Table 1

Measurement of mediators levels in sputum

MediatorSputum processingRecovery %Effect of DTT on gradient of standard curve#Mediator concentrationSubjectsAssay[Ref.]
Eosinophil proteins
ECP1NDDecreased288–1040 ng·mL−1N, A, SBRIA, Pharmacia2
2NDND49–107 ng·mL−1ARIA, Pharmacia3
3>85Not used0–1800 ng·mL−1A, CF, PRIA, Pharmacia8
4100+Decreased 30–50%12.8 ng·mL−1A, NFEIA, Pharmacia9
2NDND730–910 ng·mL−1A, NRIA, Pharmacia10
4100ND6–26 ng·mL−1A, NFEIA, Pharmacia11
Selected plus 10 vol 0.1% DTT for 30 min; 700×g for 10 min at 4°C98.6+No effect51–4605 ng·mL−1N, A, COPD, BRIA, Pharmacia12
3 (centrifuged repeatedly)NDND1200 ng·mL−1CFRIA, Pharmacia13
EPX/EDN3NDNot used100–4700 ng·mL−1A, CF, PRIA, Pharmacia8
1NDND270–1510 ng·mL−1N, A, SBRIA2
EPO5NDNo effect40–178 ng·mL−1A, COPDFEIA14
1NDND4.5–28.1 ng·mL−1NFEIA15
MBP1NDND304–1176 ng·mL−1A, N, SBIMRA2
Neutrophil proteins
HNE5NDNo effect4.2–5.2 µg·mL−1COPDEA (N‐MSN)16
2NDND0–20 µg·mL−1§ (free)A, N, COPDEA (N‐MSN)17
0–52 µg·mL−1§ (total)ELISA
Unselected plus 1 vol PBS; 25000×g for 20 minNDNot used2.6–5.9 µMCFEA (N‐MSN)18
2NDNo effect16 µMCFEA (N‐MSN)19
6NDNot used36.5–384.5 µg·mL−1§BEA (N‐MSN)20
7NDNot used0–2.1 µMCOPD, BEA (N‐MSN)21
Cathepsin G6NDNot used0.2–54.9 µg·mL−1§BEA (N‐SN)20
Cathepsin B7NDNot used65.8 U·mL−1BEA (N‐MESA)22
MPO5NDNo effect0.6–8.1 ng·mL−1A, COPDRIA, Pharmacia23
3 (centrifuged repeatedly)NDND110 ng·mL−1CFRIA, Pharmacia13
Selected plus 9 vol 0.1% DTT in shaking waterbath, at 37°CNDND1–220 ng·mL−1A (children)ELISA, R&D Systems24
478ND188–382 ng·mL−1A, NRIA, Pharmacia11
1NDND1.9 ng·mL−1ARIA, Pharmacia25
7>85ƒNot used3–15.7 U·mL−1§COPD, BEA (o‐DH2Cl)21
29–70Not used391–4557 ng·mL−1ELISA, R&D Systems
HNL5NDND1.7–10.5 mg·mL−1A, COPDFEIA23
1NDND7.1–12.4 mg·mL−1NFEIA15
5NDND3.7–5.1 mg·mL−1AFEIA14
Mast cell products
Histamine2100ƒND7–10 ng·mL−1A, NRIA, AMAC, Inc.26
4100+No effect (but lower levels with DTE)0.95 ng·mL−1A, NRIA, Serotec, Inc.9
Tryptase1NDND12–20.8 U·L−1A, N, SBIMRA, Pharmacia2
4NDND0–7.2 ng·mL−1A, NRIA, Pharmacia11
2100ƒND1.3–6.8 U·L−1A, N, SBRIA, Pharmacia26
4100+No effect8.8 ng·mL−1A, NRIA, Pharmacia9
2NDND1–6.1 ng·mL−1§ (detectable in 18%)A, NFEIA, Pharmacia27
Plasma exudate markers
Albumin1NDNo effect288–704 µg·mL−1N, A, SBELISA2
2NDND52–140 µg·mL−1ANephelometry3
2NDND9.9–32.7 µg·mL−1ATurbidimetry28
8SSP 99ƒDecreased, restored by NEMSSP 29.3–3300 ng·mL−1A, COPDImmunoturbidimetry, Roche Diagnostics29
Fibrinogen1NDND0.44–2.08 µg·mL−1N, A, SBELISA2
2NDND0.17–1.29 µg·mL−1AELISA3
1NDND2.7–250 µg·mL−1A, COPDELISA30
2100ƒND11.9–44.2 µg·mL−1A, NELISA26
α2‐M8106ƒ SSPNot usedSSP 900–1100 ng·mL−1A, COPDELISA29
93 SGP (after NEM)Decreased, restored by NEMSGP 1900–2100 ng·mL−1
7NDNot used0.16–0.21 µMCOPDRadial immunodiffusion31
Frozen, ultrasonicated 15 min, and centrifuged at 32000×g for 15 minNDNot used1.66–18.38 µg·mL−1NRIA32
Cytokines
GM­CSF3>85Not used0–91.8 pg·mL−1A, CF, PELISA, R&D Systems8
468ƒNDDetectable <5%A, NELISA, R&D Systems11
977Not usedDetectable <1%A, NELISA Amersham Biosciences33
5NDND1.3–1.5 ng·mL−1AELISA14
IL­1βSelected plus 7 vol 0.05% DTTNDND4.0–9.0 ng·mL−1AELISA, R&D Systems34
10NDNot used0.269–12.22 ng·mL−1COPD, CF, NELISA, R&D Systems35
980.9Not used0–0.40 ng·mL−1A, NELISA, Amersham Biosciences33
Unselected, frozen, thawed and centrifuged at 100000×g for 30 min at 4°CNDNot used8.21–17.96 ng·mL−1BELISA, R&D Systems36
IL‐2977.8Not used40–70 pg·mL−1A, NELISA, Amersham Biosciences33
IL‐33>85Not used0–39 pg·mL−1A, CF, PELISA, R&D Systems8
IL‐51NDNo effect9.4–46 pg·mL−1 (detectable 26.5%)N, A, SBELISA, R&D Systems2
3>85Not used0–0.17 ng·mL−1A, CF, PELISA, R&D Systems8
1NDND0–0.16 ng·mL−1AELISA, R&D Systems25
5NDND0.11–0.16 ng·mL−1 (detectable <30%)A, COPDELISA, R&D Systems14
Selected plus 9 vol 0.1% DTT in shaking waterbath at 37°CNDNDDetectable <5%A (children)ELISA, R&D Systems24
480ƒNDDetectable <5%A, NELISA, R&D Systems11
1NDNDDetectable 0%A, NELISA, R&D Systems30
1NDND44–67 pg·mL−1 (detectable 62.5%)AELISA, R&D Systems37
Unselected plus 3 vol PBS centrifuged at 40000×g for 30 min at 4°CNDNot used1.18 ng·mL−1AELISA, GIF38
5NDNo effect2.1–3.6 ng·mL−1AELISA14
IL‐62NDND79–187 ng·mL−1AELISA28
2NDND27–45 pg·mL−1NELISA, R&D Systems39
1NDND64–120 pg·mL−1COPDELISA, R&D Systems40
10NDNot used26–620 pg·mL−1COPD, CF, NELISA, R&D Systems35
980Not used50–310 pg·mL−1A, NELISA, Amersham Biosciences33
IL­101NDND0–68 pg·mL−1A, N, COPDELISA, R&D Systems41
10NDNot used24–177 pg·mL−1COPD, CF, NELISA, R&D Systems35
TNF‐α5NDND1.02–1.73 ng·mL−1AELISA14
752–60+Not used0–7.5 nM§N, A, COPDELISA42
367–75ƒNot used0.10–0.15 ng·mL−1CFELISA, R&D Systems43
Unselected, frozen, thawed, and centrifuged at 100,000 g, for 30 min at 4°CNDNot used44–120 pg·mL−1BELISA, R&D Systems36
TGF‐β1Unselected plus 3 vol PBS centrifuged at 40000×g for 30 min at 4°C. Acid activationNDNot used21.7 ng·mL−1AELISA, R&D Systems38
ET‐1Selected, centrifuged at 13000 rpm, for 15 min. HPLC, on C18 column88Decreased, therefore not used11–16 pg·mL−1N, ARIA, Nichols Institute Diagnostics44
IFN‐γ1NDNDDetectable 0%A, NELISA, R&D Systems30
975.3Not used30–70 pg·mL−1 (detectable 23%)A, NELISA, Amersham Biosciences33
Chemokines
IL‐83>85Not used1.27–4.95 ng·mL−1A, P, CFELISA, Bender MedSystsems8
2NDND2.1–2.7 ng·mL−1AELISA3
5NDNo effect2.1–3.5 nMARIA23
7>90ƒNot used0–3.5 nM§A, N, COPDRIA42
398ƒNot used30–49 ng·mL−1CFELISA, R&D Systems43
421ƒND41–87 pg·mL−1 (free; detectable 45%)A, NELISA11
438.3 (free)+; 100 (total)+No effect0 ng·mL−1 (free)A, NELISA9
1NDND2.88–16 ng·mL−1A, NELISA, R&D Systems30
5NDDecreased6.9–10.9 ng·mL−1 (total)COPDELISA, Amersham Biosciences16
RANTES3>85Not used15.6–151.5 ng·mL−1A, P, CFELISA, R&D Systems8
482ƒNDDetectable <5%A, NELISA, R&D Systems11
974.6Not used40–251 pg·mL−1A, NELISA, Amersham Biosciences33
Eotaxin10NDNot used14–530 pg·mL−1A, NELISA45
1 (on ice)NDND0.31 ng·mL−1 (detectable 86%)AELISA, R&D Systems46
MCP‐12NDND1.6–3.9 µg·mL−1AELISA, R&D Systems28
3 (centrifuged 1000×g for 10 min)NDNot used0–25 mg·mL−1§AELISA47
MIP‐1α3 (centrifuged 1000×g for 10 min)NDNot used0–25 mg·mL−1§AELISA47
Soluble cytokine receptors
IL­1RA10NDNot used31–94 ng·mL−1COPD, CF, NELISA, R&D Systems35
sTNFRII10NDNot used0.44–4.9 ng·mL−1COPD, CF, NELISA, R&D Systems35
sICAM‐1335–50ƒNot used0.63–1.9 ng·mL−1CFELISA, British Biotech43
2 (1 mM DTT)NDND1.1–11.9 ng·mL−1A, NELISA, Boehringer Mannheim48
486ƒND12–52 ng·mL−1A, NELISA, R&D Systems11
Proteases/inhibitors
MMP‐15NDDecreased19.1–20.5 ng·mL−1COPDELISA, Amersham Biosciences16
MMP‐92NDND23–80.5 ng·mL−1A, N, COPDELISA, Amersham Biosciences; zymography49
5NDDecreased13.2–13.7 ng·mL−1COPDELISA, Amersham Biosciences16
3NDNot used500–3000 ng·mL−1AELISA, Amersham Biosciences50
TIMP‐12NDND60–475 ng·mL−1A, N, COPDELISA, Amersham Biosciences50
5NDDecreased14.7–15.1 ng·mL−1COPDELISA, Amersham Biosciences16
3NDNot used200–550 ng·mL−1AELISA, Amersham Biosciences; zymography50
SLPI5NDDecreased5.3–5.7 µg·mL−1COPDELISA, Amersham Biosciences16
7>85ƒNot usedNot givenCOPD, BELISA, R&D Systems21
7NDNot used1.5–1.8 µMCOPDELISA31
α1‐AT2NDND0–78 µg·mL−1§A, N, COPDNephelometry17
7NDNot used2.4–2.6 µMCOPDRadial immunodiffusion31
Eicosanoids
LTB412 (with HPLC)27.8ƒ (radiolabelled, all stages assessed)Not used44.3 nMCFRIA51
7>85ƒNot usedNot givenCOPD, BELISA, Amersham Biosciences21
Unselected, frozen, centrifuged at 100000×g for 30 min at 4°CNDNot used1.62–3.17 ng·mL−1BELISA, R&D Systems36
LTC4, LTD4, LTE412 (with HPLC)19–27.2+ (radiolabelled, all stages assessed)Not used74.4 nMCFRIA51
1 (on ice); C18 column80–85ƒ (radiolabelled)ND6.4–13 ng·mL−1A, NELISA, Cayman Chemicals52
1 (on ice); C18 column80–85ƒ (radiolabelled)ND3.45–11.95 ng·mL−1AELISA, Cayman Chemicals53
12NDNot used1.0–5.7 nMB, COPD, CFRIA54
Ethanol, C18 columnNDNot used2.31 ng·mL−1A, NELISA, Cayman Chemicals55
PGD21 (on ice)NDND0.15–0.4 ng·mL−1A, NMS52
1 (on ice)NDND0.36–0.51 ng·mL−1AMS53
PGE21 (on ice)NDND1.2–1.6 ng·mL−1A, NMS52
1 (on ice)NDND1.09–1.11 ng·mL−1AMS53
12NDNot used19.3–45.7 nMB, COPD, CFRIA54
TxB21 (on ice)NDND0.7–1.3 ng·mL−1A, NMS52
12NDNot used6.3–10.5 nMB, COPD, CFRIA54
Ethanol, C18 columnNDNot used0.88 ng·mL−1A, NELISA, Cayman Chemicals55
PGF2α1 (on ice)NDND0.3–0.7 ng·mL−1A, NMS52
12NDNot used4.5–10.4 nMB, COPD, CFRIA54
Miscellaneous
NO productsSelected plus 7 vol 0.05% DTTNDND330–400 µMAGreiss reaction34
Unselected plus 4 vol 0.005% DTT, centrifuged at 13000×g for 30 minNDND387–774 μMN, CFGreiss reaction56
Lactoferrin2NDND35.2–118.9 µg·mL−1A, NELISA57
MLG2NDND562–2574 µg·mL−1A, NELISA57
DNA2NDND3.6–7.1 µg·mL−1A, NMicrofluorimetry, Calbiochem­Behring57
Total IgA4NDND10.5–18.4 µg·mL−1A, NELISA11
SC4NDND3.2–4.1 µg·mL−1A, NELISA11

  • 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.