CHAPTER FIVE
5.4 Binding characteristics of complement components
5.7.3 Results
Little TIMP-1, release was seen. The TIMP-1 that was released seem to be degraded to 14.5 kDa (Figure 5.5 B). Degradation may be due to the release of primary granule enzymes, especially elastase (It oh and Nagase, 1995), and/or oxidative processes (Stricklin and Hoidal, 1992).Degradation was possibly due to oxidative process as PMN samples incubated in buffer only showed no elastase release (results not shown), but a TIMP-1 band at 14.5 kDa (Figure 5.5 B). Other samples also showed low level elastase release (results not shown). Though levels of proteins loaded varied slightly most TIMP- 1 was released when PMNs were incubated in storage buffer (Figure 5.5 B, f) and to a lesser extent upon uptake of coated beads (Figure 5.5 B, c) or uncoated beads in the absence of serum (Figure 5.5 B, d).
kDa kDa
94- 94
66- 66
45- 45
30- 30
16- 16
14- 14
a b c d e f 9 h
A
kDa kDa
94 94
66 66
45 45
30 30
16 16
14 14
.--
a b
c
d e f 9 hB
Figure 5.5 TIMP-l release during Clq-coated latex bead uptake studies. Freshly isolated PMNs were re-suspended in PBSG buffer and 1 x 106 cells were added to a) PBSG (75 Ill) + PMA (90 ng),b) serum (25 Ill)+coated beads (2 x 107) +PBSG (50 Ill),c) PBSG (75 Ill) +coated beads (2 x 10\ d) PBSG (75 Ill)+uncoated beads (2 x 10\ e) serum (25 Ills)+ PBSG (50 Ill)+uncoated beads (2 x 10\ f) PBSG (75 Ill), g) serum (25 Ill)+PBSG (50 Ills). Controls included h) diluted serum i) PMN homogenates. Suspensions were incubated for 20 min RT, centrifuged(1300 g, 1 min) and supematant (2.5 III each) were reduced with equal amount of treatment buffer and run in (A) Tris-tricine gel as follows a)0.07 ug,b)1.54 ug, c) 0.09 ug,d)0.067 ug, e) 1.48 ug, f) 0.067 ug, g) 1.02 ug,h)2.12 Ilg, i) 0.096 ug, j) molecular weight markers.(B) Gel was blotted into nitrocellulose for 16 h and detected with 7.2ug/ml chicken anti-TIMP-l antibody followed by rabbit anti- chicken antibody (1/100000). Colour was developed by NBT/BCIP system.
kDa kDa
94 94
66 66
45 45
30 30
16 16
14 14
a b c d e f 9 h
A
kDa kDa
94 66 45 30 16 14
a b c d e
B
f 9 h
-45 -30 -16 14
Figure 5.6 MMP-9 release during Clq-coated latex bead uptake studies. Freshly isolated PMNs were resuspended in PBSG buffer and 1 x 106 cells were added to a) PBSG (75 ul)+ PMA (90 ng),b) serum (25 ul)+coated beads (2 x 107) +PBSG (50 ul),c) PBSG (75 ul) +coated beads (2 x 10\ d) PBSG (75 ul)+uncoated beads (2 x 10\ e) serum (25 ul)+ PBSG (50~1)+uncoated beads (2 x 10\t)PBSG (75 ul), g) serum (25~1)+PBSG (50
~1).Controls included h) diluted serum i) PMN homogenate. Suspensions were incubated for 20millRT,centrifuged(1300g, 1min) and supematants (2.5 ~1each) reduced with equal amount of treatment buffer and run in (A) Tris-tricine gel as follows a) 0.07~g,b) 1.54 ug,c) 0.09ug,d) 0.067ug,e) 1.48 ug,t)0.067ug, g) 1.02 ug,h) 2.12ug, i) 0.096 ug,j) molecular weight markers. B) blotted into nitrocellulosefor 16 h and detected with 219 ug/ml chicken anti-MMP-9antibody followed by rabbit anti-chickenantibody (1/100 000) and 1\1J3TIBCIP system.
The 94 kDa pro-MMP-9 form of MMP-9 seemed to be released due to the binding of Clq-coated beads by PMNs (Figure 5.6 B, a-g, compare with serum control lane h).
Increased release of MMP-9 was, however, triggered in the experiments where PMNs were incubated with coated beads in the presence of serum (Figure 5.6 B, b).
kDa
66 45 30
a b
c
d e f 9 hkDa
66 45 30
Figure 5.7 Zymographic results of MMP-9 release during Clq-coated latex bead uptake studies. Freshly isolated PMNs were re-suspended in PBSG buffer and 1 x 106cells were added to a) PBSG (75 Ill)+PMA (90 ng), b) serum (25 Ill)+coated beads (2 x 107) + PBSG (50 Ill), c) PBSG (75 Ill)+ coated beads (2 x 10\ d) PBSG (75 Ill)+uncoated beads (2 x 10\ e) serum (25 Ill)+PBSG (50 Ill)+uncoated beads (2 x 10\ f) PBSG (75 Ill), g) serum (25 Ill)+ PBSG (50 Ill). Two controls were included h) diluted serum i) PMN homogenates. The suspensions (2.5 ug each) were incubated for 20 min at room temperature and centrifuged (1 300 g, 1 min). Supematants were treated with equal amount of non-reducing treatment buffer and run in (A) Laemmli gel containing 1% (w/v) gelatin as follows a) 0.07 ug, b) 1.54 ug, c) 0.09ug, d) 0.067 ug, e) 1.48 ug, f) 0.067 ug, g) 1.02 ug, h) 2.12 ug, i) 0.096 ug, j) molecular weight markers. The gel was subsequently renatured in Triton X-lOO (2.5 % v/v) for 1 h and developed in development buffer (0.05M Tris-HC1, 5mMcsci,2mMPMSF, and 0.02% (v/v) Brij-35, pH 8.8) for
16 h. The gel was subsequently stained with 0.1 % Commassie blue and destained.
Zymogram results for MMP-9 showed that only the 2: 94 kDa forms of pro-MMP-9 secreted or in the PMN homogenatewere active (Figure 5.7 a-g and i, respectively). No activity was detected in serum (Figure 5.7 h) and activity comparable to that seen in non-PMA stimulated cells was observed in the sample in which PMNs were incubated with PMA (Figure 5.7, a).
kDa kDa
94- 94
66- 66
45- 45
30- 30
16 14
MW a b
c
de
f 9 MWkDa
A
kDa
94 66 45 30
16 14
94 66 45 30
16 14
9 f
e
dc
b aB
Figure 5.8 MMP-9 in the pellet from during Cl.q-coated latex bead uptake studies. Freshly isolated
6 .
PMNs were re-suspended in PBSG buffer and 1 x 10 cells were added to a) PBSG (75 Ill)+ PMA (90 ng),b) serum (25 Ill)+coated beads (2 x 107) +PBSG (50 Ill), c) PBSG (75 Ill)+ coated beads (2 x 10\ d) PBSG (75 Ill)+uncoated beads (2 x 10\ e) serum (25 Ill)+PBSG (50 Ill)+uncoated beads (2 x 10\ f) PBSG (75 Ill),g) serum (25 Ill)+PBSG (50 Ill),(MW) molecular weight markers.The suspensions were incubated for 20 min at room temperature and centrifuged (1 300 g, 1 min). The pellets were reduced with equal amount of treatment buffer and run in (A) Tris-tricine gel (B) The gel was blotted into nitrocellulose for 16 hours and detected with 219 ug/ml chicken anti-TIMP-l antibody followed by rabbit anti-chicken antibody (l/100 000). Colour was developed by NBTIBCIP system.
MMP-8 is also able to degrade gelatin. However, MMP-8 activity was absent in zyrnograrns showing that there was no significantrelease of specific granules (Figure5.7).
No TIMP-l was detected in pelletsamples indicating that, under the test conditions, all TIMP-l was released(results not shown). Detection for MMP-9 ,however, revealed a>
94 kDa, a 65 kDa, 25 kDa and a 15 kDa band in most samples (Figure 5.8 B). As maximum release of MMP-9 was observed in PMNs incubated with coated beads and serum, low level of MMP-9 was detected in the pellet confirming that MMP-9 was mostly released (Figure 5.8 B, b). Like in supernatant samples, lower bands were also observed in pellets which wereincubated inserum and may represent degradedproducts of MMP-9.
5.8 Bead processing for uptake studies
In order to have a clear picture of the effect ofMMP-9 and TIMP-l release on Cl q- mediated uptake of latex beads in the test system, the third test in the triplicate set of tests set up as required in 5.7.2 was processed for electron microscopy. Robert and Quastel (1963) had previously showen theuptakeofIgG coated or uncoated polystyrene beads by PMNs. However, uptake of Clq coatedpolystyrene beads had not been studied before.
5.8.1 Reagents
Manipulations involvingOS04wereall carried outin a fume hood.
Sodium cacodylate buffer CO.2 M (CHiliCAsOzNa)). Sodium cacodylate (2.14 g) was dissolved in 80 ml of purified water, the pH adjusted to pH 7.2-7.4 by the addition of HCI and the solution was made up to 100ml,
3% (w/v) Glutaraldehyde in phosphate buffer. Glutaraldehyde (3 g) was dissolved in 12 ml of purified water, stock phosphate buffer (0.2 M), 25 ml, was added and made up to 80 ml with purified water. The pH was adjusted to pH 6.8 - 7.4 and made up to 100 ml with purified water.
Fixative solution, 3% (m/v) glutaraldehyde in 0.05 M sodium cacodylate buffer. 0.2 M cacodylate buffer (25 ml) and 25% (w/v) glutaraldehyde (12 ml) were made up to 80 ml with purified water, the pH was adjusted to pH 6.8-7.4 with HCl and the volume made up to 100 ml.
4% (w/v) Osmium tetroxide.OS04 (1 g) was dissolved in 25 ml of purified water.
2% (w/v) Osmium tetroxide in 0.05 M sodium cacodylate buffer. 1 ml of (0.2 M stock sodium cacodylate buffer, 2 ml of 4% OS04 and 1 ml of purified water) was mixed in a fume hood.
EPON-ARALDITE. EPON 812 (1 part) and ARALDITE CY212 (1 part) (Inbed, Electron Microscopy, Washington, US.A.) and dodecenyl succinic anhydride (DDSA) (3 parts) (Agar Scientific Limited, Cambridge, U.K.)were mixed by dissolving.