by DTT, but affected by proteases, in accordance with the experience of previous investigators (14). Sputum is rich in neutrophil elastase (45) and matrix metalloproteases (46), and along with other proteases these enzymes have the capacity to cause significant proteolysis of chemokines and cytokines in sputum during processing before immunoassay.

There may be high intrasubject variability in detectable levels of chemokines and cytokines when treating sputum with DTT because there can be variable time of exposure of these proteins to DTT during the liquefaction process. Hence, use of a PI cocktail and optimized dialysis results in lower variability in detectable levels of chemokines and cytokines on repeat sputum sampling in the same individual with mild asthma. In the current study we have added PIs only after dithiothreitol dispersion of sputum samples, adding to the supernatant after thawing. This is because our aim was to increase the use of supernatants obtained after conventional DTT dispersion. However, it is probable that earlier use of PIs, by adding the cocktail immediately after sputum expectoration, may further increase detectable levels of cytokines such as IL-5.

Assessment of induced sputum is always likely to have the inherent problems of sampling error, because only a fraction of the pooled inspissated secretions in the airways is likely to be expectorated. Induced sputum represents a particular respiratory compartment, and within the airways there may be considerable cell death and release of intracellular cytokines and proteases, especially in the presence of infection or airway obstruction. Influence of proteases, mucus, infection, and salivary contamination are all possible further confounding factors for the assessment of inflammatory mediators.

Use of optimized dialysis partially overcomes the denaturant effects of DTT and has permitted the identification of particular cytokines and chemokines associated with severe asthma. Sputum supernatants from subjects with severe asthma contain elevated amounts of particular cytokines and chemokines; and this may have important diagnostic, monitoring, and therapeutic implications. In addition, it would be of interest to assess levels of neuropeptides and proteins involved in remodeling in severe asthma. Particular immunoassays should be validated with respect to the effects of DTT and the need for PIs, and this validation procedure should be performed with sputum samples from various respiratory diseases.


   Acknowledgments
 
The authors thank Mrs. Jackie Turner for expert help with statistical analysis.


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200603-311OC on October 25, 2007

Conflict of Interest Statement: E.M.E. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.R.J. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. O.M.K. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.S.Z. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.C.N. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.N. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.C.T. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.J.T. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.R.L. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.B. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.J.J. has no financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.J.B. has received research funding and lecture fees, and has served on scientific advisory boards for GlaxoSmithKline (GSK), AstraZeneca, Boehringer-Ingelheim, Novartis, Altana, and Pfizer, all of which have an interest in new therapies for chronic obstructive pulmonary disease. T.T.H. has received funding from GSK, Pfizer, Novartis, IMMD, Oxagen, and CMP Therapeutics for research work carried out in the NHLI Clinical Studies in the last 3 years.

Received in original form March 1, 2006; accepted in final form October 25, 2007


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