Mediator | Sputum processing | Recovery % | Effect of DTT on gradient of standard curve# | Mediator concentration¶ | Subjects | Assay | [Ref.] |
Eosinophil proteins | |||||||
ECP | 1 | ND | Decreased | 288–1040 ng·mL−1 | N, A, SB | RIA, Pharmacia | 2 |
2 | ND | ND | 49–107 ng·mL−1 | A | RIA, Pharmacia | 3 | |
3 | >85 | Not used | 0–1800 ng·mL−1 | A, CF, P | RIA, Pharmacia | 8 | |
4 | 100+ | Decreased 30–50% | 12.8 ng·mL−1 | A, N | FEIA, Pharmacia | 9 | |
2 | ND | ND | 730–910 ng·mL−1 | A, N | RIA, Pharmacia | 10 | |
4 | 100 | ND | 6–26 ng·mL−1 | A, N | FEIA, Pharmacia | 11 | |
Selected plus 10 vol 0.1% DTT for 30 min; 700×g for 10 min at 4°C | 98.6+ | No effect | 51–4605 ng·mL−1 | N, A, COPD, B | RIA, Pharmacia | 12 | |
3 (centrifuged repeatedly) | ND | ND | 1200 ng·mL−1 | CF | RIA, Pharmacia | 13 | |
EPX/EDN | 3 | ND | Not used | 100–4700 ng·mL−1 | A, CF, P | RIA, Pharmacia | 8 |
1 | ND | ND | 270–1510 ng·mL−1 | N, A, SB | RIA | 2 | |
EPO | 5 | ND | No effect | 40–178 ng·mL−1 | A, COPD | FEIA | 14 |
1 | ND | ND | 4.5–28.1 ng·mL−1 | N | FEIA | 15 | |
MBP | 1 | ND | ND | 304–1176 ng·mL−1 | A, N, SB | IMRA | 2 |
Neutrophil proteins | |||||||
HNE | 5 | ND | No effect | 4.2–5.2 µg·mL−1 | COPD | EA (N‐MSN) | 16 |
2 | ND | ND | 0–20 µg·mL−1§ (free) | A, N, COPD | EA (N‐MSN) | 17 | |
0–52 µg·mL−1§ (total) | ELISA | ||||||
Unselected plus 1 vol PBS; 25000×g for 20 min | ND | Not used | 2.6–5.9 µM | CF | EA (N‐MSN) | 18 | |
2 | ND | No effect | 16 µM | CF | EA (N‐MSN) | 19 | |
6 | ND | Not used | 36.5–384.5 µg·mL−1§ | B | EA (N‐MSN) | 20 | |
7 | ND | Not used | 0–2.1 µM | COPD, B | EA (N‐MSN) | 21 | |
Cathepsin G | 6 | ND | Not used | 0.2–54.9 µg·mL−1§ | B | EA (N‐SN) | 20 |
Cathepsin B | 7 | ND | Not used | 65.8 U·mL−1 | B | EA (N‐MESA) | 22 |
MPO | 5 | ND | No effect | 0.6–8.1 ng·mL−1 | A, COPD | RIA, Pharmacia | 23 |
3 (centrifuged repeatedly) | ND | ND | 110 ng·mL−1 | CF | RIA, Pharmacia | 13 | |
Selected plus 9 vol 0.1% DTT in shaking waterbath, at 37°C | ND | ND | 1–220 ng·mL−1 | A (children) | ELISA, R&D Systems | 24 | |
4 | 78 | ND | 188–382 ng·mL−1 | A, N | RIA, Pharmacia | 11 | |
1 | ND | ND | 1.9 ng·mL−1 | A | RIA, Pharmacia | 25 | |
7 | >85ƒ | Not used | 3–15.7 U·mL−1§ | COPD, B | EA (o‐DH2Cl) | 21 | |
29–70 | Not used | 391–4557 ng·mL−1 | ELISA, R&D Systems | ||||
HNL | 5 | ND | ND | 1.7–10.5 mg·mL−1 | A, COPD | FEIA | 23 |
1 | ND | ND | 7.1–12.4 mg·mL−1 | N | FEIA | 15 | |
5 | ND | ND | 3.7–5.1 mg·mL−1 | A | FEIA | 14 | |
Mast cell products | |||||||
Histamine | 2 | 100ƒ | ND | 7–10 ng·mL−1 | A, N | RIA, AMAC, Inc. | 26 |
4 | 100+ | No effect (but lower levels with DTE) | 0.95 ng·mL−1 | A, N | RIA, Serotec, Inc. | 9 | |
Tryptase | 1 | ND | ND | 12–20.8 U·L−1 | A, N, SB | IMRA, Pharmacia | 2 |
4 | ND | ND | 0–7.2 ng·mL−1 | A, N | RIA, Pharmacia | 11 | |
2 | 100ƒ | ND | 1.3–6.8 U·L−1 | A, N, SB | RIA, Pharmacia | 26 | |
4 | 100+ | No effect | 8.8 ng·mL−1 | A, N | RIA, Pharmacia | 9 | |
2 | ND | ND | 1–6.1 ng·mL−1§ (detectable in 18%) | A, N | FEIA, Pharmacia | 27 | |
Plasma exudate markers | |||||||
Albumin | 1 | ND | No effect | 288–704 µg·mL−1 | N, A, SB | ELISA | 2 |
2 | ND | ND | 52–140 µg·mL−1 | A | Nephelometry | 3 | |
2 | ND | ND | 9.9–32.7 µg·mL−1 | A | Turbidimetry | 28 | |
8 | SSP 99ƒ | Decreased, restored by NEM | SSP 29.3–3300 ng·mL−1 | A, COPD | Immunoturbidimetry, Roche Diagnostics | 29 | |
Fibrinogen | 1 | ND | ND | 0.44–2.08 µg·mL−1 | N, A, SB | ELISA | 2 |
2 | ND | ND | 0.17–1.29 µg·mL−1 | A | ELISA | 3 | |
1 | ND | ND | 2.7–250 µg·mL−1 | A, COPD | ELISA | 30 | |
2 | 100ƒ | ND | 11.9–44.2 µg·mL−1 | A, N | ELISA | 26 | |
α2‐M | 8 | 106ƒ SSP | Not used | SSP 900–1100 ng·mL−1 | A, COPD | ELISA | 29 |
93 SGP (after NEM) | Decreased, restored by NEM | SGP 1900–2100 ng·mL−1 | |||||
7 | ND | Not used | 0.16–0.21 µM | COPD | Radial immunodiffusion | 31 | |
Frozen, ultrasonicated 15 min, and centrifuged at 32000×g for 15 min | ND | Not used | 1.66–18.38 µg·mL−1 | N | RIA | 32 | |
Cytokines | |||||||
GMCSF | 3 | >85 | Not used | 0–91.8 pg·mL−1 | A, CF, P | ELISA, R&D Systems | 8 |
4 | 68ƒ | ND | Detectable <5% | A, N | ELISA, R&D Systems | 11 | |
9 | 77 | Not used | Detectable <1% | A, N | ELISA Amersham Biosciences | 33 | |
5 | ND | ND | 1.3–1.5 ng·mL−1 | A | ELISA | 14 | |
IL1β | Selected plus 7 vol 0.05% DTT | ND | ND | 4.0–9.0 ng·mL−1 | A | ELISA, R&D Systems | 34 |
10 | ND | Not used | 0.269–12.22 ng·mL−1 | COPD, CF, N | ELISA, R&D Systems | 35 | |
9 | 80.9 | Not used | 0–0.40 ng·mL−1 | A, N | ELISA, Amersham Biosciences | 33 | |
Unselected, frozen, thawed and centrifuged at 100000×g for 30 min at 4°C | ND | Not used | 8.21–17.96 ng·mL−1 | B | ELISA, R&D Systems | 36 | |
IL‐2 | 9 | 77.8 | Not used | 40–70 pg·mL−1 | A, N | ELISA, Amersham Biosciences | 33 |
IL‐3 | 3 | >85 | Not used | 0–39 pg·mL−1 | A, CF, P | ELISA, R&D Systems | 8 |
IL‐5 | 1 | ND | No effect | 9.4–46 pg·mL−1 (detectable 26.5%) | N, A, SB | ELISA, R&D Systems | 2 |
3 | >85 | Not used | 0–0.17 ng·mL−1 | A, CF, P | ELISA, R&D Systems | 8 | |
1 | ND | ND | 0–0.16 ng·mL−1 | A | ELISA, R&D Systems | 25 | |
5 | ND | ND | 0.11–0.16 ng·mL−1 (detectable <30%) | A, COPD | ELISA, R&D Systems | 14 | |
Selected plus 9 vol 0.1% DTT in shaking waterbath at 37°C | ND | ND | Detectable <5% | A (children) | ELISA, R&D Systems | 24 | |
4 | 80ƒ | ND | Detectable <5% | A, N | ELISA, R&D Systems | 11 | |
1 | ND | ND | Detectable 0% | A, N | ELISA, R&D Systems | 30 | |
1 | ND | ND | 44–67 pg·mL−1 (detectable 62.5%) | A | ELISA, R&D Systems | 37 | |
Unselected plus 3 vol PBS centrifuged at 40000×g for 30 min at 4°C | ND | Not used | 1.18 ng·mL−1 | A | ELISA, GIF | 38 | |
5 | ND | No effect | 2.1–3.6 ng·mL−1 | A | ELISA | 14 | |
IL‐6 | 2 | ND | ND | 79–187 ng·mL−1 | A | ELISA | 28 |
2 | ND | ND | 27–45 pg·mL−1 | N | ELISA, R&D Systems | 39 | |
1 | ND | ND | 64–120 pg·mL−1 | COPD | ELISA, R&D Systems | 40 | |
10 | ND | Not used | 26–620 pg·mL−1 | COPD, CF, N | ELISA, R&D Systems | 35 | |
9 | 80 | Not used | 50–310 pg·mL−1 | A, N | ELISA, Amersham Biosciences | 33 | |
IL10 | 1 | ND | ND | 0–68 pg·mL−1 | A, N, COPD | ELISA, R&D Systems | 41 |
10 | ND | Not used | 24–177 pg·mL−1 | COPD, CF, N | ELISA, R&D Systems | 35 | |
TNF‐α | 5 | ND | ND | 1.02–1.73 ng·mL−1 | A | ELISA | 14 |
7 | 52–60+ | Not used | 0–7.5 nM§ | N, A, COPD | ELISA | 42 | |
3 | 67–75ƒ | Not used | 0.10–0.15 ng·mL−1 | CF | ELISA, R&D Systems | 43 | |
Unselected, frozen, thawed, and centrifuged at 100,000 g, for 30 min at 4°C | ND | Not used | 44–120 pg·mL−1 | B | ELISA, R&D Systems | 36 | |
TGF‐β1 | Unselected plus 3 vol PBS centrifuged at 40000×g for 30 min at 4°C. Acid activation | ND | Not used | 21.7 ng·mL−1 | A | ELISA, R&D Systems | 38 |
ET‐1 | Selected, centrifuged at 13000 rpm, for 15 min. HPLC, on C18 column | 88 | Decreased, therefore not used | 11–16 pg·mL−1 | N, A | RIA, Nichols Institute Diagnostics | 44 |
IFN‐γ | 1 | ND | ND | Detectable 0% | A, N | ELISA, R&D Systems | 30 |
9 | 75.3 | Not used | 30–70 pg·mL−1 (detectable 23%) | A, N | ELISA, Amersham Biosciences | 33 | |
Chemokines | |||||||
IL‐8 | 3 | >85 | Not used | 1.27–4.95 ng·mL−1 | A, P, CF | ELISA, Bender MedSystsems | 8 |
2 | ND | ND | 2.1–2.7 ng·mL−1 | A | ELISA | 3 | |
5 | ND | No effect | 2.1–3.5 nM | A | RIA | 23 | |
7 | >90ƒ | Not used | 0–3.5 nM§ | A, N, COPD | RIA | 42 | |
3 | 98ƒ | Not used | 30–49 ng·mL−1 | CF | ELISA, R&D Systems | 43 | |
4 | 21ƒ | ND | 41–87 pg·mL−1 (free; detectable 45%) | A, N | ELISA | 11 | |
4 | 38.3 (free)+; 100 (total)+ | No effect | 0 ng·mL−1 (free) | A, N | ELISA | 9 | |
1 | ND | ND | 2.88–16 ng·mL−1 | A, N | ELISA, R&D Systems | 30 | |
5 | ND | Decreased | 6.9–10.9 ng·mL−1 (total) | COPD | ELISA, Amersham Biosciences | 16 | |
RANTES | 3 | >85 | Not used | 15.6–151.5 ng·mL−1 | A, P, CF | ELISA, R&D Systems | 8 |
4 | 82ƒ | ND | Detectable <5% | A, N | ELISA, R&D Systems | 11 | |
9 | 74.6 | Not used | 40–251 pg·mL−1 | A, N | ELISA, Amersham Biosciences | 33 | |
Eotaxin | 10 | ND | Not used | 14–530 pg·mL−1 | A, N | ELISA | 45 |
1 (on ice) | ND | ND | 0.31 ng·mL−1 (detectable 86%) | A | ELISA, R&D Systems | 46 | |
MCP‐1 | 2 | ND | ND | 1.6–3.9 µg·mL−1 | A | ELISA, R&D Systems | 28 |
3 (centrifuged 1000×g for 10 min) | ND | Not used | 0–25 mg·mL−1§ | A | ELISA | 47 | |
MIP‐1α | 3 (centrifuged 1000×g for 10 min) | ND | Not used | 0–25 mg·mL−1§ | A | ELISA | 47 |
Soluble cytokine receptors | |||||||
IL1RA | 10 | ND | Not used | 31–94 ng·mL−1 | COPD, CF, N | ELISA, R&D Systems | 35 |
sTNFRII | 10 | ND | Not used | 0.44–4.9 ng·mL−1 | COPD, CF, N | ELISA, R&D Systems | 35 |
sICAM‐1 | 3 | 35–50ƒ | Not used | 0.63–1.9 ng·mL−1 | CF | ELISA, British Biotech | 43 |
2 (1 mM DTT) | ND | ND | 1.1–11.9 ng·mL−1 | A, N | ELISA, Boehringer Mannheim | 48 | |
4 | 86ƒ | ND | 12–52 ng·mL−1 | A, N | ELISA, R&D Systems | 11 | |
Proteases/inhibitors | |||||||
MMP‐1 | 5 | ND | Decreased | 19.1–20.5 ng·mL−1 | COPD | ELISA, Amersham Biosciences | 16 |
MMP‐9 | 2 | ND | ND | 23–80.5 ng·mL−1 | A, N, COPD | ELISA, Amersham Biosciences; zymography | 49 |
5 | ND | Decreased | 13.2–13.7 ng·mL−1 | COPD | ELISA, Amersham Biosciences | 16 | |
3 | ND | Not used | 500–3000 ng·mL−1 | A | ELISA, Amersham Biosciences | 50 | |
TIMP‐1 | 2 | ND | ND | 60–475 ng·mL−1 | A, N, COPD | ELISA, Amersham Biosciences | 50 |
5 | ND | Decreased | 14.7–15.1 ng·mL−1 | COPD | ELISA, Amersham Biosciences | 16 | |
3 | ND | Not used | 200–550 ng·mL−1 | A | ELISA, Amersham Biosciences; zymography | 50 | |
SLPI | 5 | ND | Decreased | 5.3–5.7 µg·mL−1 | COPD | ELISA, Amersham Biosciences | 16 |
7 | >85ƒ | Not used | Not given | COPD, B | ELISA, R&D Systems | 21 | |
7 | ND | Not used | 1.5–1.8 µM | COPD | ELISA | 31 | |
α1‐AT | 2 | ND | ND | 0–78 µg·mL−1§ | A, N, COPD | Nephelometry | 17 |
7 | ND | Not used | 2.4–2.6 µM | COPD | Radial immunodiffusion | 31 | |
Eicosanoids | |||||||
LTB4 | 12 (with HPLC) | 27.8ƒ (radiolabelled, all stages assessed) | Not used | 44.3 nM | CF | RIA | 51 |
7 | >85ƒ | Not used | Not given | COPD, B | ELISA, Amersham Biosciences | 21 | |
Unselected, frozen, centrifuged at 100000×g for 30 min at 4°C | ND | Not used | 1.62–3.17 ng·mL−1 | B | ELISA, R&D Systems | 36 | |
LTC4, LTD4, LTE4 | 12 (with HPLC) | 19–27.2+ (radiolabelled, all stages assessed) | Not used | 74.4 nM | CF | RIA | 51 |
1 (on ice); C18 column | 80–85ƒ (radiolabelled) | ND | 6.4–13 ng·mL−1 | A, N | ELISA, Cayman Chemicals | 52 | |
1 (on ice); C18 column | 80–85ƒ (radiolabelled) | ND | 3.45–11.95 ng·mL−1 | A | ELISA, Cayman Chemicals | 53 | |
12 | ND | Not used | 1.0–5.7 nM | B, COPD, CF | RIA | 54 | |
Ethanol, C18 column | ND | Not used | 2.31 ng·mL−1 | A, N | ELISA, Cayman Chemicals | 55 | |
PGD2 | 1 (on ice) | ND | ND | 0.15–0.4 ng·mL−1 | A, N | MS | 52 |
1 (on ice) | ND | ND | 0.36–0.51 ng·mL−1 | A | MS | 53 | |
PGE2 | 1 (on ice) | ND | ND | 1.2–1.6 ng·mL−1 | A, N | MS | 52 |
1 (on ice) | ND | ND | 1.09–1.11 ng·mL−1 | A | MS | 53 | |
12 | ND | Not used | 19.3–45.7 nM | B, COPD, CF | RIA | 54 | |
TxB2 | 1 (on ice) | ND | ND | 0.7–1.3 ng·mL−1 | A, N | MS | 52 |
12 | ND | Not used | 6.3–10.5 nM | B, COPD, CF | RIA | 54 | |
Ethanol, C18 column | ND | Not used | 0.88 ng·mL−1 | A, N | ELISA, Cayman Chemicals | 55 | |
PGF2α | 1 (on ice) | ND | ND | 0.3–0.7 ng·mL−1 | A, N | MS | 52 |
12 | ND | Not used | 4.5–10.4 nM | B, COPD, CF | RIA | 54 | |
Miscellaneous | |||||||
NO products | Selected plus 7 vol 0.05% DTT | ND | ND | 330–400 µM | A | Greiss reaction | 34 |
Unselected plus 4 vol 0.005% DTT, centrifuged at 13000×g for 30 min | ND | ND | 387–774 μM | N, CF | Greiss reaction | 56 | |
Lactoferrin | 2 | ND | ND | 35.2–118.9 µg·mL−1 | A, N | ELISA | 57 |
MLG | 2 | ND | ND | 562–2574 µg·mL−1 | A, N | ELISA | 57 |
DNA | 2 | ND | ND | 3.6–7.1 µg·mL−1 | A, N | Microfluorimetry, CalbiochemBehring | 57 |
Total IgA | 4 | ND | ND | 10.5–18.4 µg·mL−1 | A, N | ELISA | 11 |
SC | 4 | ND | ND | 3.2–4.1 µg·mL−1 | A, N | ELISA | 11 |
Sputum was processed as follows: 1) selected sputum, 4 volumes (vol) 0.1% (6.5 mM) dithiothreitol (DTT) and 4 vol phosphatebuffered saline (PBS) centrifuged at 500×g for 10 min; 2) unselected sputum and 1 vol 0.1% DTT in shaking waterbath at 37°C for 15 min, centrifuged at 2,000 revolutions per minute (rpm) for 5 min; 3) unselected sputum and 1–4 vol PBS centrifuged at 2000×g for 20–30 min at 4°C; 4) unselected sputum and 1 vol 10 mM dithioerythritol (DTE) centrifuged at 400×g for 10 min at 4°C; 5) unselected sputum and 2 mL 1% DTT centrifuged at 300×g for 10 min; 6) unselected sputum and 3 vol PBS centrifuged at 8,800×g for 20 min; 7) unselected sputum centrifuged at 50,000–60,000×g for 60–90 min at 4°C; 8) thawed frozen spontaneous sputum centrifuged at 50,000×g for 90 min at 4°C (sputum sol phase (SSP)); rest mixed with 1 vol 10 mM DTT and 150 U DNAse type IV (Sigma, St Louis, MO, USA) for 60 min at 4°C and then centrifuged at 50,000×g for 90 min at 4°C (sputum gel phase (SGP)); 9) unselected sputum and 10 vol PBS glass homogenised at 4°C and then centrifuged at 3,000 rpm for 15 min at 4°C; 10) selected sputum and 1 vol PBS centrifuged at 8,800×g for 20 min; and 11) unselected sputum collected into ethanol on ice and centrifuged at 12,000×g for 20 min, evaporated to dryness and separated on C18 column
The locations of the companies from which commercial assays were obtained are as follows: Pharmacia: Uppsala, Sweden
R&D Systems: Minneapolis, MN, USA;
AMAC, Inc.: Westbrook, ME, USA
Serotec, Inc.: Raleigh, NC, USA
Roche Diagnostics: Grenzacherstrasse, Basle, Switzerland
Amersham Biosciences: Little Chalfont, UK
GIF: Munster, Germany
Nichols Institute Diagnostics: San Clemente, CA, USA
Bender Medsystems: Vienna, Austria
British Biotech: Oxford, UK; Boehringer Mannheim: Mannheim, Germany
Cayman Chemicals: Ann Arbor, MI, USA; and CalbiochemBehring: La Jolla, CA, USA
ECP: eosinophil cationic protein
EPX/EDN: eosinophil protein X or eosinophilderived neurotoxin
EPO: eosinophil peroxidase
MBP: major basic protein
HNE: human neutrophil elastase
MPO: myeloperoxidase
HNL: human neutrophil lipocalin
α2‐M; α2‐macroglobulin
GMCSF: granulocytemacrophage colonystimulating factor
IL: interleukin
TNF: tumour necrosis factor
TGF‐β1: transforming growth factor‐β1
ET‐1: endothelin‐1
IFN‐γ: interferon gamma
RANTES: regulated on activation, normal T‐cell expressed and secreted
MCP‐1: monocyte chemotactic protein‐1
MIP‐1α: macrophage inflammatory protein‐1α
IL1RA: IL‐1 receptor antagonist
sTNFRII: type II soluble TNF receptor
sICAM‐1: soluble intercellular adhesion molecule‐1
MMP: matrix metalloproteinase
TIMP: tissue inhibitor of metalloproteinase
SLPI: secretory leucocyte protease inhibitor
α1‐AT: α1‐antitrypsin
LT: leukotriene
PG: prostaglandin
TxB2: thromboxane B2
MLG: mucinlike glycoprotein
DNA: deoxyribonucleic acid
Ig; immunoglobulin
SC: secretory component of IgA
ND: not determined
N: normal control
A: asthmatic
SB: smokers' bronchitis
RIA: radioimmunoassay
CF: cystic fibrosis
P: pneumonia
FEIA: fluorimetric enzyme immunoassay
COPD: chronic obstructive pulmonary disease
B: bronchiectasis
IMRA: immunoradiometric assay
EA: enzyme assay (specified substrate)
N‐MSN: N‐methoxysuccinylAlaAlaProVal p‐nitroanilide
ELISA: enzymelinked immunosorbent assay
N‐SN: N‐succinyl‐AlaAlaProPhe p‐nitroanilide
N‐MESA: 2‐(N‐morpholino)ethanesulphonic acid
o‐DH2Cl: o‐dianisidine dihydrochloride
NEM: N‐ethylmaleimide
HPLC: highperformance liquid chromatography (reverse phase)
MS: mass spectroscopy
#: ND indicates that the effect of DTT on the standard curve was not investigated despite DTT being used to process the sputum; not used indicates that DTT was not used to process the sputum
¶: data are presented as range of mean/median values
+: sputum spiked preprocess (no label indicates that it is not known when the sputum was spiked)
§: range (not mean range)
ƒ: sputum supernatant spiked.