1
Supplemental data
HPLC
HPLC flow rates and protocols for different columns were as follows: Zobrax GP250:
120 mM NaCl and 20 mM phosphate at pH 7, 1ml/min, isocratic elution; Mono Q: 10
mM monoethanolamine at pH 9.65 (buffer A) and 10 mM monoethanolamine with 1.5 M
NaCl (buffer B) at pH 9.65, 0.5 ml/min, 0-5 min, 100% buffer A, 5-25 min, 100 % buffer
B; Superdex 200, 120 uM NaCl at pH 7 or water, 0.5 ml/min, isocratic elution; C18:
unbuffered water (buffer A) and 80:20 acetonitrile:water (buffer B), 1 ml/min, 0-5 min,
100% buffer A, 5-20, 0-60% buffer B. For large liver cell preparations, Superdex 75 HR
10/30 column (Pharmacia) was used in place of GF-250 with identical conditions except
for a flow rate of 0.5 ml/min. The cytosol and fractions were lyophilized and suspended
in smaller volumes of water, filtered through 0.45 and 0.2 um membranes before
injection into the HPLC or animals, except where noted. All reagents were HPLC grade.
Mass Spectrometry
Trimethylsilyl (TMS) derivatives were formed by addition of 25 µl of silylating reagent
(N,O-bis-(trimethylsilyl)trifluoroacetamide,
pyridine,
hexamethyldisilazane
and
trimethylchlorosilane; 13:2:1:10 v:v) and heating at 100oC for 30 minutes. For GC EIMS,
two µl of this solution was analyzed by GC-MS using a Waters Quattro-II triple
quadrupole GC-MS system with a J & W Scientific DB-23 fused silica capillary column
2
(0.25 mm X 30 m, 0.25 µm phase thickness) in the splitless injection mode with He as
the carrier gas at 0.9 ml/min. The column temperature was programmed from 100oC to
255oC at 10oC/min. Injector and transfer lines were maintained at 225oC. Positive ion
electron impact ionization was used with the source temperature at 200oC and the
ionization energy at 70 eV. Full mass spectra were acquired from m/z 40 to 800 at 1 sec.
intervals. For ESI MS, one twentieth liver equivalent or 4 x 107 UV-treated 3T3 active
fraction from the C18 column was suspended in 50/50 methanol: water for the injection.
Negative ion electrospray mass spectra were acquired by direct infusion at 3 µl/min using
a Finnigan LCQ quadrupole ion trap mass spectrometry system with the electrospray
voltage at 4 kV and the capillary at 175 V. MS2 and MS3 product ion spectra of m/z 167
and 124 were acquired with normalized collision energies of 42% and 30%, respectively.
Preparations of other crystals and particles
Alum and 1 um latex beads (polysciences) were purchased. Basic calcium phosphate
crystals were precipitated from saturated solutions of calcium phosphate followed by
multiple boiling and washing in NaOH; this procedure produces crystals very similar to
those formed in human joints27. Allopurinol crystals were prepared by dissolving its
powder in 0.1 M borate (pH8.5) at 55oC. Filtered solutions were gradually cooled to RT
during which time 0.1N NaCl was added dropwise.
washed with ethanol and acetone and then air dried.
The precipitated crystals were
3
27.
Evans, R. W., Cheung, H. S. & McCarty, D. J. Cultured human monocytes and
fibroblasts solubilize calcium phosphate crystals. Calcif. Tissue Int. 36, 645–650 (1984).
4
Supplemental figures
Supp Fig 1.
Electrospray ionization MS analysis of commercial uric acid.
The
parameters were identical to Fig 2. A. m/z of -335 was a homodimer of uric acid in this
preparation. B, C and D were identical to Fig 2 C, D and E.
Supp Fig 2. Uric acid adjuvant effect in C57BL/6 mice. The assay was performed as in
Fig 3, A, B and C, except that 2 ug of ovalbumin/latex beads, SIINFEKL peptide, and
OVA-transfected EL4 (EG7, triangles)/EL4 (squares) cells were used in place of gp120
beads, gp120 peptide, and 15-12/18neo target cells respectively.
Supp Fig 3. 100 ul of plasma from heparinized blood from mice treated with uricase and
allopurinol (see methods) or control mice was mixed with 100 ul PBS and filtered
sequentially through 0.45 um, 0.2 um and 10 Kd cutoff filters. 150 ul of the final filtrate
was injected into Superdex 75 HR 10/30 HPLC column. The data show the UV
absorption at 292 nM of the uric acid containing time point (53.75th minute).
Supp Fig 4. Five ul of FITC-conjugated latex beads was added to Balb/c dendritic cell
cultures (in 12 well plates) in the presence (heavy line) or absence (thin line) of MSU
crystals (500 ug /ml, see methods) and incubated for 5 hours. Internalization of the FITC
beads was quantified by flow cytometry in the presence of Trypan blue (1% final
concentration) to quench the fluorescence of extracellular beads, as previously
reported11,25. Untreated DCs are shown as the shaded curve.
5
Supp Fig 5. MSU crystals stimulate C57BL/6 and C.C3H lps-d (LPS-nonresponsive)
dendritic cells. Same as Fig 4 C, except that C.C3H lps-d or C57BL/6 dendritic cells
were used in place of Balb/c dendritic cells.