Figure 3.

CRP recruits C1 and MASP1 to the surface of MSU crystals. (a) 40 µg/ml purified CRP or vehicle was added to serum and plasma (hirudin) from the same healthy male donor (CRP concentration of 0.7 µg/ml). Serum and plasma was incubated with MSU crystals (90 mg/ml) for 45 min at 37 °C. Crystals were washed extensively and bound proteins were eluted in SDS buffer, separated on a polyacrylamide gel and stained with coomassie. (b) 40 µg/ml purified CRP or vehicle was added to plasma of three donors, two male (M), one female (F). Numbers indicate original CRP concentration in µg/ml. Each plasma was incubated with MSU or PC-agarose and bound proteins were eluted as in a. Eluted proteins were subjected to Western blot analysis using the indicated antibodies. Protein names are indicated at the expected molecular weight. Cleaved/active forms of proteins are indicated with an *. Background signals due to inefficient stripping are indicated with a #. SAP antibody shows a background band at the position of CRP (o), which is likely due to cross-reactivity. (c) Five distinct preparations (4 lots, one of which untreated and sonicated (s)) of MSU crystals were incubated with pool serum containing vehicle or 40 µg/ml purified CRP. Bound proteins were eluted and analyzed as in b. (d) Purified CRP was added to NHS (originally containing 0.4 µg/ml CRP) to a concentration of 0, 30, or 100 µg/ml and was incubated with four distinct preparations of MSU crystals (lots 1-4) for 30 min at 37 °C. Bound proteins were eluted as in a and subjected to Western blot analysis using C3 antibody. Signal for full length C3 (>170 kDa) and its degradation product C3c α2 (39 kDa) were quantified by densitometry and normalized to the intensity of full length C3 in the absence of added CRP. (The corresponding Western blot is shown in Fig. S5). A paired two-tailed t-test was used to compare vehicle (0) with 30 µg/ml purified CRP. (e) Two distinct lots of MSU crystals were incubated in 4 individual human sera with 0 or 40 µg/ml purified CRP added for 30 min at 37 °C, extensively washed and stained with rabbit anti SC5b-9 plus anti rabbit PE. MSU crystals were analyzed using a flow cytometer. Median fluorescence intensity (MFI) of PE / 1000 is shown. A paired two-tailed t-test was used to compare vehicle (0) with 40 µg/ml purified CRP. Experiments from b-e are representative of at least 2 independent experiments. Uncropped images of the gel and Western blots are shown in Fig. S4 and S5.